Horse Genetics Module Two Modified and Complex Inheritance

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Night Owl Education and Equestrian Module Two: modified and complex inheritance

Lesson four: Modified Ratios Introduction Partial dominance Co-dominance Lethal alleles Lethal White Overo Epistasis Penetrance and expressivity Summary Reference

Lesson five: Genetic Linkage Introduction Linkage and the gene for equine combined immune deficiency disorder Partial Linkage example of partial linkage Sex Linkage Summary References

Lesson six: Complex Traits and Polygenic Inheritance Introduction Continuous Traits Describing the variation for a quantitative trait Continuous Traits are Polygenic How can quantitative genetics be useful to horse-breeders? Running Speed in Race Horses Personality traits, and their possible connections with colour and pattern Summary References

Module two assignment

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Lesson Four: Modified Ratios

Introduction

Mono- and di-hybrid crosses do not always give the classical 3:1 and 9:3:3:1 Mendelian ratios.

In the examples so far only simple gene action was considered: for each gene one of the alleles has always been completely dominant over the other in the heterozygote. In the case of two genes the genes control two different characters and act separately from one another. Often the situation is more complex than in these examples. The different ways genes are expressed in the phenotype can affect genetic hybrid ratios, especially where there’s an interaction between genes.

Modified ratios can also occur because the genes being considered are linked on the same chromosome (for a di-hybrid cross) or because they’re on the sex chromosomes (sex linked). These situations of linkage and sex-linkage are considered in lesson five.

Remember: the different ways genes are expressed in the phenotype can affect genetic ratios, especially where there’s an interaction between genes

Partial dominance

In cases of complete dominance the heterozygote has the same phenotype as the dominant homozygote. This relationship between a pair of alleles provides the simplest situation for study. With partial dominance (also called semi-dominance or incomplete dominance) the heterozygote exhibits a phenotype which is intermediate between the homozygous forms. There’s a well known example in horse genetics, that of the cream dilution gene (the C locus).

Alleles at the C locus are responsible for the palomino, buckskin, smokey black, cremello, perlino and smokey cream. The two known alleles are designated C+ and CCr. CCr shows partial dominance. It dilutes red pigment to yellow in a single dose and to pale cream in a double dose. Night Owl Education and Equestrian

Horses with a chestnut base colour and genotype C+C+ are chestnut, while those of genotype CCrCCr are cremello. Horses of genotype C+CCr are palomino, a colour intermediate between the phenotypes for the homozygous forms. Hence there are three phenotypes, with heterozygous horses having an “in-between” phenotype.

Similarly horses with a brown or bay base colour and genotype C+C+ are brown or bay, while those of genotype CCrCCr are perlino. Horses of genotype C+CCr are buckskin, a colour intermediate between the two. Cream dilution can have a very subtle effect on black pigment and horses with a black base colour are diluted to smokey black (C+CCr), which can look almost black or sometimes brown or liver, or to smokey cream (CCrCCr) which can be a very attractive fawn colour.

You should notice that while the CCr is semi-dominant the wild-type C+ allele is effectively recessive since it needs to be homozygous for there to be no dilution of the base colour.

The 3:1 phenotypic ratio for a monohybrid cross is modified to 1:2:1 ratio. For example a cross between two palominos gives an expected theoretical ratio of 1 chestnut: 2 palomino: 1 cremello. This is the same as the genotypic ratio since the heterozygotes are of a separate phenotype (i.e. heterozygotes are palomino and not cremello, as they would be in the case of complete dominance of the CCr allele). This ratio is important for palomino breeders, especially those wishing to avoid producing cremello foals. Cremello horses were formerly discriminated against by some of the breed societies, including the American Quarter Horse Association (AQHA) who until recently didn’t allow their registration. However it can be seen that breeding together palominos will always result in some cremello foals, which will make up about ¼ of foals produced from such crosses. Night Owl Education and Equestrian

Genetic contributions from sire from dam ↓

C+ CCr

C+ 25% chance: C+C+ 25% chance: C+ CCr chestnut palomino

CCr 25% chance: C+ CCr 25% chance: CCr CCr palomino cremello

Remember

● With partial dominance the heterozygote exhibits a phenotype which is intermediate between the homozygous forms. ● the 3:1 phenotypic ratio for a monohybrid cross is modified to 1:2:1

Co-dominance

In co-dominance both alleles are expressed in the phenotype and the heterozygote has the characters of both parents. The inheritance patterns for co-dominance are similar to those for partial dominance.

