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Does chromosome cross occur over male and female or vice versa?

Does chromosome cross occur over male and female or vice versa?


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My understanding is homologous chromosome pair, which means male and female chromosome inside the DNA. So if that's homologous how does male do with female? Does it flip over and change direction, that could not happen as male chromosome 1 (say) is indistinguishable from female? And what about the XY chromosome in male,say? I know some are pseudoautosomal, but most are normal sex chromosome nucleotides. So what about them?


The question is unclear to me but hopefully that will help a little bit.

My understanding is homologous chromosome pair, which means male and female chromosome

There is no male and female chromosome. Chromosomes have no sex. In humans, to the exception of chromosomeYall chromosome can be found in individuals of any sex.

There are paternally inherited chromosomes and maternally inherited chromosomes though. As you said the maternally inherited chromosome is indistinguishable from the paternally inherited chromosome when comparing their sequence (they may well differ in terms of epigenetic changes).

chromosome inside the DNA.

A chromosome is not inside the DNA. DNA is the name of the very long molecule from which a chromosome is (mainly) made of. DNA can be organized in different forms one of which is into chromosomes (another one would be a plasmid for example). While it would still sound funny, it is more correct to talk about DNA inside a chromosome than talking about chromosome inside DNA.

So if that's homologous how does male do with female?

This is very unclear… do what? Why would the paternally inherited chromosome (assuming this is what you mean by male chromosome) do anything with the maternally inherited chromosome rather than the other way around? While it is clear from the what follows that you would like to talk about recombination, I don't get where you're heading.

does it flip over and change direction, that could not happen as male chromosome 1(say) is indistinguishable from female?

I don't get what kind of "flip" you are thinking about. You probably should have a look at the wikipedia article for cross-over.

In short, synapsis are formed during prophase I of meiosis and when segregation happen synapsis are resolved with a probability of $frac{1}{2}$ of causing a recombination event. Synapsis occur as they allow matching pairs of homologous chromosomes together that will ensure their separation during anaphase 1. You should probably take some time to review the meaning of this paragraph.

And what about the XY chromosome in male,say?

Sorry, that's a bit unclear too.

I know some are pseudoautosomal, but most are normal sex chromosome nucleotides. So what about them?

Sounds like you misunderstand what a sex chromosome is and what pseudoautosomal mean.

In short, in humans, there is only one pair of sex chromosomes (XXorXYdepending on the sex). The other 22 pairs of chromosomes are autosomes. There is no such thing as a pseudo-autosomal chromosome in humans but there is a pseudo-autosomal region on theY-chromosome.

Most of theYchromosome never recombine (while theXchromosome recombine in females only) as it is never present with anotherYchromosome in the same cell. There is only a short sequence on top of theYchromosome that recombines with theXchromosome, this region is called the pseudo-autosomal region. This region probably still exist to allow separation ofXandYchromosomes during anaphase I.


Please also note that you seem very focus on humans (without saying it and maybe without realizing it). The diversity of sex-determination is huge. You might want to have a look at Do males of all sexual species have Y chromosomes?


There is a difference between the processes of meioses and mitoses. Meiosis typically leads to 23 chromosomes in a new cell while mitoses typically leads to 46 chromosomes in a new cell. Because every normal human being must have 46 chromosomes, you need to get 23 from your father and 23 from your mother. Meiosis creates cells that are necessary for humans to procreate. Mitosis creates cells that help an individual grow.

Almost every cell in your body has its own 46 chromosomes. Red blood cells, for example, are cells that don't have chromosomes. When a cell has 23 chromosomes it is called a haploid cell, and when it has twice that much it's called a diploid cell.

Chromosomes are not specific to a gender. The sex chromosomes only confer gender depending on whether you get one copy of each of X and Y, or two copies of the X chromosome. So you could have a man passing 23 genes to his daughter and then the daughter passes some of those chromosomes ,after genetic recombination, to her son or daughter. It doesn't matter. The separation is more or less random.

During genetic recombination the homologous chromosomes cross-over by DNA strands being cut by biomolecule processes. The image of this process is called a Holliday junction. So they do flip over and change direction, but they are only strands (aka strings) of DNA.


Changes in Number and Arrangement of Genes

A single chromosome may result in a loss of small piece at the terminal end is said as terminal deficiency or deficiency. Sometimes a chromosome may break, however, at any two points, releasing an intercalary segment which may result ring or rod shaped, if its broken ends are fused.

If a centromere is present in the broken segment, it survives as a small but deficient chromosome. If there is no centromere with the broken segment, it is said to be deletion and is soon lost during the nuclear division.

A chromosome which shows deletion, becomes a deficient chromosome. In the case of diffuse centromere, the deleted segment of the chromosome retains its survivality and is added as an extra-chromosome to the original set.

In this type of structural change, the total gene content of the organism is affected. If ABCDEFG are the genes in a chromosome, then ABCDE is deficiency and ABFG is deletion. According to Roberts (1975) all deficiencies are really interstitial or deletion because of universal presence of telomere.

Cytological effect of deletion:

In the deletion heterozygote, pairing of chromosomes takes place normally but in the region of the deletion where a number of genes are missing, the normal chromosomes is pushed out in the form of a loop and this is generally referred to as buckle formation. Deficiency reduces the amount of crossing-over.

Genotypic and phenotypic effects of deficiencies:

Deficiency is lethal due to loss of genes. Individuals with homozygous deficiency fail to survive because a complete set of genes is absent. When a segment is lost from only one member of a homologous pair, forming a deficiency heterozygote, the individual survives but shows abnormal or unusual phenotypic effect.

The loss of very small segment of a chromosome by deletion behaves like a Mendelian unit in inheritance. Therefore, very small deficiencies are some times mistaken for gene mutation.

In other words, small deletions might not prevent the development of the organism, but they produce some mutant character in the individual. A classical case of deficiency was discovered and worked out by Bridges in 1917.

A mutant character in Drosophila called notch produces a notched margin of the wings. This is due to a small deletion in a certain part of the X-chromosome. It inherited as sex-linked dominant in the female and lethal in the male.

In the F1 offsprings the females with notched wings were all white eyed, while the normal expectation would be red eyed forms, since red is a dominant character. In the absence of a dominant allele due to deletion, the recessive gene finds expression phenotypically. This kind of unexpected expression of a recessive character which is caused by the absence of a dominant allele is called psendodominance.

(Deletion may be terminal in which missing segments are at the end of chromosome and they may be intercalary in which missing parts are in the middle of the chromosome.)

(ii) Duplication:

The presence of a part of a chromosome in excess of the normal complement is known as a duplication or sometimes a segment or a part of the chromosome becomes repeated in the same chromosome. These additional duplicated segments are called duplications.

Occasionally a nucleus is found to be aberrant in that it has extra material beyond that found in the normal chromosomal complement. The extra material may be either in the form of whole chromosomes or extra sets of chromosomes (if the broken piece has a centromere, it is included as an extra chromosome) or it may be just a part of the chromosome.

The last one is referred to as duplication which is different from the first two. That is, addition of one or more genes as a result of which the organism carries the same segment of the chromosome repeated in its haploid chromosome complement.

(Under deletion and deficiency loss of one or more genes is exhibited. In duplication addition of one or more genes, as a result of which the organism carries the same gene repeated in its haploid chromosome complement).

Kinds of Duplications:

(a) Extra-chromosomal Duplication:

If a centromere is present in the broken out piece, it behaves like an independent additional or extra chromosome.

The duplicated segment lies by the side of the same genes, in the chromosome. The duplicated genes lie in the same order as in the normal chromosome e.g., if the order of genes in the normal chromosome is ABCDEFGH.IJKL the tandem duplication shall be ABCDECDEFGH.IJKL (full stop represents the centromere).

(c) Reverse tandem Duplication:

In this case the order of the genes in the duplicated segment of a chromosome is just the reverse of the original sequence. For example, in the above case, it would be ABCDEEDCFGH.IJKL.

(d) Displaced Duplication:

In some cases the duplicated segment does not lie adjacent or near to the normal segment. Depending on, whether the duplicated portion, is on the same side of the centromere (homo-brachial) or on the other side as the original (heterobrachial). In this case we would have ABCDEFGCDEH.IJKL.

(e) Translocation or Transposed duplication:

In this case the duplicate segment is attached to a non-homologous chromosome. The duplicated region can be transposed to a non-homologous chromosome interstitially (or intercalary) or terminally (terminal). In this case, it would be MNOPCDEQ.RSTUV.

In Drosophila Bridges found that some individuals which are definitely homozygous for recessive genes were found to exhibit the dominant characters. This on analysis, has been found due to extra chromosomal material carrying the dominant gene.

If the extra chromosomal material is provided with a centromere it might exist as a separate chromosome, but when it is without a centromere, it is attached to one of the normal chromosome. Duplications are less harmful than those of the effects of deletions.

A small segment of a chromosome might some times be duplicated as a result of unequal crossing over and such a duplication is referred to as repeat. In Drosophila mutant bar eyes which are narrow and constricted are due to small duplicated gene. Duplication has helped in evolution. Due to increase in number of genes, it is possible for different mutations to appear in the same gene without affecting the normal functions of the organism.

Changes in the Arrangement of Gene Loci:

(i) Translocation:

The term translocation was used by Bridges and Morgan in 1923 to indicate the unusual behaviour of chromosomes during which a segment from one of them becomes attached to another chromosome. This is the important and the most complex of all the chromosomal aberrations. During this process, a deleted segment moves from its normal position in one chromosome to a new situation in a different chromosome.

In translocation, segments of equal or unequal lengths are exchanged between two members of a homologous pair, or between non-homologous chromosomes. This exchange between chromosomes is called a mutual or reciprocal translocation.

In simple translocation only a piece of chromosome becomes attached to other chromosome without exchange. It is, however, suspected that simple translocation is also reciprocal but one of the segments is very small.

Besides simple and reciprocal translocations one more type of translocation is visible which involves breakage at three points but union at two points known as Shift translocation. Furthermore in allelosomal translocation interchange of segments occur between non-homologous chromosomes.

Translocations do not involve any loss or addition of chromosomal parts but simply rearrangement of its parts or genes in the chromosomes, not the quality or quantity of the genes.

For this reason, they are some times referred to as chromosomal rearrangements. They reduce the crossing-over by hindering chromosome pairing. Individuals carrying the rearrangements are phenotypically normal unless the relations of a gene or genes to adjascent genes, affect the phenotypic expression i.e., “position effect.”

Another rare type of “lateral translocation” has also been observed, in which a trans-located segment becomes attached to the sides of the receiving chromosome.

Translocation was referred to as “illegitimate” crossing-over in the beginning, indicating the two processes, resemble each other. The chief similarity between the both is as (i) there is breakage of chromosomes followed by union and exchange of segments.

But translocation and crossing-over differ from each other in the following respects or points:

(i) Crossing-over occurs as a natural and normal process during which segments of non-sister chromatids of equal size are exchanged between homologous chromosomes. The non-sister chromatids show breakage at corresponding points and new combination of genes are formed but no new gene is introduced. The percentage of crossing over may also be predicted in most cases.

(ii) In a typical translocation there is an exchange of non-homologous chromosomes. Thus genes from outside are introduced into the linkage group. Translocations are never under prediction and do not follow any set rule. It is not necessary that the exchanged segments may be of equal size.

Cytological effect of translocation:

The genetic techniques for detecting and studying translocations will be more easily understood when the cytological phenomena produced by translocations are known. Suppose that two chromosomes having respectively the genes ABCDE.FGHI and LMNOPQ.RST exchange segments and give rise to translocation chromosome LMNDE.FGHI and ABCOPQ.RST.

The individual thus formed receives from one of its parents the normal and from the other parent the translocation chromosomes. Such an individual is a translocation heterozygote. Since the chromosome pairing (synapsis) at the Meiotic prophase I, zygotene, is caused by specific attraction of homologous segments containing allelic genes, a translocation heterozygote may be expected to produce a cross shaped pairing.

Occurrence of crossing over in each of the four arms of the cross will result in formation of chiasmata in each arm. Instead of bivalents i.e., a pair of synapsed homologous chromosomes there will be formed a quadrivalent.

A group of four associated chromosomes, each member of the group being partially homologous to two other chromosomes in the group. The quadrivalent will appear at diakinesis and at metaphase I of the first meiotic division as a circle or ring of four chromosomes, which may be either twisted as shown in figure, left, or open as in the centre drawing of the same figure.

If chiasmata fail to be formed in one arm of the pachytene or diplotene cross, the ring is transformed into an open chain of four chromosomes. Such rings or chains of chromosome were observed and interpreted by Belling at Meiosis in the Datura and were thereafter found in maize, peas, wheat, Tradescantia and other plants and in some animals. They occur regularly in many evening primroses plant which bear yellow flowers.

There are different ways in which the chromosomes associated in a ring or chain may be distributed to the gametes formed as a result of meiosis. The two original chromosomes, ABCDE.FGHI and LMNOPQ.RST may go to the same gamete, and the translocation chromosome, ABCOPQ.RST and LMNDE.FGHI, to another gamete.