The best known examples of co-dominance are those of the blood groups and blood protein types, for example the AB blood group where animals with blood group AB have both A and B type antibodies in their blood. Co-dominant traits are used especially for parentage testing and for research, including for conservation purposes and for finding important genes involved in genetic disorders. They are discussed further in later parts of the course. Night Owl Education and Equestrian

There are also loci with alleles made up of non-coding DNA, i.e. DNA that isn’t responsible for any external phenotype characters. Such loci can have lots of different alleles (because they don’t “do” anything mutations aren’t harmful to them and lots of variation can build up without natural selection throwing it away). Some of these loci are useful as genetic markers in molecular genetic studies. They have, for example, been used to find genes for some important horse genetic disorders, such as equine combined immune deficiency disorder (equine CID, discussed elsewhere in relation to linkage). These loci have phenotypes only at the level of a molecular genetics test, but their inheritance is nevertheless co-dominant in as much as both alleles at a locus can be detected.

Remember ● In co-dominance both alleles are expressed in the phenotype and the heterozygote has the characters of both parents. ● The inheritance patterns for co-dominance are similar to those for partial dominance. ● Co-dominant traits are useful for parentage testing and for research, including for conservation and for finding genes involved in genetic disorders.

Lethal alleles

There are a few well known examples of lethal alleles in horses, including the white allele and the overo allele. The roan allele was formerly thought to be lethal but has recently been shown not to be. Lethal alleles result in modified ratios among surviving foals. It can be important for horse breeders to know about lethal alleles, especially those breeding paint, pinto or coloured horses.

Two alleles are known for the gene for white coat colour, symbolised WW and W+. Most horses are not white and have genotype W+W+. The WW allele is rare in most breeds of horse, but occurs in Tennessee Walking Horses, American Albinos and Miniatures, and rarely in Arabians, Standardbreds and Thoroughbreds. Horses with the WW allele are dark-eyed horses with white coats. WW is dominant over W+, so that horses of genotype W+WW are white. These horses are not the same as white sabinos or other pintos or paints, nor are they cremellos (which have blue eyes). No horses are known with the genotype WWWW. Breeding between white horses always produces some coloured foals, indicating that the horses are heterozygous. It would seem that embryos or Night Owl Education and Equestrian foetuses homozygous for allele WW die early in gestation and are then either resorbed or miscarried. WW is therefore acting as a recessive lethal allele. We say that the allele WW is dominant visible and recessive lethal.

The following diagram shows how the standard 3:1 phenotypic ratio of a monohybrid cross between white horses is modified to a 2:1 ratio typical of recessive lethal genes.

Genetic contributions from stallion

from mare ↓ WW W+

WW WWWW WWW+ Dies in utero White

W+ WWW+ W+W+ White coloured

There appears to be another lethal gene that occasionally causes death in new born Arabian horses. The foals are born a dilute colour (“lavender”) and often result from a difficult foaling. They have neurological problems and fail to stand and nurse.

Remember: ● Horse breeders should know about lethal alleles, especially those breeding paint, pied or coloured horses. ● Lethal alleles result in modified ratios among surviving foals. ● For recessive lethal genes the standard 3:1 phenotypic ratio of a monohybrid cross is modified to a 2:1 ratio. Night Owl Education and Equestrian

Lethal White Overo

A second well known lethal gene is that which causes the white pattern in overo horses. There are various different genes that cause white coat patterning in paints or “coloured” or pied horses, and overo is genetically distinct from other patterns such as tobiano and sabino. Unfortunately the term “overo” is sometimes unhelpfully used to describe any horse with white spotting patterns not due to appaloosa.

Overos are heterozygous for a gene that is lethal when homozygous. Thus the overo allele (OO) is dominant for colour pattern but has a recessive lethal effect.