It may be noted that in each of these gametes, every gene symbolized by a letter occurs once only, as gametes are formed by normal individuals. On other hand, if chromosomes adjascent in the ring go to the same pole at the meiotic division, the four kinds of gametes are formed.

The common property of these four kinds of gametes is that they carry certain genes twice and do not have some genes at all. In other words, they carry duplications for some and deficiencies for other genes.

Translocations result into changed linkage relationships between genes. New linkage groups are established. Independently assorting genes becomes linked and linked genes begin to show independent assortment, provided their linkage groups are changed because of translocation.

[Heterozygous translocations are semi-sterile. In animals, some times, the unbalanced gametes are visible but the zygotes formed by them are not able to undergo normal development and differentiation. In plants like Rhoeo and Oenothera translocation heterozygotes have become stable due to the presence of balanced lethals or lethals that are not expressed under heterozygous conditions],

(ii) Inversion:

There are translocations occurring within single chromosome. It was primarily demonstrated by Sturtevant and Dobzhansky in the salivary gland chromosome of Drosophila.

Inversion involves a rotation of a part of a chromosome or a sets of genes by 180° on its own axis. Breakage and reunion is essential for reversion to occur and the net result is neither a gain nor a loss in the genetic material but simply a rearrangement of the gene sequence. They may be either terminal (occurrence at the end of chromosome) or intercalary when changes occur in the middle of chromosome.

Inversions that include the centromere are known as pericentric inversions where as those which do not involve the centromere are known as paracentric ones. If the normal order of genes in a chromosome is ABC.DEFG the sequence or order in paracentric and pericentric inversions will be ABC.DGFE and AED.CBFG respectively.

In individuals which are homozygous for inversions, zygotene and pachytene is normal because of similarities of abnormalities in the homologous chromosomes. But heterozygous inversion is small enough, the opposing inverted regions fail to pair and crossing-over is prevented in this part of the chromosome.

A crossing-over in the inverted region of heterozygous paracentric inversion results in to a chromosome with two centromeres and an acentric segment. As the two centromeres of dicentric chromosome move towards two opposite poles, a chromatid bridge is formed at anaphase I. The acentric segment is not attached to the spindle, lies free in the cytoplasm and is not free properly. Thus, meiotic separations are usually abnormal.

In case meiotic separation is normal, four types of gametes are formed-one with a normal gene sequence, second with an inverted gene sequence, third with a dicentric chromosome and duplication of some genes and fourth with an acentric chromosome and deletion of some genes. The later two types of gametes are usually not viable with the result that heterozygotes for paracentric inversions are highly sterile and only parent like a progeny are produced.

In other words, crossing-over is prevented due to a paracentric inversion. Crossing-over in a heterozygous pericentric inversion does not result into a chromatid bridge, but results in to deletions and duplications in the gametes. Therefore, pericentric inversions also apparently prevent crossing over.

Pericentric inversions involving unequal arms result in to drastic changes in the morphology of chromosomes. For example, metacentric (V-shaped) chromosomes can be transformed in to rod shaped (acrocentric) ones or vice-versa.

As inversion homozygotes are fertile and inversion heterozygotes are sterile, leads to the establishment of two groups of organisms within same species.

Thus, inversion has been useful in maintaining a heterozygous condition, thus useful in the origin of new species. In inversion, crossing over is suppressed and only parental progenies are produced. Recessive lethals can be of added advantage because heterozygotes for them will be viable but homozygous non viable.

The various chromosomal rearrangements described above often causes visible changes in the organisms. These visible phenotypic changes produced as a result of change in chromosomal segments constitute position effects.

This changed behaviour of genes due to rearrangement has been studied in Drosophila and first discovered by Sturtevant and Bridges in 1925. They found that the formation of Bar eye in Drosophila is due to position effect.

Lewis has classified position effects into two categories:

(i) Variegated type:

This type of position effects have been studied by Muller and others. These effect result in somatic instability of gene action. Variegated position effects result in the diversification of a character usually seen in a particular structure or area of the body specks of different colours, for e.g., may occur in the eyes of Drosophila following rearrangement of the ‘w’ (white eye) locus.

Inversions or translocations that place ‘w’ near the heterochromatin may cause white variegation or mosaicism for eye colour. This concept of heterochomatinisation producing variegated position effects hold goods in various eye colour examples.

A large number of structural rearrangement in Drosophila produce somatic stable position effects. These include barred eye, hairy wings etc. The classical examples of bar eye was done by Sturtevant and Bridges. In this bar character, eyes become narrow and number of facets have reduced. This arise as a result of duplication of genes.

There is an approximate quantitative relation between number of chromosome segment and the size of the eye. When segment was duplicated in various ways the barred character became prominent with the reduction in facets producing barred, double barred and ultra barred eyes. Two hypothesis have been put forward to explain the mechanism of position effect.

This theory assumes that position effects are caused as a result of local interaction between the gene products of adjascent loci or is based on the change in the chemical environment in which the gene is placed after the rearrangement. It implies that single gene is not completely independent in producing the position effects but action is influenced by the neighbouring genes.

2. Structural hypothesis:

According to this view, there is produced a sort of physical change in the gene locus itself where break occurs, nucleoprotein molecules may be deformed during the physical change.

Non-Disjunction of Chromosomes:

Non-disjunction means non-separation of pairs of homologous chromosomes during meiosis. Such case was first reported by Bridges (1913) in Drosophila. He found that sometimes in Drosophila egg the two chromosomes do not separate after synapsis and pass to the same pole, leaving the other without and ‘X’ chromosome.

Hence three kinds of eggs are produced as given below:

(i) Normal eggs, each containing one X chromosome,

(ii) Eggs containing two X chromosomes.

(iii) Eggs without the X chromosomes.

Thus, a quantitative abnormality or unusuality of the X chromosome occurs. White eye colour in Drosophila is a recessive sex-linked character. When a white eyed female is crossed with red eyed male, there is criss-cross of inheritance.

In the F1 offsprings, males have white eyes and female red eyes. But there are some exceptions and about one in 2000- 3000 F1 offsprings show red eyes in male and white in female. This has shown to be due to non-disjunction of the X chromosomes.

2. Secondary Non-Disjunction:

All the exceptional males without the Y chromosome are sterile although they are quite males in appearance and in behaviour. However, the white eyed ‘X X Y’ females are normal and fertile. Bridges crossed them to normal red-eyed males and observed in their offspring the secondary non-disjunction. In their progeny about 96% of the daughters have red eyes and 4% white eyes. Among the sons, about 96% are white eyed and 4% red eyed.

During the reduction division in XXY females, the X and Y chromosomes are distributed in different ways and four kinds of eggs are formed.

(i) Eggs with a single X chromosome

(ii) Eggs with a X and a Y chromosome

(iii) Eggs with 2X chromosome

(iv) Eggs with a Y chromosome.

Fertilized by sperms of normal red eyed male, three eggs should produce eight different types of zygotes. 3/8 types of zygotes will not occur with equal frequency. Estimation of the figure shows several ways of testing the validity of this complicated working hypothesis.

All the white eyed female and some of the red eyed ones must carry not only two X chromosomes but also a Y chromosome. Bridges not only made these predictions but verified them by cytological examination of the various classes of the flies.

Non-disjunction of X chromosomes in Drosophila leads to the appearance of zygotes which have one chromosome more (XXX, XXY, XYY) or one chromosome less than normal flies, since the Y chromosome contains relatively few genes, flies with extra Y chromosomes appear to be normal, while males which lack a Y chromosome, differ from normal only in being sterile. On the contrary, the presence of an extra X chromosome (XXY) is usually lethal.

Non-disjunction occurs occasionally not only for the X and Y chromosomes but for other chromosomes as well. It results in the production of zygotes which have one of the chromosomes of the normal complement in triplicate (Trisomies, 2n+1 types) which have chromosomes represented only one instead of twice (Monosomies, 2n-1 types).


Abstract

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.


Top 3 Fundamental Laws of Genetics

The following points highlight the three fundamental laws of genetics proposed by Mendel. The laws are: 1. Law of Segregation 2. Law of Dominance 3. Law of Independent Assortment and Di-Hybrid Cross.

1. Law of Segregation:

According to Altenburg, this law may be defined as “Non-mixing of alleles i.e., the allele for tallness does not mix with the allele for dwarfness in the hybrids.” Offspring’s arising from two parents receive contributions of hereditary characteristics from them through gametes. These gametes are the connecting links between successive generations.

The contrasting characters such as tall and dwarf stems of peas are determined by something that is transmitted from the parents to the offspring through the gametes are called factors or genes. The important point is that different factors such as those for tallness and dwarfness (D and d) do not blend, contaminate or mix with each other while they remain together in the hybrid.

Instead, the different factors separate or segregate pure and uncontaminated passing to two different gametes produced by the hybrid and then transmit to the different individuals or the offspring’s of the hybrid. Each gamete carries one of the two members of a pair of contrasting or alternative factors i.e., either for tallness or dwarfness (D or d) and never both.

D d (F1 hybrid tall) → factor D and d remain together pure

The simplest conventional or custom method of denoting these Mendelian factors is to give each a letter, the dominant factor being represented by capital letter and recessive by small letter. In the cross of pure bred tall and dwarf plants let D stand for the gene for tallness and d for alternate form of this gene which results in dwarfness of the stem. D and d are called alleles or allelomorphs.

Since an individual develops from the union of two gametes produced by the male and female parents. It receives two alleles D and d. The true breeding tall plant may be represented as DD and its gamete as D and the true breeding dwarf plant as dd and its gamete as d.

When the two plants are crossed an egg (D) is fertilized by the male gamete (d) or vice- versa. The resulting hybrid zygote will have both D and d. Thus the two alleles of a gene are represented by the same gene symbol and are differentiated from each other by their first letter being in capital or small (D or d).

A gene can be represented by a symbol derived from the name of the character it governs. The gene controlling length of stem as dwarf in pea may be represented by the small letter ‘d’ and the symbol for the allele producing the dominant form of character is the same as that for the recessive allele, but the first letter of this symbol is in capital. For example, tall stem is dominant and is assigned D

According to the principle of segregation the alleles borne by the heterozygous tall plant (Dd) do not mix, fuse, blend or contaminate with each other, despite the fact that the phenotype of the F1 hybrid shows only the tall character, and it fails to give any visible indication of the presence of the gene (d) in the genotype. The alleles segregate when the hybrid organism produces gametes so that approximately half of the gametes will carry D and the other half d.

In fertilization the gametes combines at random. There is an equal opportunity for the different types of gametes to unite with each other. The male gamete may unite or fuse with female gamete with either D or d. The other kind of male gamete ‘d’ may also have an equal opportunity to unite or fuse with the female gamete D or d. Hence four recombination’s occur. One fourth (1/4) of them are homozygous tall plants having only the allele for tallness (DD).

The other half of them (two out of four) are heterozygous having both the alleles D and d. Since D is dominant over d, these plants are tall. One fourth (1/4) of them are homozygous plants having only the allele for dwarfness (dd). In F2 generation, tall and dwarf plants appear in the ratio 3 : 1 (3/4 tall and 1/4 dwarf plants).

Mendel tested the validity of factor hypothesis by applying further strict method by means of which it could be confirmed or disproved. In the F2 of his cross of tall plants with dwarf plants there were tall and dwarf plants approximately in the ratio of 3:1. Mendel’s interpretation of these results by means of the law of segregation shows that there are two kinds of F2 tall plants.

About 1/3 of them should be genotypically homozygous for tallness (DD). About 2/3 should be heterozygous (Dd) carrying both the dominant and recessive alleles (D and d). The validity of these predictions can be tested in actual experiments. The homozygous dwarf plants should breed true through all subsequent generations if self- fertilized or crossed with other.

All the plants although they look alike would not behave in the same way. About 1/3 of them homozygous with the genetic formula (DD) should breed true. But 2/3 of the F2 tall plants, the heterozygotes (Dd) should breed exactly like the F1 hybrid plants. They should produce tall and dwarf plants in the phenotypic ratio 3: 1 and the genotypic ratio 1:2:1. This is what Mendel obtained in his experiments. Thus the law of segregation has been confirmed in actual experiments.

Characters become separated or segregated in the second filial (F2) generation. Thus the factors responsible for hereditary characters are independent units, which although enter the crosses together but segregate out again as distinct characters. This law is by far the most important of Mendel’s discoveries. This law is some times called as the law of purity of gametes or law of the splitting of hybrids.

(Law of segregation means that when a pair of allelomorphs are brought together in the hybrid (F1), they remain together in the hybrid without blending and in F2 generation they separate complete and pure during gamete formation. This law is also known as law of the purity of gametes).

(The two alleles present in the F1 are able to separate and pass in to separate gametes in their original form producing two different types of gametes in equal frequencies this is known as segregation).

Main facts about segregations:

To summarize Mendel’s monohybrid cross experiment, following cardinal points are notable:

The hereditary differences among the individuals depend upon the difference in cellular units of genes or factors. These genes are hereditary units, control a particular character and are present at a fixed place in the chromosomes called loci. Thus genes for tall character in the peas shown by ‘D’ in chromosome is at a fixed locus and genes for dwarf character ‘d’ is at the same locus in the other chromosome.

Law of segregation itself shows the purity of gametes and their freedom from mixing or blending with each other. The gametes contain only one factor or gene and are pure for a particular trait or character governed by the same factor or gene of gamete.

3. Non-mixing of alleles in hybrids:

These genes or factors of heredity, whatever the nature may be, unite when derived from different parental sources in the hybrids from which they may be separated out during successive or subsequent generation and unmodified with the presence of other alleles in hybrids.

In summary, the cross between tall and dwarf pea is as follows:

The original tall and dwarf variety of pea constitute the first parental generation (P1). The hybrids produced by their cross constitute first filial generation (F1) and offspring of the hybrids constitute second filial or F2 generation.

Johansen (1911) proposed the following four terms to distinguish individuals among themselves:

An organism or hybrid or zygote in which both members of a pair of genes are alike (DD or dd) are referred to as homozygous (Greek: Homos = alike = zygos, yoke (bond or under bondage of another).

Individuals having identical genes (DD or dd) are called homozygous. Homozygous are always pure.

An organism or hybrid or zygote in which both members of a pair of genes are unlike (Dd) are termed as heterozygous (heteros = dissimilar). Heterozygous individuals are always hybrid. In the F2 generation, there is a ratio of 3 tall and 1 dwarf plant apparently but genetically, this ratio is 1 DD tall: 2 Dd tall: 1 dd dwarf.

3. Genotype and Phenotype:

Genotype is the term used to denote genetic constitution of an organism. It represents the total hereditary possibilities within the individual. In the monohybrid cross experiments, the hybrid plant of F1 generation is phenotypically tall but genetically it is a hybrid (Dd).

The external morphological feature of an organism constitute its phenotype or it is the term used to denote the visible characteristics of an organism or individual. It represents the sum total of all apparent characteristics of an organism regardless of it genetic make up or genotype.

In the F2 generation, 3 out of 4 (3/4) are phenotypically tall but genotypically one third (1/3) of them is pure tall and two third (2/3) hybrid tall with two contrasting allele.

What we observe or which is visible or otherwise measurable are called phenotypes. While the genetic factors responsible for creating the phenotype are called genotype. Phenotype is determined by the dominant alleles.

Monohybrid Back cross or Test Cross:

The cross between the F1 hybrid (Dd) to one of its parents (DD or dd) is called back cross while cross between F1 hybrid (Dd) and homozygous recessive parent (dd) is called test cross since it confirms the purity of gametes.

(i) The above cross between homozygous dominant (DD) and hybrid (Dd) is called dominant back cross and (ii) Cross between homozygous recessive (dd) and hybrid (Dd) is called recessive back cross. This recessive back cross has great importance in experimentation because phenotypic and genotypic ratios are identical. Hence recessive back cross is termed test cross to identify or test gamete nature or whether an individual is homozygous or heterozygous as shown below.

In case of Back cross:

Diagram showing Monohybrid back cross between F1 hybrid and dominant homozygous parent

Phenotype – 2 Tail: 2 dwarf (50% tall and 50% dwarf)

Genotype – 2 Tall: 2 dwarf (50% tall and 50% dwarf)

Diagram showing Monohybrid test cross between F1 hybrid and recessive homozygous parent (1 : 1).

2. Law of Dominance:

Mendel’s first experiments were crosses between pea varieties differing in only one visible character. These are monohybrid cross experiments.

A heterozygote (F1 hybrid) contains two contrasting genes, but only one of the two is able to express itself, while the other remain hidden. The gene which is able to express itself in F1 hybrid is known as dominant gene, while the other gene which is unable to express itself in presence of the dominant gene is the recessive gene. No doubt recessive gene is unable to express itself, but is transmitted to the next generation without change.

When Mendel crossed true breeding tall peas, with true breeding dwarf peas the first offspring’s formed were all tall plants.

The dwarf character appears to have been suppressed and tallness seems to dominate. Such characters like tallness, redness, roundness of seeds, yellow coloured cotyledons, inflated seed pods, green unripe pods and axial flowers, were called dominants and their respective alleles as dwarfness, whiteness, wrinkledness of seeds, green coloured cotyledons, constricted seed pods, yellow unripe pods and terminal flowers were called recessives.

The law of dominance, thus states that out of a pair of a allelomorphic characters (= alternative or contrasting characters) one is dominant and other recessive. Mendel found this fact to be true between all the seven pairs of characters studied by him. The pair of contrasting or alternative characters are called allelic pair or allelomorphic pair and each member of the pair may be regarded the allele of the other.

Thus the tallness and dwarfness are alleles of each other. The hereditary units which are responsible for the appearance of character in the offsprings or progenies have been called factors or determiners. Now these are called genes.

Four types of dominance are seen:

The phenomenon in which both alleles are expressed in the hybrid (F1) is called co-dominance. Blood group antigens of man is one of the best example of Co-dominance. It produces 1:2:1 ratio in F2.

2. Complete dominance or simple dominance:

It is the ability of one allele to mask or inhibit the presence of another allele at the same locus in the heterozygote or F1 hybrid.

3. Incomplete dominance:

If the F1 hybrids or heterozygotes are phenotypically intermediate between both homozygous type.

4. Over dominance:

The superiority of heterozygote or hybrid over its both homozygotes or parents (DD and dd) is termed as over dominance. Unlike complete, partial and co-dominance, over-dominance is not the characteristics of an allele but is the consequence of the heterozygous condition of the related gene.

3. Law of Independent Assortment and Di-Hybrid Cross:

Mendel discovered not only crosses in which the parent differed in single pair or characters, but also others in which the parent differed in two pairs. Such a cross, which includes two pair of contrasting characters at a time is called di-hybrid cross. The law of independent assortment is applicable to the inheritance of two or more pair of characters.

For a di-hybrid experiment, Mendel crossed two pea plants, one of which was homozygous for yellow and round seeds and the other for green and wrinkled seeds. Genes for yellow and round characters were dominant over the green and wrinkled characters described by the Mendel. The F1 hybrids produced as a result of this cross were yellow round which were heterozygous for both the alleles known as Di-hybrid.

Genotypes and Phenotypes of F2 offsprings:


The above phenotypic ratio, which Mendel obtained may be thought of as a monohybrid phenotypic ratio 3 : 1 multiplied algebraically by 3 : 1 that means (3: 1) x (3: 1) = 9: 3: 3: 1.

Although Mendel was not aware with the behaviour of chromosomes during meiosis even then he assumed that the members of each two pairs of factors (WW, ww) for the two pairs of contrasting characters (round/wrinkled) are separated independently or freely of the members of the other pair.

In brief, according to Mendel at the time of reduction division during gamete formation, the members of each chromosome (= genes or factors) pair segregate (or separate) from one another.

They do not dilute or affect the other pair and behave independently. The separation of chromosomes or genes belonging to one pair without reference to those belonging to the other pair at reduction division is known as independent assortment (or separation) of genes.

The dihybrid (GgWw) produces four kinds of gametes (parental or non-parental types or crossover or non-crossover types) namely GW, Gw, gW, gw which by self fertilization produced F2 generation in 16 possible ways. Since G (Yellow) and W (round) are dominant characters so whatever genes (G or W) will be, the seeds will show dominant characters.

Genotypically, typical di-hybrid will show following ratio:

1GGWW : 2 GgWW : 2 GGWw : 4 GgWw : 1 ggWW : 2 ggWw : 1 GGww : 2 Ggww : 1 ggww. Their phenotypic ratio will be 9 Yellow round: 3 Yellow wrinkled: 3 Green round: 1 Green wrinkled.

Fractional Method of Calculated Ratio:

The checker board method of determining Mendelian ratio given by Punnet is useful in certain aspects. It represents graphically all the essential steps like formation of gametes, their union to form zygotes and resulting phenotypes. But its disadvantage is that it is time consuming and many other errors may come in it. Therefore, M.D. Jones (1947) described fractional method to determine ratios which is algebraic in nature.

(ii) F2 di-hybrid phenotypes:

The genotypic ratios may be obtained by dividing the dominants in to homo and heterozygotes i.e.,

If we cross the di-hybrid (GgWw) with the homozygous recessive parent (ggww) then di-hybrid will produce four types of gametes (GW, Gw, gW, gw) while green wrinkled seeds will form only one type of gamete (gw).

This gamete becomes fused with four types of gametes thus producing four classes of offsprings as follows:

1 Yellow round: 1 Yellow wrinkled: 1 Green round: 1 Green wrinkled

Thus, a dihybrid test cross will give a genotypic and phenotypic ratio of 1: 1: 1: 1 because four different types of gametes will be produced by the F1 hybrid in equal numbers.

In case of di-hybrid cross, Mendel demonstrated the independent assortment (or segregation) of factors or genes. Likewise, tri-hybrid experiments were carried out by Mendel involving three pairs of characters.

For instance, he took yellow round grey seeds and crossed them with green wrinkled white seeds, the F1 progeny will be heterozygous for three genes and will phenotypically resemble the dominant parent. Each of these F1 progeny will produce 8 types of gametes and therefore 64 combinations of F2 progeny.

Results of tri-hybrid cross worked out by the forked line method:

Genotypes of F2 and their relative proportions:

Phenotypes of F2 and their relative proportions:

A tri-hybrid test cross will give a phenotypic and genotypic ratio of 1: 1: 1: 1: 1: 1: 1: 1, because 8 different types of gametes and in equal numbers will be produced by the F1 hybrid. Test crosses are of great importance since they yield or produce same genotypic and phenotypic ratios.

It is obvious from foregoing descriptions that the number of heterozygous genes involved in a cross increases the number of types of gametes and the number of types of F2 progeny.

Phenotypes GgWwCc, GgWwcc, GgwwCc, Ggwwcc, ggWwCc, ggWwcc, ggwwCc, ggwwcc.


Results

Mapping Strategy

We studied sex-specific recombination rates along the entirety of mouse Chr 1 as they occurred in the meioses of C57BL/6J (B6) and CAST/EiJ (CAST) F1 hybrids of both sexes at an average resolution of 225 kb, and further refined the extended subtelomeric region of 24.7 Mb. To test for potential effects parental imprinting might have on recombination, the F1 animals were produced by reciprocal crosses, and then backcrossed to C57BL/6J. Mapping the location of crossovers in these backcross progeny provided information on the recombination events arising in the F1 hybrids. A total of 6028 progeny were genotyped, of which 1465 were offspring of female B6xCAST, 1537 of female CASTxB6, 1479 of male B6xCAST, and 1547 of male CASTxB6. In all, we detected and localized 5472 crossover events on Chr 1, reaching a genetic resolution of 0.017 cM in the combined offspring. The frequency with which chromosomes with different numbers of crossovers were observed is summarized in Table 1 . We found significantly more multiple crossovers in female compared to male meiosis (p㰐 � by χ 2 test) as described before [22].

Table 1

Number of Crossovers per Chromosome01234Total Samples Tested
Female B6xCAST3637503311921465
Female CASTxB64327353422801537
Total Female 795 1485 673 47 2 3002
Male B6xCAST517731226501479
Male CASTxB6516770259201547
Total Male 1033 1501 485 7 0 3026

Backcross offspring were genotyped in two consecutive rounds with single nucleotide polymorphism (SNP) assays developed using the Amplifluor system (see Materials and Methods). In the first round, all progeny DNAs were mapped over the entire chromosome at 10-Mb resolution. This was sufficient to detect virtually all crossovers, given the strong interference in mouse meiosis [33]. In the second round, the crossovers occurring in each interval were mapped using additional SNP markers to an average physical resolution of 225 Kb. To provide a sample of even more detailed information, recombinants in the subtelomeric 24.7 Mb were subjected to additional rounds of testing using a combination of SNP and simple sequence length polymorphism (SSLP) markers. Among the crossovers occurring in this region, 81.4% were mapped to under 100 kb resolution: 8.2% at 50� kb resolution, 33.5% at 20� kb resolution, 8.6% to a nearly hotspot resolution of 5� kb and 31.1% were mapped to υ kb, ensuring hotspot level resolution. All markers used in this study, their positions according to NCBI Build 36, physical resolution and the number of crossovers in each interval are included in Table S1. Individual crossovers in five of the newly identified hotspots (shown in Table S1) were sequenced to determine exact locations of the chromatid exchange points within the limits of resolution provided by the locations of internal SNPs.

Regional Variation of Recombination Activity along Chr 1 at 225 kb Resolution

In total, the sex-averaged genetic map length of Chr 1 in the B6xCAST cross was 90.9 cM, which represents an average rate of 0.469 cM/Mb across 193.8 Mb, excluding the centromere adjacent 3 Mb for which no sequence information is available according to NCBI sequence build 36.

At 225 kb resolution, recombination activity was distributed very unevenly along the chromosome, forming alternating domains of higher and lower activity ( Figure 1A ). Recombination activity was found in only 64% of all intervals along the chromosome, the remaining 36% being completely devoid of recombination. In several places along the chromosome, recombination activity tended to be clustered in runs of consecutive intervals all of which were active, forming “torrid zones”. The most concentrated of them were 1.4𠄶.1 Mb long and were located at 37� Mb, 51�.4 Mb, 72�.8 Mb, 81.6� Mb, 131.4�.8 Mb, and 189.5�.6 Mb (red boxes in Figure 1A ).