The allele also shows something called pleiotropy. This means it has more than one effect on the phenotype (i.e. it affects more than one character). Homozygous foals (OOOO) are all-white with blue eyes and die of complications from intestinal tract abnormalities. Both melanocytes (pigment cells) and ganglia (nerve cells) are migratory cells that originate from the same area of the developing foetus known as the neural crest. The all-white foals lack both pigmentation and nerve cells in the intestinal tract (aganglionosis). It is the lack of nerve cells that ultimately causes the condition to be lethal, with foals not being able to pass food through the digestive system. When two heterozygous overo horses are bred together there is a 25% chance of a lethal white foal. Surviving offspring are either overo or solid coloured. Matings between solid and overo horses result in solid and overo foals in approximately equal numbers, with no lethal white foals.

Occasionally horses without noticeable body spotting patterns have sired or produced lethal white foals. The overo spotting pattern is phenotypically heterogeneous (i.e. it varies a lot) and it is possible that such horses show insufficient white spotting for registration purposes, even though they have the overo genotype. (Another possibility is that the overo mutation occurs “de novo” in the gametes of one parent, but this isn’t a likely explanation in most cases.) The risk is greater in breeds where white markings have traditionally been discriminated against, such as the American Quarter Horse. Selection in those breeds has led to some genetically overo horses with minimal markings.

Overo can occur in combination with other patterns. Then overo may not be apparent, either because it is minimal or because the other pattern is relatively extensive (or both). I know of one Night Owl Education and Equestrian person who bred her apparently non-overo sabino quarter horse stallion to overo mares, only to later discover that he did in fact have an overo gene too. She’s now nervously awaiting the outcome of these matings. Let’s hope she is lucky.

Breeders would obviously like to be able to recognize horses at risk of producing lethal white foals. Until recently, there has been no reliable way to identify which horses have the gene associated with lethal white overo (LWO). Now, however, there is a molecular genetics test (discussed later in the course). This test can identify horses with an overo allele even when their phenotype is cryptic.

A defect similar to lethal white syndrome has been reported in Clydesdale foals (Dyke et al, 1990), which also were lacking intestinal ganglia. The foals were not white and lived for between 4 and 9 months. However they were sabino, which is another white spotting pattern. It is possible that, during development, insufficient numbers of ganglia cells migrated to the intestine region, possibly as a result of the actions of the sabino genes. However the reason may be more complicated or different from this.

Remember

● Overo is genetically distinct from other similar white spotting patterns such as tobiano and sabino. ● Overos are heterozygous for a gene that is lethal when homozygous.

● The overo allele (OO) is dominant for colour pattern but has a recessive lethal effect. ● Overo may not be apparent in the phenotype if a horse is minimally marked or if overo is occurring in combination with other patterns, such as tobiano. There is, however, now a molecular genetics test that can positively identify whether a horse has the overo allele. ● Pleiotropy occurs when a gene has more than one effect on the phenotype (i.e. it affects more than one character). Night Owl Education and Equestrian

Epistasis

Epistasis is a gene interaction where an allele or alleles at one gene masks the phenotypic expression of alleles at another gene or genes. The phenotype is governed by the masking gene when both are present together in the genotype. A genotype that masks another’s expression is said to be epistatic, while the gene or genes whose expression is masked are said to be hypostatic. There are several examples of epistatic relationships in horse genetics, especially between genes that govern coat colours.

Epistatic alleles may be recessive or dominant. If they are recessive then individuals homozygous for the epistatic allele are of the same phenotype regardless of the genotype at the other gene or genes. Alternatively epistasis may result from the presence of a dominant allele, which also conceals the genotype at the second or more loci.

An example of recessive epistasis is shown by the extension and agouti genes, which account for the differences between black, bay, brown and chestnut horses. The recessive alleles of the extension gene are epistatic, all the alleles of the agouti gene are hypostatic.