A. Sex-averaged recombination map of Chr 1 in C57BL/6J퟊ST/EiJ cross. Boxes represent runs of consecutive intervals showing recombination (red) or no recombination (blue). B. Cytological map of Chr 1 (from ENSEMBL). C. Correlation between recombination rates in C57BL/6J퟊ST/EiJ backcross and HS mice at different resolution. The red line represents the best fitting logarithmic trend extrapolated to zero correlation. The best fitting function and its correlation coefficient are shown, indicating that correlation between the two crosses approaches zero at distances around 0.05 Mb.

Correspondingly, intervals devoid of recombination activity tended to cluster in 𠇌old zones”, the largest of which was over 6 Mb long. These were most prominent around 44.6�.8 Mb, 48.6� Mb, 84.8�.0 Mb, 96�.8 Mb, 102.6�.6 Mb, 110� Mb, 119�.6 Mb, 149.2�.4 Mb, 158.6�.2 Mb (blue boxes in Figure 1A ).

We did not detect any significant correlation along the chromosome between the locations of torrid and cold zones and traditional cytological banding patterns ( Figure 1B ).

Conservation of Regional but not Local Variation in Recombination Rates

To test the extent to which the recombination properties of a chromosome are evolutionarily conserved, we compared our results, obtained in a cross of only two strains, with the recombination map of Shifman et. al. [17]. The Shifman map was prepared at an average 550 kb resolution using the progeny of heterogeneous stock (HS) mice which merge the genetic backgrounds of eight mouse strains, including C57BL/6J but not CAST/EiJ. The two crosses have similar regional distribution of recombination along the chromosome, but do not share a substantial fraction of hotspots, if any.

Regional conservation between the two crosses was indicated by the significant correlation of recombination rates along the chromosome when tested at long intervals (r =𠂠.87 at 8.75 Mb resolution, Pearson correlation). However, this correlation decreased markedly when smaller intervals (4.4 Mb, 2.2 Mb, 1.1 Mb and at the maximum resolution of 0.55 Mb) were compared ( Figure 1C ). At the half-megabase scale, we found only a weak regional correlation (r =𠂠.38).

These estimated correlations are somewhat attenuated by the sampling variation in the estimates of recombination rates, and this attenuation increases at higher resolution, since the sampling variation is greater at higher resolution (due to smaller numbers of observed recombination events in smaller intervals). But for the sample sizes in these studies, the attenuation in the estimated correlations is negligible (on the order of 1/1000), and so cannot account for the large observed decrease in correlation from the 8.75 Mb scale to the 0.55 Mb scale.

Long regions of very low or no recombination were evident in both crosses and provided the strongest parallels between the crosses. These regions include those around 43� Mb, 96� Mb, 111� Mb and several smaller regions between 141� Mb. The lack of recombination in these regions cannot be attributed to inversions, which would prevent the survival of recombinants. Two main reasons speak against this possibility. First, some parents in the mixed genetic background will inevitably have the same orientation of the region in question if it were inverted in some of the eight strains, and therefore recombination would be detected in their progeny. Second, some intervals in these regions are not totally devoid of recombination in both crosses but have very low rates.

Effects of Genetic Background on Overall Recombination Rates

In addition to local variation in recombination rates, genetic background also plays a role in determining overall recombination rates. The genetic map length of Chr 1 was �% higher in HS mice than in our two-strain cross. The reasons for this significant difference are uncertain. The lack of local correlation indicates that this difference is not simply due to an increased use of the same hotspots in HS mice. The present genetic data [22] agree with counts of the average number of chiasmata per meiosis during spermatogenesis among inbred strains [34] and counts of MLH1 foci marking sites of crossing over on Chr 1 [35]. It might be possible that recombination in a very heterogeneous genetic background is quite different from that seen in crosses of inbred strains. The importance of genetic background in recombination is also suggested by substantial differences between the crosses' recombination rates at specific intervals. For example, in the 24.7 Mb region that was mapped at considerably greater resolution (see below), recombinational activity was often present in one mouse cross (B6xCAST or HS) but not the other.

Positioning Relative to Genes, Exons and Transcription Start Sites

We found an overall positive correlation between gene density and recombination along the entire chromosome over megabase distances (r =𠂠.557 at 10 Mb). However, this effect diminished over shorter distances (r =𠂠.164 at 500 kb) ( Table 2 ). At 200 kb, the correlation was low (r =𠂠.079) but statistically significant. Moreover, this positive correlation was not uniform along the chromosome but was restricted to only some regions, and statistically significant only for the region between 100� Mb (maximum correlation r  =𠂠.877 at 5 Mb for the sex-average data). In this region, the positive correlation was still detected, and statistically significant, at 200 kb (r =𠂠.278). For the first and second 50-Mb segment (3� and 50� Mb), the correlation was positive but not statistically significant, whereas the correlation for the last region (150� Mb) was slightly negative up to 2Mb but not statistically significant. The 24.7-Mb part of the last segment was mapped to higher resolution (see below) and showed slightly negative correlation between gene density and recombination at 200 kb which disappeared at 50 kb.

Table 2

Sex200kb500kb1mb2mb5mb10mb
Chr 1 entire
r p r p r p r p r p r p
Female 0.093 0.000 0.184 0.001 0.206 0.001 0.308 0.000 0.482 0.000 0.678 0.001
Male 0.055 0.045 0.126 0.011 0.141 0.023 0.255 0.0050.2500.0640.3270.107
Sex-Average 0.079 0.007 0.164 0.001 0.187 0.005 0.304 0.001 0.379 0.009 0.557 0.005
3� Mb
Female 0.131 0.0300.1650.0510.1550.1280.0960.3040.3770.131
Male0.0790.1150.1720.0530.1460.1420.1200.2510.0620.352
Sex-Average0.1150.051 0.180 0.0360.1610.1210.1170.2850.2090.238
50� Mb
Female0.0190.7710.0720.4870.0750.6140.1660.4380.6260.053
Male0.0740.2510.0840.4140.1120.4490.3360.1090.6000.067
Sex-Average0.0550.3960.0850.4090.1030.4870.2860.176 0.674 0.032
100� Mb
Female 0.295 0.000 0.405 0.000 0.495 0.001 0.696 0.000 0.876 0.000
Male 0.191 0.007 0.299 0.006 0.419 0.003 0.558 0.003 0.821 0.003
Sex-Average 0.278 0.000 0.403 0.000 0.505 0.000 0.689 0.000 0.877 0.001
150� Mb
Female𢄠.0350.3170.0510.321𢄠.0100.469𢄠.0190.4990.5760.068
Male𢄠.0580.2250.0070.440𢄠.0960.305𢄠.2340.1650.2070.251
Sex-Average𢄠.0530.2180.0250.403𢄠.0710.351𢄠.1750.2480.4330.138
168.8�.5 Mb
50kb100kb200kb
Female𢄠.0070.427𢄠.0370.303𢄠.0670.238
Male0.0180.3350.0100.430𢄠.0800.204
Sex-Average0.0100.412𢄠.0080.460𢄠.0810.201

r represents correlation coefficient, p is the probability calculated by bootstrapping. The correlations with pπ.05 are shown in bold.

Recombination tended to avoid gene deserts larger than 1.5 Mb but showed a tendency of clustering at their borders. The average rate in large gene deserts totaling 59.77 Mb (shown in Figure S1) was 0.26 cM/Mb compared to 0.55 cM/Mb in the remaining 134.02 Mb of non-deserts (p㰐 � by χ 2 test) and 0.467 cM/Mb over the entire chromosome. The average rate was 0.80 cM/Mb in the 0.5𠄰.7 Mb border regions surrounding large gene deserts (p㰐 � ) and rapidly decreased beyond that to become statistically indistinguishable from the average chromosome rate (p =𠂠.596).

Similar correlation was found over the entire chromosome between exon density and recombination (r =𠂠.566 at 10 Mb and r =𠂠.126 at 500 kb, Table S2) and transcription start sites and recombination (r =𠂠.585 at 10 Mb and r =𠂠.121 at 500 kb, Table S3). However, the correlation was not statistically significant at 200 kb (r =𠂠.043, p =𠂠.101 for exons and r =𠂠.026, p =𠂠.204 for transcription start sites). In these two comparisons, most of the positive correlation was statistically significant for the region between 100� Mb but not for the rest of the chromosome. In the 24.7-Mb region mapped to higher resolution, both exon density and transcription start sites were slightly negatively correlated with recombination down to 50 kb (r =  𢄠.045 and r =  𢄠.071, respectively) and this effect was statistically significant for transcription start sites (p =𠂠.021).

Two striking examples of torrid zones that occur in large introns provide evidence that recombination is not restricted to intergenic regions. The first one consists of at least six hotspots in the 218-kb long second intron of Pbx1 (pre B-cell leukemia transcription factor 1, located at 169.995�.268 Mb, NCBI Build 36), which is also a hotspot for translocations associated with acute lymphoblastic leukemia in humans [36],[37]. The second torrid zone includes at least three hotspots in the 80-kb long third intron of Esrrg (Estrogen receptor-like receptor gamma, located at 189.309�.915 Mb).

Relative Abundance of Intervals with Differing Recombination Rates

We observed a simple, negative exponential relationship between the crossover rate among intervals and the likelihood of seeing hotspots of that activity. Among intervals averaging 225 Kb in length, recombination rates (expressed as cM/Mb to correct for variations in interval length) varied continuously over almost three orders of magnitude, from 0.017 cM/Mb (the lower limit of detection in this cross) up to 10 cM/Mb. Intervals with differing recombination rate were not equally likely instead, when they were placed in rank order of recombination activity, the rates were distributed in a simple exponential manner where Rn, the recombination rate in the nth ranked interval was equal to ke cn , where k and c are constants ( Figure 2A ). Figure 2B , which is also an exponential function, describes the cumulative recombination rate among rank-ordered intervals. A similar exponential relationship for the cumulative recombination rate was reported by McVean et al [38] for the human genome.

A. Distribution of recombination in intervals of increasing rates (intervals lacking recombination are not included). The rates are presented in logarithmic scale to emphasize the exponential shape of the distribution. The deviation at the lower end of the distribution represents low-activity intervals mapped to a lower resolution. Red line represents the best fitting exponential function. The exponential function and its correlation coefficient are shown. B. Cumulative recombination as a function of chromosomal size. Both recombination rates and chromosomal length are expressed as fractions of the total. The intervals are in rank order of increasing recombination rate.

These exponential relationships indicate that nearly 50% of all recombination activity occurred in only 7.6% of the intervals while 22.2% of the intervals accounted for 80% of all recombination activity. Similar findings that a high percentage of all recombination is concentrated in a small fraction of chromosome intervals have recently been reported for the human genome [39]. The interval fractions become even smaller with decrease in interval size (see below). This result, which suggests that the majority of all recombination events occur in a relatively small fraction of the chromosome, has important practical implications for genetic mapping strategies. The conclusion that follows is that a moderate size cross should be optimal for mapping genes and QTLs because adding more offspring will not substantially increase the resolution power. The result provides an experimental ground to something that mouse geneticists have known intuitively for some time-if a gene cannot be mapped with the first few hundred offspring, the best strategy is to move to another cross if that is at all possible.

High Resolution Mapping in the Telomere-Proximal 24.7 Mb

High-resolution mapping further emphasizes the uneven distribution of recombination activities among intervals ( Figure 3A and Figure S2).

A. Sex-averaged map of the region of 168.8�.5 on Chr 1. Recombination rates in intervals that are off scale are shown as numbers over each interval. The red circles mark newly identified hotspots full circles, hotspots that were sequenced through to determine the fine positioning of crossover exchanges. B. Hotspots in the third intron of Esrrg (189.75�.8 Mb). C. Number of intervals containing recombination activity higher than given thresholds at different interval size. The threshold levels are shown in the legend.

The 24.7-Mb telomere-proximal segment between 168.8�.5 Mb had a genetic length of 22.7 cM. This accounts for a relative recombination rate of 0.92 cM/Mb, which is about twice the average rate of the entire chromosome. When it was mapped further to an average resolution of 75 kb, the distribution of recombination activities among intervals remained continuously variable as in the 225 kb intervals. However, as expected from the punctate location of hotspots, a smaller fraction of the genome-52% compared to 64% at 225 kb resolution𠄼ontained all recombination. Indeed, 50 percent of all recombination occurred in 16 intervals spanning only 1.8% of the segment length, with each of these intervals having an activity of 0.34 cM or more.

Recombinations in eight of these sixteen most active intervals were mapped down to 20� kb resolution while those in the remaining eight intervals marked with red circles on Figure 3A were mapped down to 𢏃 kb resolution. All but one of the eight intervals contained a single hotspot, which was separated from the closest adjacent hotspot by at least 30 kb of sequence. The notable exception was the presence of two hotspots only 5 kb apart in the third intron of the Esrrg gene ( Figure 3B ).

Distances between adjacent intervals with recombination rates of 0.34 cM or more varied over three orders of magnitude in genomic terms, ranging from 5 kb to 5 Mb (1.52 Mb on average). The variation was much smaller in genetic terms, from 0.37 to 2.44 cM, or an average of 1.26 cM.

Total Number of Hotspots in the Mouse Genome

As interval sizes become smaller, it becomes increasingly likely that an interval contains only one hotspot. This provides a means of estimating the total number of hotspots in this 24.7-Mb segment, and by extension the total number in the genome. For this, the number of intervals showing any recombination activity was plotted as a function of interval size and the resulting trend lines extrapolated to a 5kb interval size, the minimal distance we found between adjacent individual hotspots ( Figure 3C , results summarized in Table S4). This yielded an estimate of one hotspot per 108 kb on average, or about 228 hotspots accounting for all recombination in this segment among 6028 meioses. As expected from the exponential relationship described above, more active hotspots occur less frequently. On average, those with rates higher than 0.1 cM are likely to occur once per 425 kb, and those with rates higher than 0.2 cM, about once per megabase. These results are obviously tempered by the fact that they were obtained for one genetic combination in a region of the genome whose recombination rate is higher than the genome wide average.

To the extent this region is representative of the rest of the genome, its hotspot density provides an estimate of the total number of hotspots in the entire mouse genome that are active in this B6xCAST cross. We have made this estimation by relating the genetic length of the 24.7-Mb region to the total genetic length of the mouse genome. We assume that genetic lengths (measured in cM) will be more relevant than physical lengths (measured in Mb) because of the uneven distribution of recombination along the chromosome and the existence of long regions devoid of recombination. This calculation, using the Dietrich et al [30] sex-average map length of 1361 cM for the same C57BL/6JxCAST/EiJ cross, results in an estimate of about 13,670 hotspots (228/22.7�) across the mouse genome.

A recent study [40] typing 8.23 million SNP markers detected about 40,000 haplotype blocks in 12 classical inbred mouse strains based on ancestry inferred from representative strains of the four main mouse subspecies. Although the haplotype block boundaries were not always well defined, to the extent that they represent bona fide historical sites of recombination, the scales of these two estimates are not far apart. Our study should be considered a minimum estimate as it measured recombination from contemporary hotspots in one generation of a cross involving only two inbred strains, and was limited by the sensitivity of detection of 6028 meioses. The estimate of Frazer et al [40] suggested a higher number of hotspots in the genome of classical mouse inbred strains because it is not limited to contemporary hotspots and reflects the behavior of historical hotspots generating recombination over many generations in a variety of genetic backgrounds.

The most recent estimate [41] using more than 3.1 million SNPs has identified 32,996 hotspots in the human population, which is in the range of these estimates for the mouse genome.

Sex Specificity of Recombination

The two sexes differed at all levels of organization of recombination. Overall recombination rates were higher in females than males recombination was distributed differently along the chromosome in males and females, and there were also sex-specific hotspots.

The female recombination map of Chr 1 was 99.5 cM, or 1.21 times longer than the male map which was 82.3 cM, with average recombination rates over the entire chromosome of 0.51 and 0.42 cM/Mb, respectively. These differences were statistically significant (p㰐 𢄦 by Fisher's exact test). Among 225 Kb intervals, there was an overall positive correlation between female and male rates (r =𠂠.64) along the chromosome. This correlation did not change significantly at larger interval sizes up to 8 Mb. The underlying reason why the correlation did not increase with interval size was the substantial variation in distribution of recombination along the chromosome ( Figure 4A ), which included differences in both the number and relative recombination activity of intervals.

A. Sex-specific recombination map of Chr 1. Red line, female recombination rates blue line, male recombination rates. B. Female:male ratio along the chromosome. Dark blue line: female:male ratio purple line: sex-averaged recombination rate over the entire Chr 1.

Recombination activity was spread over a larger fraction of the chromosome in females than in males. In females, 57.1% of intervals were recombinationally active compared to only 42.2% in males (a ratio of 1.35). This differential was apparent at all activity levels 80% of all activity occurred in 23.2% of female versus 13.6% of male intervals, and 50% occurred in 8.23% of female versus 4.65% of male intervals.

These sex differences in the relative rates of recombination were regionally controlled ( Figure 4B ). Female recombination rates were higher in the centromere-proximal 27 Mb and in the region between 79� Mb, whereas male recombination rates were higher in the telomere-proximal 178� Mb region and generally, but not in the entirety, of the region between 27� Mb.

To study regional effects in more detail, we examined the switch between higher female and higher male recombination found in the fine-mapped 24.7 Mb sub-telomeric region. Female recombination rates were generally higher than those in males in the region between 169� Mb, with an abrupt transition to the opposite case in the adjacent region between 178� Mb where males had higher recombination ( Figure 5 and Figure S3). Interestingly, the switch occurs in a region of very low recombination in both sexes. Overall, the difference between the two sexes was highly significant over the entire region (p㰐 𢄤 ).

Recombination rates in intervals that are off scale are shown as numbers over each interval. Red arrows: hotspots predominantly active in females blue arrows: hotspots predominantly active in males.

Although the sexes share a substantial fraction of hotspots, there are many considerable differences in activity. Commonality of hotspot usage was indicated by the observation that comparisons at multiple interval sizes did not change the correlation between the two sexes (r =𠂠.62). However, there were also specific sex differences in hotspot activity that were independent of regional control. Among the 28 intervals with sufficiently high recombination (Ϡ.2cM) to provide sufficient numbers of crossovers for statistically significant analysis, 18 showed sex-specific differences after adjustment for multiple testing ( Table 3 ). Among these 18, eleven showed at least some activity in both sexes, seven being markedly more active in females and four in males (pπ.01, qπ.1). Seven of the 18 were detected in only one sex, four in females and three in males. The latter group indicates that some hotspots may be truly sex specific, or at least that the differences in their activity are so great (㸐 times) that recombination was not detected in the low-activity sex even in several thousand meioses.

Table 3

Number of RecombinantsSignificance
Hotspot Location (Mb)femalemale p * q **
171.31500.0000.000
186.317880.0000.000
189.819630.0000.000
187.40150.0000.007
190.21310.0000.007
174.42050.0010.016
176.5900.0010.016
181.30110.0010.016
175.21740.0020.023
186.41130.0030.031
179.24200.0030.031
177.71010.0030.031
171.7700.0060.049
171.91430.0070.056
191.0080.0080.063
176.7600.0100.073
170.340240.0120.085
170.6810.0150.099

*: p values are calculated by Fisher's exact test.

**: q values are calculated as described in [56].

Importantly, this sex specificity of individual hotspots is not constrained by regional controls. For example, the hotspot at 173.967 Mb is more active in males despite lying in the midst of a female predominant region, and the hotspot at 190.204 Mb, which is considerably more active in females, nevertheless lies in a male predominant region.

To address the broader question of how the total numbers and relative activity of hotspots differ between male and female meioses, we compared the two sexes across the female and male predominant segments of the subtelomeric 24.7 Mb region by extrapolating the resolution dependent trend lines for activity down to 5 Kb. Interestingly, the two regions gave distinct answers greater female recombination in the proximal segment largely resulted from an increased number of hotspots, whereas in the distal segment, greater male recombination was primarily the result of increased recombination in a comparable number of hotspots ( Table 4 ). In the proximal 9.8 Mb, where females had twice the recombination rate of males (9.0 cM vs. 4.2 cM), they had twice as many hotspots as well (72 vs. 34) that were somewhat more active, while in the distal 16 Mb where females have a significantly lower recombination rate than males (12.4 cM vs 19.8 cM), there were similar numbers of inferred hotspots (91 vs. 88) in the two sexes, but males had higher average recombination rates per hotspot.

Table 4

Females Males
Genomic Region (Mb)Hotspot Activity (cM)Number of Hotspots Density (HS/Mb)Number of Hotspots Density (HS/Mb)
168.8-193.5>.032163 6.6122 4.9
>.05105 4.382 3.3
Ϡ.181 3.354 2.2
Ϡ.232 1.330 1.2
Rec. Rate (cM) 21.5 24.0
168.8-178>.03272 7.334 3.5
>.0548 4.923 2.3
Ϡ.128 2.916 1.6
Ϡ.213 1.34 0.4
Rec. Rate (cM) 9.0 4.2
178-193.5>.03291 5.988 5.7
>.0557 3.759 3.8
Ϡ.133 2.138 2.5
Ϡ.219 1.226 1.7
Rec. Rate (cM) 12.4 19.8

These sex differences largely apply to lower activity hotspots, those less than 0.2 cM. The inferred numbers of hotspots with rates of up to 0.2 cM were significantly higher in females than in males over the entire 24.7 Mb ( Table 4 ). However, this inequality did not hold for higher activity hotspots both sexes had the same number of hotspots more active than 0.2 cM.

Distinct Chromatid Control at Individual Hotspots

Fine mapping of crossover exchange points within hotspots made it possible to identify the parental chromosome initiating recombination and thereby show that the two parental chromatids are under independent recombinational control.

The locations of all 457 crossover events in five of the nine hotspots mapped to σ kb resolution (marked with full red circles on Figure 3A ) were further mapped using all available SNPs. In each case, the sites of crossing over were distributed over distances ranging from 500 to 2000 bp, which is a typical size for a hotspot [3] (Dataset S1). In some cases, recombination activities were distributed along the entirety of the hotspots regions following a single normal distribution, but in others they appeared to be the sum of two overlapping bimodal distributions. Distinguishing between the two distributions depended on the availability of SNPs for precisely mapping recombination events near the hotspot center. When such conveniently positioned SNPs were available, we observed that crossover events were predominantly located at the two sides of the hotspot, with very little or no recombination at the center ( Figure 6B ). According to the currently valid models of recombination, bimodal distribution will be observed when double strand breaks initiate in very narrow regions, and the crossover exchange points which are located at the sites of resolution of the Holliday junctions migrate sufficiently away from the initial sites of double strand breaks. Our finding that a bimodal distribution was observed when the necessary SNPs were available for detection suggests that this is likely to be the case for most hotspots.

A. Physical positions of the SNPs used to determine the crossover exchange points according to NCBI Build 36. In panels B, C and D, the left end (0) corresponds to 186,316,643 A/G. B. Distribution of crossover exchange points in female and male progeny. The number of crossovers in each interval is shown. Red, females blue, males. C. Distribution of reciprocal crossovers (B-C and C-B) in female progeny. The number of crossovers in each interval is shown. Red, B-C tan, C-B. D. Distribution of reciprocal crossovers (B-C and C-B) in male progeny. The number of crossovers in each interval is shown. Blue, B-C green, C-B.

For the hotspot at 186.3 Mb, the availability of particularly suitable SNPs ( Figure 6A ) allowed us to deduce that for this hotspot the B6 and CAST chromatids are under independent, sex-specific recombinational control. The sites of crossing over within the hotspot were quite different when the crossover products were B proximal-C distal v. C proximal-B distal. This was true for F1 animals derived from both reciprocal crosses, i.e. there were no imprinting effects. Among the 16 crossovers arising in female meioses, all B-C exchange points were positioned centromere-proximal to the center of the hotspot, whereas all C-B recombinants crossed over in the centromere-distal part. Thus, the center of the hotspot was of CAST origin in all crossovers ( Figure 6C ), indicating that, in this cross, recombination events in females only initiated on the B6 chromosome [5]. In males, which have 5.6 times higher recombination at this hotspot, there was also a strong bias towards initiation on the B6 chromosome, although the effect was not absolute. Crossover events of both types were distributed on both sides of the central region, indicating that recombination could initiate on either parental chromatid ( Figure 6D ). However, initiation on the B6 chromatid was 2.5 times more frequent than on the CAST chromatid.

Our results for the 186.3 hotspot clearly show that the overall control of recombination at a hotspot is the sum of distinct controls for each chromatid, and that this distinction applies to issues of both sex specificity and absolute recombination rates.

Imprinting of Recombination Activities

Examining 225 Kb intervals over the entire chromosome to compare F1 hybrids derived from the reciprocal crosses of B6xCAST and CASTxB6 provided statistically significant evidence for parent-of-origin effects on recombination activities in both sexes (p =𠂠.013 for reciprocal males and p =𠂠.009 for reciprocal females). The direction of imprinting was not uniform, and imprinting was only detected by finding a statistically significant excess of hotspots showing a preference for recombination in one direction of the cross or the other. In no case did we find absolute imprinting, where recombinants were significantly absent from one direction of the cross. A statistically significant difference was also detected in the fine mapped 24.7-Mb region of the chromosome in males (p =𠂠.001), but the difference was only marginally significant in females (p =𠂠.07). None of the higher activity hotspots in this region showed significant parent-of-origin effects after correction for multiple testing rather, imprinting effects were restricted to medium- and low-activity hotspots. (See Tables S5 and S6).

However, although we detected slight but significant cumulative differences between reciprocal crosses in 225 Kb intervals in both female and male meiosis, and in male meiosis in the telomere-proximal 24.7 Mb, no one interval gave significant evidence for a difference in recombination rate between the reciprocal crosses. It is likely that the effects may be subtle and only recognizable statistically when data is accumulated across large chromosomal regions. Individual intervals, when considered on their own, showed recombination rate differences between the reciprocal crosses that could reasonably be explained by chance variation, but overall there were many more intervals with suggestions of recombination rate differences than could reasonably be explained by chance variation.