The alleles of the extension gene extend (E+) or diminish (e and ea) the amount of the black eumelanin pigment in the coat, with opposite effect on the amount of the red pigment phaeomelanin. The dominant alleles of the agouti locus cause the distribution of black hairs to be restricted to the points (e.g. lower legs, mane, tail and ear rims). Horses homozygous for the recessive allele Aa are uniformly black. Since there are usually no black hairs in chestnut horses the agouti gene can have no effect on the distribution of black in these horses. The genotypes ee, eaea and eea all give chestnut coated horses, with the difference in the two alleles being only slight and usually only distinguishable at the molecular level. These extension locus genotypes conceal the genotype at the agouti locus: whatever the genotype at the agouti locus these horses are always chestnut (or sorrel), or some colour derived from chestnut, such as palomino, red dun or red roan.

We can demonstrate the affect of epistasis on genetic ratios through an example. Say we had a bay mare and stallion who were both of genotype E+e at the extension locus (E+ causes the production of the black eumelanin pigment) and of genotype AAAa at the agouti locus (which controls the distribution of black pigment). In genetic terms crossing together two heterozygous horses like this Night Owl Education and Equestrian is to make a dihybrid cross.

The gametes may now be of four types, any of which are equally likely: E+AA, E+Aa, eAA or eAa. The possible outcomes of the cross can be seen from a Punnett square:

Genetic contributions stallion

mare ↓ E+AA E+Aa eAA eAa

E+E+AAAA E+E+AAAa E+eAAAA E+eAAAa E+AA bay bay bay bay

E+E+AAAa E+E+AaAa E+eAAAa E+eAaAa E+Aa bay black bay black

eAA E+eAAAA E+eAAAa eeAAAA eeAAAa bay bay chestnut chestnut

eAa E+eAAAa E+eAaAa eeAAAa eeAaAa bay black chestnut chestnut

Instead of the typical 9:3:3:1 dihybrid ratio there is a 9:3:4 of bay: black: chestnut. The agouti allele in the chestnut horses is irrelevant to the phenotype since there is no black pigment to distribute, either uniformly or in the points.

A well know example of dominant epistasis is that of the grey allele. Horses with at least one copy of the allele GG go through the greying process regardless of the genotype at the other genes controlling coat colour. They may be born chestnut, bay, buckskin or any other colour or colour pattern, but they will steadily turn grey over time, and eventually may turn almost white.

Because of the grey allele being dominant heterozygous grey horses can have foals of other colours, depending on their genotype for the other colour genes. Consider, for example, that we had a grey heterozygous mare and stallion (both of genotype G+GG) who were also heterozygous for the allele e, that causes chestnut when homozygous (i.e. they are therefore both of genotype E+e). The gametes from each horse would now be of four types, any of which are equally likely: G+E+, G+e, GGE+ or GGe. Night Owl Education and Equestrian

Genetic contribution from sire dam ↓ GGE+ GGe G+E+ G+e

GGE+ GGGG E+E+ GGGG E+e G+GG E+E+ G+GG E+e grey grey grey grey

GGe GGGG E+e GGGG ee G+GG E+e G+GG ee grey grey grey grey

GGG+ E+e G+GG E+e G+G+ E+E+ G+G+ E+e G+E+ grey grey black, bay or black, bay or brown brown

G+GG E+e G+GG ee G+G+ E+e G+G+ ee G+e grey grey black, bay or chestnut brown

There is now a 12:3:1 ratio. The genotype at the extension locus makes no difference to the eventual phenotype as long as there is at least one grey allele present. Notice that about ¾ of all foals are expected to be grey, which is as it would be if we considered the grey gene on its own (i.e. for a monohybrid ratio).

Remember: ● Epistasis is a gene interaction where an allele or alleles at one gene masks the phenotypic expression of alleles at another gene or genes. ● A genotype that masks another’s expression is said to be epistatic, while the gene or genes whose expression is masked are said to be hypostatic. ● Recessive epistasis is when the homozygous recessive genotype at one locus is epistatic, hiding the effect of alleles at another hypostatic locus. ● Dominant epistasis is when the dominant allele or alleles at one locus are epistatic, hiding the effect of alleles at another hypostatic locus.