Gene Conversions and Genetic Interference

Additional data obtained from the backcross animals provided the first genetic evidence in mammals that genetic interference, which regulates the spacing of crossovers, does not affect the relative locations, one to the other, of the two distinct outcomes of the recombination process, crossing over and gene conversions not associated with crossing over.

Gene conversions arising in male meioses were detected in three of the fine-mapped hotspots by genotyping every SNP across each hotspot among 1365 male backcross progeny ( Table 5 ). Only eleven conversions were found, six conversions not associated with crossovers (noncrossovers) and five conversions associated with simultaneous crossovers at the same hotspot. In the best mapped hotspot at 186.3 Mb, all five events we detected were positioned in the central part of the hotspot. The three noncrossovers were located between positions 1135� bp on Figure 6B , and the two conversions associated with crossovers spanned between positions 877� bp. For all three hotspots, the apparent frequencies of non-crossover conversions were lower (5� times) than crossover frequencies at the same hotspots, however these ratios must be interpreted with caution as while we were able to detect all crossovers, we were only able to detect the sample of conversions occurring at sites of available SNPs. The relative ratios of crossovers to noncrossover conversions in several human and mouse hotspots have shown considerable variation, from more than 12𢍡 to 1𢍤 [2],[5],[25],[42]. Given the positions of the available markers, the actual conversion frequencies could be much higher than detected. From SNP locations we could deduce that the minimum-maximum length for noncrossover conversion tracts was 9� bp. In contrast, conversion tracts associated with crossing over at the same hotspots had a minimum-maximum span of 199� bp. Both estimates are of similar scale to those reported at the human DNA3 hotspot, 55� bp for conversion tracts not associated with crossovers and � bp for conversion tracts associated with crossing over [25].

Table 5

Hotspot186.3187.8189.78
NCR conversions312
CR conversions212
Crossovers311110
NCR Rate0.0020.0010.001
CR Rate0.0230.0080.007
NCR/CR Ratio0.0970.0910.200

The six progeny chromosomes carrying noncrossover conversions contained seven crossovers located elsewhere along the chromosomes. In four cases the distances between crossovers and conversions were significantly longer, 95� Mb, than the minimal male interference distance of 57 Mb between two crossovers observed in the 3026 male meioses used in this study [22]. However, in three cases the crossovers and conversions were only a few megabases apart, the closest distance being 1.12 Mb. We conclude that the process of genetic interference limiting the proximity of crossovers, one to another, does not limit the proximity of crossovers and non-crossover conversions. Our finding is in agreement with the lack of interference between crossovers and non-crossover conversions originally found in yeast [43].


Effects of pubertal testosterone on the developing hippocampus

The second developmental period in which steroid hormones can exert an organizing effect on the brain is during puberty, defined as a period of adolescence marked by a steep rise in circulating gonadal steroids. The most important outcome of puberty is the activation of sexually differentiated neural substrates by gonadal steroids in order to promote reproductive behavior. However, circuits mediating social and cognitive behaviors are also sculpted in a sexually differentiated manner during this time via a variety of cellular mechanisms (see [133, 134], for review). Evidence that hippocampal function and neurophysiology are both modulated and programmed by pubertal androgens is seen in animal and human studies.

Longitudinal imaging studies in children generally find no prepubertal sex differences in hippocampal size or morphology, but greater hippocampal volume in boys throughout puberty and adolescence, when corrected for total intracranial volume [135,136,137,138,139,140]. In terms of the relative growth trajectories between males and females, however, the data seemingly conflict. While some studies demonstrate parallel increases in hippocampal volume throughout puberty in both boys and girls [138, 141], others show sexually differentiated growth trajectories, where hippocampal volume increases steadily with advancing age and puberty in females, but growth slows in males during late puberty [137, 139, 142, 143]. This may indicate a sexually differentiated response to gonadal steroids in the hippocampus, or it may indicate a biphasic effect of androgens, where rising levels promote a positive growth trajectory, but high levels, as found in the circulation of late-pubertal males, have a negative impact on growth. This is supported by data from Wierenga et al. [140], which correlates high circulating testosterone in adolescent females with slower hippocampal growth. In addition, while the trajectory of hippocampal growth is predicted by advancing puberty status in males, in adolescent females hippocampal volume is not strongly associated with pubertal status, but instead is predicted by age [144] or circulating testosterone levels [139, 140, 142, 145]. Further support for a direct role of androgens in determining the sex difference in hippocampal volume during adolescence is found during adrenarche, which is characterized by elevated adrenal-derived androgens and where higher circulating testosterone is associated with larger hippocampal volume in girls [145].

A similar pattern of testosterone effects during puberty is seen functionally. In adolescents, response to fearful faces or performance on context-dependent memory tasks are positively associated with hippocampal activation. Test performance and hippocampal activation are both predicted by stage of pubertal development in boys and girls however, in girls, the predictive power of pubertal stage is due almost entirely to levels of circulating testosterone, and not age or stage of puberty [146, 147]. In males, activation of the hippocampus during emotional processing is significantly greater in young boys who have familial male-precocious puberty, compared to unaffected boys of the same age [148].

Perhaps the most compelling evidence for the role of pubertal androgens in hippocampal maturation are imaging studies in adolescents with Klinefelter syndrome. Aneuploid boys with more than one X chromosome initiate puberty but are androgen deficient [149, 150] and have smaller hippocampal grey matter volume than typically developing boys [151, 152]. Treatment with a testosterone analog during adolescence normalizes hippocampal volume in Klinefelter patients [153], indicating that testicular steroids, and not sex chromosome complement, is a primary driver of hippocampal maturation during adolescence. In spite of the impossibility of direct functional experiments in humans, what emerges is that androgens have a primary role in directing adolescent maturation of the hippocampus, and the level of circulating testosterone is an important setpoint for sexual differentiation of this brain region during puberty in both sexes. Although a similar role for ovarian steroids during puberty is not supported by the imaging data in humans, a role for brain-derived estradiol in the adolescent brain, similar to the perinatal period in rodents, cannot be ruled out [154]. Peripheral blood cells from pre- and postpubertal adolescents demonstrate female-specific methylation patterns that arise during puberty for genes involved in androgen signaling and that are proximal to estrogen response elements [155], indicating that the rise of ovarian steroids during puberty may epigenetically program the response to androgens in girls.

There are relatively few studies that shed light on potential cellular mechanisms mediating the effects of pubertal testosterone on hippocampal development. Data from rhesus macaques demonstrate a role for adolescent testosterone in regulating cell survival and differentiation in the hippocampus, as castration in early puberty increases survival and maturation of granule neurons in the dentate gyrus, while there are no changes in cell proliferation [156]. Changes in spatial reasoning and memory tasks involve alterations in synaptic plasticity, and androgens play a significant role in this regard in the adult hippocampus [21, 157]. There is also evidence that androgens modulate hippocampal synaptic plasticity during adolescence. Male mice undergo loss of dendritic spine synapses in the CA1 hippocampal subregion over the course of puberty, an effect largely reversed by gonadectomy [158], although prepubertal gonadectomy does not prevent a similar loss of synapses in female rats [159]. Functionally, adult male rats have reduced social memory compared to juveniles, coinciding with a shift from long-term potentiation to depression in response to stimulation in the CA1 region of the hippocampus. Both gonadectomy and androgen receptor antagonism at the beginning of puberty, but not later in adolescence, prevent the developmental shift to long-term depression and improve social memory in adults [160]. While these data suggest an organizational role for pubertal androgens in males, whether these are truly sexually differentiated responses is not clear, as comparative treatments using both males and females were not included in these studies. Interestingly, these cellular effects of androgen signaling in the adolescent hippocampus are opposite those in the adult, where androgens promote cell proliferation and survival in the dentate gyrus [19], and synaptogenesis in Ammon’s horn [21, 161].


Methods

Plant material and DNA isolation

Three bi-parental mapping populations of Actinidia were used to study the recombination rates along chromosome 25. Mapping population I was an interspecific bi-parental mapping family A. rufa × A. chinensis var. chinensis (A, ‘MT570001’ × ‘Guihai No4’) [22]. Mapping population II was an intraspecific diploid A. chinensis var. chinensis family developed from a cross between ‘Hort16A’ and male parent P1. Mapping population III was also an intraspecific diploid A. chinensis var. chinensis family derived from a female from seed from Henan province, and the male parent from a seed accession from Guangxi province, China [21].

Seedlings from mapping population II were raised in tissue culture and 236 individuals selected for genotyping. Young expanded leaf tissue, weighing approximately 100 mg, was harvested and stored at − 80 °C by snap freezing in liquid nitrogen. Genomic DNA extraction was performed using the CTAB method [51]. DNA quantification was carried out using Qubit™ fluorometric analysis.

Genotyping-by-sequencing (GBS), variant calling and selection of single nucleotide polymorphism (SNP) markers

The method for developing GBS libraries of population II followed [52], modified by the use of BamHI for the restriction digestion step. The libraries were individually amplified and successful preparation verified by analysis of an aliquot by agarose gel electrophoresis, before pooling the amplicons prior to sequencing [53]. Libraries prepared from 236 genotypes were sequenced over 5 lanes using Illumina™ HiSeq2000 in single-end mode, with each lane generating more than 200 million single-end 100 bp reads. Two plates of libraries (192 genotypes) were sequenced over 2 lanes and half a plate (44 genotypes) was sequenced over 1 lane. SNP calling was performed using the reference-guided TASSEL pipeline on the Red5 (version PS1.1.68.5 [54], an earlier version of the published genome [55]) and the ‘Hongyang’ reference genomes [56]. SNPs were filtered at the filtering criteria of 0.7 for coverage across all genotypes, generating

44–50 K SNP sites from each genome across 29 pseudochromosomes and unassigned scaffolds in linkage group Chr 30. SNP markers were de-convoluted for each parent using the following criteria. First, the SNP markers were selected that were heterozygous (ab) in one parent and homozygous (aa) in the other parent and vice versa. This generated a set of markers which would theoretically segregate as 1:1 < ab×aa> (pseudo-testcross) and are unique to each parent these were used for construction of the male and female genetic linkage maps individually.

Genetic map construction

Genetic maps using ‘Hort16A’ and P1 SNP markers were constructed using Joinmap3® (www.kyazma.nl). SNP marker data were processed in JoinMap using the ‘CP’ format for the population structure. Linkage groups were developed using default settings for grouping with modifications, including: a) the threshold range for Independence logarithm (base 10) of odds (LOD) started from a LOD score of 10 to 20 and, b) use of a regression mapping algorithm.

Recombination rates along chromosomes

The physical positions of segregating SNP markers on the genetic map were plotted against physical SNP locations on pseudomolecules using R 3.3.0 (https://www.R-project.org) in two populations, an interspecific Actinidia rufa × A. chinensis var. chinensis (‘MT570001’ × ‘Guihai No4‘) mapping population I and an intraspecific A. chinensis var. chinensis (‘Hort16A’ × P1) mapping population II.

Segregation of microsatellite alleles within the SDR

The inheritance pattern of SSR markers within the SDR was investigated in an intraspecific A. chinensis var. chinensis mapping population previously described [20]. Twenty-one microsatellite markers were selected for analysis eighteen of these amplify from within the terminal 6 Mb of chromosome 25, the remaining three amplify from the distal portion. The parents and eighty-seven progeny of this mapping population were screened with these markers in the same manner as previously described [57]. The sequences of these primers and annealing temperatures are given in Additional file 1: Table S1. Based on the pattern of segregation of alleles from fully informative, female-informative and male-informative markers, alleles were grouped into one of four groups depending on the chromosome from which they had originated i.e. group 1 originated from chromosome X1, group 2 originated from chromosome X2 (inherited from the female parent), group 3 originated from X3, and group 4 from Y1 (inherited from the male parent).

Whole-genome sequence alignment, variant calling and kinship analysis

Adaptors and low-quality or undetermined sequences of short-insert Illumina whole genome sequence were filtered and clipped using fastq-mcf (fastx toolkit, version 0.0.13) [58]. Paired reads at approximately 30x coverage were mapped to the ‘Hongyang’ genome [56] using bwa-mem 0.7.15 [59]. Variant calling was performed using Freebayes 1.1.0 [60], in 1-Mb windows. Variant Call Format (VCF) files were filtered using for biallelic variants with no missing data using the following vcflib (https://github.com/vcflib/vcflib) pipeline:

vcfbiallelic | vcffilter -f ‘NS = 14 & QUAL > 30 & SAR > 3 & PAIRED > 0.8 & SAF > 3.

VCF files were phased using Beagle 4.0 and default values [61]. Fst, Tajimas Pi and kinship coefficients were generated for each 1-Mb window of pseudochromosome 25 using vcftools 0.1.14 [62] and kinship determined using the vcftools relatedness2 option [63]. Rates of linkage disequilibrium (LD) decay was determined in 1-Mb windows using PopLDDecay (https://github.com/BGI-shenzhen/PopLDdecay). LD decay within each window was summarised as the median R 2 in the 1 kb–10 kb sub-interval.

Chromosome preparation

The female genotype of diploid A. chinensis var. chinensis used for this study, CK51_05, is the female parent of the mapping population III was used previously to construct a genetic map in A. chinensis var. chinensis [21]. Young flower buds at various stages were collected and placed immediately into 3:1 ethanol:acetic acid and stored at 4 °C for at least one day. If buds were to be stored for more than two weeks, they were transferred to 70% v/v ethanol and stored at − 20 °C until required.

Flower buds containing meiocytes at pachytene stage were identified by squashing an anther from each in FLP (formo:lacto:propiono) orcein and observing the meiotic stage. Once buds at pachytene had been identified, chromosome preparations were made following the method of Andras SC, Hartman TP, Marshall JA, Marchant R, Power JB, Cocking EC and Davey MR [64], using the remaining anthers in each bud and modified to use anthers rather than root tips. Anthers were hydrolysed in 1 M HCl at 37 °C for 15–20 min and then digested for 80 min in the following enzyme mixture: 4% (w/v) Onozuka R10 cellulase (Merck 102,321), 4% (w/v) cellulase (Sigma C-9442), 2% (w/v) pectoylase (Sigma P-3026) and 1% (w/v) cytohelicase (Sigma C-8274) dissolved in 0.01 M citrate buffer pH 4.5. The drop techniques of Felsenstein J [65] and Henegariu O, Heerema NA, Lowe Wright L, Bray-Ward P, Ward DC and Vance GH [66] were subsequently used to prepare chromosome spreads.

Construction and screening of BAC library

The genomic DNA BAC library from A. chinensis var. chinensis CK51_05 was prepared by Bio S&T, Quebec, Canada and printed on 23 nylon filters in a 4 × 4 printing configuration. The genes ndhA (NADH-dehydrogenase subunit A) and cox2 (cytochrome c oxidase) were employed to estimate contamination from chloroplasts and mitochondria, respectively. Only 0.6% of the BAC library contained organellar DNA. From a sample of 309 BAC clones, we determined the average insert size to be 71.32 ± 46.15 kb and that 30% of the sampled BAC clones contained large inserts of 80–260 kb.

Polymerase chain reaction (PCR) probes developed from a non-polymorphic marker derived from the sex linked SmX marker [67], and two genetic markers flanking the sex locus (Ke225 and udkac096) [21] were used to identify the SDR of the female parent. Purified PCR probes were labelled non-radioactively with digoxigenin-11-dUTP (Roche Diagnostics), hybridized at 65 °C overnight and detected as specified [68]. The corresponding BAC clones were isolated and the smallest clones for each of the three markers selected and labelled with biotin by nick-translation (Roche Diagnostics). These three clones were 47F17 - containing Ke225 (59.7 kb), 180D13 - containing SmX (49.3 kb), and 156B2 - containing udkac096 (85.2 kb).

Fluorescent in situ hybridisation (FISH)

The FISH procedure followed a previously published method [64], with some modifications. Briefly, probes (50–100 ng) were dissolved in 2X SSCP (0.3 M NaCl, 0.03 M sodium citrate, 0.04 M sodium dihydrogen phosphate, pH 6.5), 50% formamide and 10% dextran sulphate and denatured at 85 °C for ten minutes. Thirty μL of this hybridisation mixture was added to each slide and the slide was covered with a plastic cover slip. Preparations were denatured for 5 min in 0.15 M NaOH in 70% ethanol and then dehydrated through an ice-cold ethanol series (70, 85, and 96% for 3 min each). Incubation was at 37 °C for 24 h prior to a wash in 2X SSC (0.3 M NaCl, 0.03 M sodium citrate) at 42 °C, followed by two stringent washes of 0.2X SSC for 15 min each at 42 °C (Stringency

68%), and a final wash in detection buffer (0.1 M Tris, 0.15 M NaCl, pH 7.5) for 5 min at room temperature. The probe was detected using 50 ng/μL cy3-strepavidin conjugate (Sigma), 5% (w/v) bovine serum albumin (Sigma) in detection buffer for 60 min at 37 °C. Slides were then washed twice in detection buffer containing 0.05% (v/v) Tween® 20 for 15 min and the chromosome preparations were counter-stained for 5 min in 1 mM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma) in 1X PBS pH 7.4 and mounted in 40 μL of mounting solution (0.2% (v/v) 1, 4-diazabicyclo-[2.2.2]octane (DABCO) (Sigma), 50% (v/v) glycerol in 1X PBS pH 7.4). Following storage at 4 °C for 2–3 days before observation, chromosome spreads were observed with an Olympus Vanox AHT3 light microscope using epi-fluorescence, and images were captured with an RS Photometrics CoolSNAP digital camera. Two images were captured per cell, at excitation wavelengths of 358 nm and 550 nm. Entire images were then manipulated using Adobe® Photoshop Version 6.0. Chromosome measurements were made using the computer application MicroMeasure version 3.3 [69].


Selection On Sex Cells Favors A Recombination Gender Gap

Males and females of the same species can be strikingly different. Peacocks strut around with flashy feathers to attract mates, while peahens blend into their surroundings with more subdued colors. But differences are not always as obvious or easily explainable as in this classic example. Even the amount of genetic reshuffling that goes on during egg and sperm production differs between males and females in most species. An evolutionary reason for this has eluded researchers since the phenomenon was originally discovered in fruitflies, Chinese silk worms, and amphipods almost 100 years ago.

Genetic diversity among organisms is promoted when genetic information is rearranged during meiosis, the cell division process that yields sperm and eggs (generically called gametes). During this genetic reshuffling, chromosome pairs overlap, forming structures called chiasmata (&ldquocrosses&rdquo in Greek), and physically recombine. This process does not just create diversity, it is also an example of diversity&mdashrecombination rates vary across chromosomes, sexes, and species.

An early 20th century hypothesis to explain the sex difference in recombination proposed that recombination is restrained within a pair of unlike sex chromosomes (X and Y, for example) and that the suppression spills over to the rest of the chromosomes. Under this idea, the sex with dissimilar sex chromosomes (XY instead of XX, for example) should be the one with the least amount of recombination in all chromosomes. But that is not always the case. Some hermaphroditic species of flatworms, for example, lack sex chromosomes altogether but still display marked differences in male and female recombination rates. In one salamander genus, more reshuffling unexpectedly occurs in the sex with two different sex chromosomes.

In a new study analyzing an updated dataset of 107 plants and animals, Thomas Lenormand and Julien Dutheil bolster the argument against the recombination suppression hypothesis by showing that in species with sex chromosomes, the sex with two dissimilar sex chromosomes doesn&rsquot necessarily have a reduced recombination rate. Additionally, they found that, as a trait, the sex difference in recombination rate is not a lot more similar between two species in the same genus than between two species in different genera, suggesting that the difference evolves quickly.

An alternative hypothesis suggests that sexual selection might play a role in recombination differences. Reproductive success among males is often highly influenced by selection, so mixing up successful genetic combinations in males could be evolutionarily counterproductive. But in past studies, sexual selection was not related to variation in recombination rates.

Putting a new twist on this hypothesis, Lenormand and Dutheil realized that selection was not necessarily limited to the adult stage and that differences in selection among eggs or sperm might help account for recombination differences between the sexes. The authors reasoned that more opportunity for selection on sperm than egg should correspond to less recombination during sperm than egg production (and vice versa), consistent with the idea that genetic combinations surviving selection should remain more intact in the sex experiencing the strongest selection at the gametic stage.

Though male gametes might be expected to be under stronger selection in many species, in true pines it seems to be the female gametes. The ovules compete with each other for resources over an entire year before being fertilized, and, indeed, from the dataset analysis, ovule production involves low recombination rates compared with male pollen in this group. In males, the opportunity for pollen competition was indirectly estimated using self-fertilization rates. The authors assumed that pollen grains competing for ovules of a self-fertilizing plant would be genetically similar and therefore experience less selection. Again, in the analysis, low selection correlated with less recombination in female gamete production, as predicted.

Is selection among eggs and sperm the evolutionary force generating sex-based variation in genetic shuffling? By demonstrating that differences may be influenced by gamete selection in plants, this work has added clarity to otherwise contradictory observations.

Citation: Lenormand T, Dutheil J (2005) Recombination difference between sexes: A role for haploid selection. PLoS Biol 3(3): e63.


Hermaphrodites

The Adaptive Significance of Having Two Routes to Fitness

Arguably the biggest difference between gonochorists and hermaphrodites is that in the latter each individual has (at least potentially) access to both the male and female routes to fitness ( Figure 1 ). It is currently unclear whether the evolution of anisogamy originally leads to gonochorism or hermaphroditism ( Schärer et al., 2014 ), and the answer may well depend on the specific organismal group. Despite this, many existing models for the evolution of anisogamy make assumptions that necessarily lead to the evolution of gonochorism (e.g., Lehtonen and Kokko, 2011 Parker, 2011 ), so broadening this theory base remains a significant challenge (as does conducting empirical work in extant groups where ongoing evolution of anisogamy can be studied). From a hermaphrodite perspective, it could be argued that gonochorists are a special case, where some individuals have lost (or given up) their ability to reproduce via one of the two routes, and an important aspect of understanding hermaphroditism is therefore to understand the conditions under which it may or may not be advantageous for individuals to maintain two routes to fitness.

Figure 1 . The two routes to fitness in hermaphrodites. In hermaphrodites, survivorship, fecundity, and mating success will often contribute to fitness separately via the male and female sex functions. Nevertheless, we can expect important feedback effects between these different fitness components, only some of which are illustrated here. For example, mating success in one sex function may impact upon reproductive success in the other sex function (so-called cross-sex effects stippled arrows), as can occur if mating is reciprocal such that additional matings in the male role automatically correspond to more matings in the female role, with potentially negative consequences (see Anthes et al., 2010 for further discussion). Note also that for simplicity only a single fecundity component has been plotted, but this could again be split into male and female components and these may often trade off against each other due to a sex allocation trade-off.

Modified from Schärer, L., Janicke, T., Ramm, S. A., 2014. Sexual conflict in hermaphrodites. Cold Spring Harbor Perspectives in Biology doi: 10.1101/cshperspect.a017673, based on an original figure for gonochorists in Arnqvist and Rowe (2005) .

Current thinking about the evolution of sequential hermaphroditism considers that the male and female functions may have differing optimal body sizes, such that an individual may maximize its total fitness by first exhibiting one sex and later changing to the other (the ‘size-advantage model’ Ghiselin, 1969 Figure 2 ). Stated more broadly, the size-advantage model predicts that an individual should want to change sex whenever it can increase its ‘reproductive value’ by doing so, emphasizing that social and ecological factors come into play in determining the optimal sex-change strategy ( Warner, 1975, 1988 Charnov, 1982 Munday et al., 2006 ). We consider the rationale of the size-advantage model in more detail in Section ‘Local Competition and Sex Allocation,’ and then provide examples in Section ‘Sex in Sequential Hermaphrodites’ below.

Figure 2 . The size-advantage model for sequential hermaphroditism. If the expected fitness returns on operating as either a male or a female change predictably with size (or age), this may – provided that it is physiologically possible and the costs of doing so are not too high (see Kazancıoğlu and Alonzo, 2009 ) – favor reproductive strategies that involve sex change. (a) Protogyny (female-to-male sex change) is favored when the relationship between size and fecundity is shallower for females than males, for example, because large males are more successful at holding a territory in which they can mate with multiple females. (b) By contrast, if the size–fecundity relationship is steeper for females, for example, because larger females are more fecund, while a male's size is relatively unimportant for its fecundity, this may favor protandry (male-to-female sex change).

Modified from Munday, P. L., Buston, P. M., Warner, R. R., 2006. Diversity and flexibility of sex-change strategies in animals. Trends in Ecology &amp Evolution 21, 89–95.

For simultaneous hermaphrodites, the benefits of dual sexuality may stem from reproductive assurance when the rate of encountering potential mates is low, for example under low population density ( Ghiselin, 1969 Schärer, 2009 ). In contrast to gonochorists, for a simultaneous hermaphrodite each encountered conspecific is a potential mating partner ( Tomlinson, 1966 ). Moreover, even in the complete absence of access to mating partners, exhibiting both sexes simultaneously – or in short sequence, such as in some Caenorhabditis nematodes – has the additional benefit of permitting self-fertilization ( Charlesworth and Morgan, 1991 Jarne and Charlesworth, 1993 Jarne and Auld, 2006 ). However, simultaneous hermaphroditism is clearly not restricted to only organisms occurring at low density, and more generally, this sexual system is expected to be stable whenever there are strong diminishing fitness returns on investment into one of the sex functions ( Charnov, 1982 Schärer, 2009 ), an argument that we develop more fully in Section ‘Local Competition and Sex Allocation.’

Whilst these adaptive explanations for hermaphroditism are undoubtedly important in understanding how it can evolve, the current taxonomic distribution of the different sexual systems among animals also reveals a strong degree of phylogenetic inertia (see Renner and Ricklefs, 1995 for data on plants). While some groups are (almost) entirely hermaphroditic (e.g., flatworms, arrow worms, and gastrotrichs) there are other groups that are (almost) entirely gonochoristic (e.g., insects, nematodes, and acanthocephalans), while yet other groups show more variable sexual systems (e.g., coelenterates, polychaetes, and molluscs) (see also Ghiselin, 1969 Schärer, 2009 Weeks, 2012 Collin, 2013 ).

Irrespective of the exact conditions under which hermaphroditism is favored or maintained, once two routes to fitness are present in the same individual, this leaves open the possibility of strategically varying the amount of resources invested into the two sex functions, both in terms of overall quantity and with respect to the timing during an individual's life history (i.e., simultaneously vs. sequentially). These are questions about ‘sex allocation,’ which we discuss next.


The gonad is an amazingly labile organ where male and female signals vie for dominance in the developing embryo.

he recent controversy over the South African runner Caster Semenya's gender illustrates the complexity of how sex is assigned in humans. Experts must decide whether DNA, genitalia, or hormones should serve as the determining characteristic. Although there are cases of genetically XX females with male genitalia and vice versa, the three sex identifiers are aligned in most people. This is because, in humans and most mammals, genetic sex (i.e., whether you are XX or XY) controls development of a testis or ovary during fetal life, and all secondary sex characteristics (genitalia, musculature, sex ducts) are controlled by hormones and other secretions from the testis or ovary. 1

In many animals, sexual characteristics are quite plastic&mdasheven in adult life. In some species of fish, all it takes is a glance.

In many animals, sexual characteristics are quite plastic—even in adult life. In some species of fish, all it takes is a glance, or lack thereof, to cause an adult female to change her sex and become male. When the dominant male goes out of sight from the school, one of the females will undergo a sex change, taking on the coloration and behavior of the alpha male, and transition from making eggs to making sperm instead. A more subtle example is a species of mole that maintains “ovotestes” in adult life, changing from female to male characteristics and back again, depending on the season and whether it's more advantageous to be submissive or to produce high levels of testosterone and exhibit aggressive behavior. 2

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What accounts for the remarkable sexual plasticity seen in many animals? Perhaps it is the inherent plasticity of the gonad. For most developmental processes, there is only one possible outcome. For example, a kidney primordium can only make a kidney, and a lung primordium can only make a lung. In contrast, the gonad can develop into either a testis or an ovary. This choice, “sex determination,” occurs during fetal life and is stable thereafter, but in other animals like some fish, this choice may be reconsidered later.

Another striking difference between sex determination and other developmental processes is that the genes that control most developmental mechanisms are tightly conserved across the animal kingdom. However, the mechanisms controlling sex determination seem to vary wildly across the animal kingdom. In some animals, the sex of offspring depends on population density, whereas in others, it depends on temperature. Humans develop inside the uterus, where they are (for the most part) protected from the vagaries of the environment. They use a genetic mechanism to determine sex based on their X and Y chromosomes. No unifying mechanism has been found that controls sex determination in all vertebrates, yet it seems impossible that such an essential process isn't tightly conserved at some level.

When I began research in my own lab, it seemed to me that insight into this problem might come from a better understanding of how sex determination occurs at the level of the cell biology of organ development. How do the cells of the gonad decide to form a testis or ovary, and how do the different mechanisms of sex determination seen across the animal kingdom regulate this process? Recent work from my lab and many others suggests that there may be a common underlying mechanism after all.

F or me, the story began in 1991, with the discovery of the gene that governs sex determination in mammals. I remember it as an eventful week in Robin Lovell-Badge’s lab at the National Institute for Medical Research in London, where I was a postdoc. We had journalists and photographers putting us in "busy scientist" poses and film crews grilling us about the details of our work. We had taken our candidate gene, the mouse Sry gene on the Y chromosome, and inserted it into the genome of an XX (female) mouse embryo, turning it into a male. In a play on that experiment, one newspaper sported a cartoon of a sex-reversing Minnie Mouse.

In the reciprocal experiment, we showed that removal of the Sry gene from the Y chromosome of male embryos caused genetically XY male animals to develop as females. 3 In collaboration with the Peter Goodfellow lab at the Imperial Cancer Research Fund (ICRF) in London (which worked on the human SRY gene), we paid a visit to the London Zoo to collect DNA samples from male and female horses, chimps, rabbits, pigs, cattle, and tigers. We found that all these animals carried the SRY gene on their Y chromosome, reflecting the wide conservation of this mechanism of sex determination in mammals. 4

With the Sry work behind me, I started my own lab at Duke University in 1993. While other groups coming out of the Lovell-Badge and Goodfellow labs continued to characterize the Sry gene and other genes immediately downstream, I wanted to study the earliest cellular mechanisms that trigger the decision to develop a testis or an ovary, at the point when the SRY transcription factor is expressed in the gonad.

The first challenge was to set up a system where I could study the gonads as they developed in a controlled environment. It was no small task to figure out the right conditions to keep embryonic mouse gonads viable in a dish for several days while they made their fate decision.

Robin Lovell-Badge and a former post-doc of his, Katarina Nordqvist, came to visit my new lab in 1995. The three of us were very interested in testing an old idea that a population of cells from the mesonephros—a nearby tissue that is closely associated with the gonad at this stage—migrates into the gonad. We made a recombinant organ by combining a mesonephros carrying a beta-galactosidasegene that makes all cells blue, with a “white” unlabeled gonad, and cultured the two pieces together for several days. To our excitement, blue cells from the mesonephros migrated into the unlabeled gonads—but only into male XY gonads, never into XX female gonads. Once in the male gonad, the mesonephric cells surrounded the Sry-expressing Sertoli cells and formed testicular cords, the first morphological change that signals a commitment to testis development. 5

We could see that cells had migrated, but it wasn't clear whether that really mattered. One of my students, Christopher Tilmann, devised an experiment to test the importance of cell migration. He placed a membrane barrier between the cultured mesonephros and gonad, demonstrating that blocking the mesonephric cells from migrating prevented the early steps of testis development. We wondered what would happen if we induced migration into an XX gonad—could we make it develop more like a testis than an ovary?

After many failed efforts to test this idea, it finally dawned on me that we could make a "sandwich" organ culture. We would place a developing XX female gonad in between an XY male gonad on one side and a blue mesonephros on the other. We were excited to see that cells from the mesonephros crossed over the XX female gonad on their way to the XY male gonad. Along the way, these traveling cells induced the developing female gonad to activate some genes associated with male development and to form male-like structures resembling testis cords—all in the absence of the master Sry gene. 6

These experiments and others in the lab were gradually changing the way we viewed the problem of sex determination. Although SRY lies at the top of the sex determination cascade in mammals, it was becoming clear that the pathways downstream of SRY are critical in controlling testis morphogenesis, and without a testis, the embryo develops all female secondary sex characteristics.

B y the late 90s we had identified several developmental processes essential for gonad development, but still lacked a clear picture of the genes that controlled it. Luck took a hand when David Ornitz at Washington University Medical School called to tell me about a mutant mouse. A post-doc in his lab, Jenny Colvin, had generated mice incapable of producing fibroblast growth factor 9 (FGF9). Mice lacking Fgf9 died at birth because their lungs could not form properly. However, Jenny noticed that all of the embryos developed as females. This was a very exciting finding because it suggested that Fgf9 was one of the genes that control developmental processes important for testis development. While SRY—a transcription factor—can only act on the cell that expresses the protein, FGF9 is a secreted protein and acts as a signaling molecule to nearby cells. It sounded like it might be just the sort of signal that controls proliferation or attracts the migration of cells from the mesonephros.

Further work in my lab showed that during the bipotential stage of gonad development—before the critical fate decision—Fgf9 is expressed in both XX and XY gonads. But after Sry is expressed, Fgf9 is strongly up-regulated in XY male gonads, and down-regulated in XX female gonads. In XY male gonads that lacked Fgf9, testis development was completely blocked and some aspects of ovary development could be detected (see graphic below).

Since FGF9 is a secreted factor, we wondered what would happen if we added it to the culture medium for female gonads. To our delight, soluble FGF9 induced mesonephric cells to migrate into the XX female gonads, pushing their development toward the testis pathway. 7,8

All indicators were pointing to the idea that Fgf9 played an important role in testis development. But what controlled female development? To the great irritation of many female investigators in the field, female development had classically been referred to as the “default pathway”—suggesting a passive process. To most of us, this was not an attractive idea.

The first evidence for an active female pathway came in 1999, when Andy McMahon's group at Harvard generated a mouse incapable of producing WNT4. Like FGF9, WNT4 is a secreted signaling molecule that can affect cells at a distance. In mice lacking the Wnt4 gene, even those that were genetically XX female, gonads developed with some characteristics of testes. For example, XX gonads from these mutants showed patterns of cell migration similar to XY gonads and, later in development, produced testosterone. 9,10 This was particularly interesting because it was consistent with reported cases of genetically XX female humans who develop a testis in the complete absence of SRY. One explanation suggested for these patients was that something had gone wrong with their active ovary-determining pathway—a pathway necessary to block testis development.

We found that, like Fgf9, Wnt4 is expressed in both sexes while the gonad is still bipotential, but it is up-regulated in XX gonads and down-regulated in XY gonads precisely at the time when the gonadal fate decision occurs—the opposite of Fgf9 expression.

About this time, we remembered a piece of evidence from organ-culture experiments done earlier in my lab suggesting that FGF9 could block expression of Wnt4. Could these two signaling pathways be acting antagonistically, staging the battle of the sexes in the gonad? Yuna Kim, another graduate student in my lab, planned a set of experiments to test this idea.

Other researchers had shown that the primary role of SRY is to up-regulate a closely related transcription factor, Sox9. Various experiments showed that SOX9 is capable of substituting for SRY in activating testis development. The question was how WNT4 and FGF9 fit into the story. Yuna found that FGF9 and SOX9 reinforce each other's signaling to establish the testis pathway in XY gonads. She showed that when Fgf9 is eliminated, XY male gonads switch sex and activate ovarian genes. But our most exciting finding was when she discovered that SOX9 and FGF9 are both up-regulated in an XX female gonad when Wnt4 is absent. This clearly showed how the male pathway could be activated in an XX genetic female, in the complete absence of the Sry gene—just as those human XX male patients had predicted. 11

Based on these experiments, we proposed a new model for mammalian sex determination. In both XX and XY primordial gonads, Fgf9, Sox9, and Wnt4 are all expressed simultaneously early in development, when the fate of the gonad is still undetermined. In an XX gonad, WNT4 dominates and turns off the testis pathway. However, in an XY gonad, SOX9 and FGF9 get an extra boost from SRY, which allows them to dominate and repress WNT4.

T he animal kingdom has many means of determining sex, from population density and behavioral cues in fish, to temperature in turtles, alligators and other reptiles, and hormonal influences in many egg-laying species. Yet, surely a process as important as sex determination must be conserved at some level.

I and others have begun to suspect that although the primary gene controlling sex determination varies among species, perhaps what is conserved is an underlying pattern of antagonistic signals—such as the ones we've seen in mice with FGF9 and WNT4. This fundamental sex-determining mechanism could easily operate in response to a genetic switch (such as Sry in mammals) or to an environmental cue (such as temperature in turtles), as long as the initial decision is amplified and reinforced by downstream pathways that recruit all the cells of the gonad to one game plan. 12

In an effort to learn from another species, we began to work with red-eared slider turtles, which determine sex via temperature. When their eggs are incubated at 26 degrees Celsius, 100% become male but when incubated at 31 degrees, 100% become female. (At temperatures in between, mixed sex ratios occur.) We have begun to explore the cellular basis for the development of the testis and ovary in the turtle, and to search for similar control signals by returning to our organ-culture methods.

This work has led us to suspect that the antagonistic signaling system that we uncovered is just the tip of the iceberg—that we should be looking at the workings of the entire complex system of signals that underlie sex determination and gonad development rather than at single genes. We are very excited about a new project to do just that, employing many of the new techniques and computational skills of systems biology.

Our understanding of sexual development is evolving along with our ability to test and measure the process. We have only begun to clarify the early genetic and cellular processes that influence the initial stages of gonad differentiation. The subsequent effects of hormones, environment, and neurological wiring all have critical roles in the eventual identification of an individual as “male” or “female.”

In the face of this complexity, tests used by many athletic organizations for the presence of SRY as the sole means of classifying contestants as male or female seem very simplistic. Among other things, this assessment does not provide a category for qualified individuals who possess some combination of male and female characteristics. Yet these individuals also represent the spectrum of human abilities. In the case of Caster Semenya, it is a pity that her impressive achievements might be overshadowed by accusations that may simply stem from her misalignment with Western standards of beauty rather than from purposeful deception with respect to her sex.

Blanche Capel is a Professor in the Department of Cell Biology at Duke University Medical Center. She thanks the many former and current members of her lab for their wonderful work, especially Lindsey Barske and Jonah Cool, who have helped edit this article.

Correction (15 September 2009): This article originally identified Blanche Capel as an associate professor. She is actually a full professor. The Scientist regrets the error.