Penetrance and expressivity

Some genotypes only exhibit themselves in particular environmental conditions, and/or in the Night Owl Education and Equestrian presence of other genes. Such genotypes are said to show reduced penetrance since the genotype doesn’t always “penetrate” to the phenotype. When this occurs it is not always possible to tell that the genes are present by observing the phenotype, causing modified (i.e. non-Mendelian) ratios among phenotypes. Penetrance is measured as the proportion of individuals with a particular genotype in which the phenotype is expressed. It is probable that there are various horse genes that aren’t always expressed, although I don’t know of any research that explicitly shows this.

Candidates include some of the genes that modify the shades of coat colours, which are known to vary with nutrition, for example. Also it seems likely that some genes for genetic disorders show variable penetrance, including those associated with cancer: skin cancer, for example, is known to be more prevalent in grey horses.

Other genes may show varying degrees of phenotype according to the environment and/or genetic background, both between individuals and within the same individual at different times. This is called variable expressivity since the gene is expressed differently in different individuals. Many coat colour and pattern genes show variable degrees of phenotype. Appaloosas, for example, may show only a few characteristics of the type, such as striped hooves, or they may be spotted all over, as in leopard spots. There are several genes for appaloosa and several corresponding phenotypes, but all the genes show variable expressivity, as do the genes that cause white spotting in paint horses. Another example of variably expressed gene is that which causes hyperkalemic periodic paralysis (HYPP), a muscle disorder that occurs most commonly in well muscled Quarter Horses. The disorder varies between horses and can be managed to reduce the effects in sufferers. This disorder, which probably also shows variable penetrance, is discussed further later in the course.

Remember: ● Some genotypes only exhibit themselves in particular environmental conditions, and/or in the presence of other genes. Such genotypes show reduced penetrance. ● Penetrance is measured as the proportion of individuals with a particular genotype in which the phenotype is expressed. ● Genes that show varying degrees of phenotype according to the environment and/or genetic background show variable expressivity. Night Owl Education and Equestrian

Summary

The different ways genes are expressed in the phenotype can affect genetic ratios, especially where there are interactions between genes, or between genes and the environment.

Dominance relations aren’t always restricted to fully dominant and recessive alleles. Where there’s partial dominance the heterozygote has a phenotype intermediate between the homozygous forms, as palomino is between cremello and chestnut. In co-dominance both alleles are expressed in the phenotype and the heterozygote has the characters of both parents. The 3:1 phenotypic ratio for a monohybrid cross involving partial or co-dominance is modified to 1:2:1 .

Lethal alleles result in modified ratios among surviving foals. For recessive lethal genes the standard 3:1 phenotypic ratio of a monohybrid cross is modified to a 2:1 ratio. Overos are heterozygous for a gene that is lethal when homozygous. There is now a molecular genetics test that can positively identify whether a horse has the overo allele.

Pleiotropy occurs when a gene has more than one effect on the phenotype (i.e. it affects more than one character). Epistasis is a gene interaction where an allele or alleles at one gene masks the phenotypic expression of alleles at another gene or genes.

Some genotypes only exhibit themselves in particular environmental conditions, and/or in the presence of other genes. Such genotypes are show reduced penetrance. Genes that show varying degrees of phenotype according to the environment and/or genetic background show variable expressivity.

Further examples of all the phenomena introduced in this lesson will be pointed out and discussed during the remainder of the course.

Reference

Dyke, T.M., Laing, E.A. and Hutchins, D.R. 1990. Megacolon in two related Clydesdale foals. Australian Vetinary Journal 67: 463-464. Night Owl Education and Equestrian Lesson five: Genetic Linkage

Introduction

The Mendelian segregation of two or more pairs of characters occurs because the genes controlling those characters are located in different chromosomes pairs. At meiosis the alleles of one gene separate independently of the alleles of the other gene (or genes) so that any one particular allele is as likely to be recombined with either of the possible alternative alleles at the other locus (or loci). For example if we had a grullo stallion heterozygous for both black (E +e) and dun (D +DD), then the E + allele is as likely to occur with the D + allele as it is with the D D allele. The same is true for allele e. Put another way the frequencies of genotypes E +D+, E +DD, eD + and eD D among the stallions sperm would be expected to be roughly equal.

If all genes were on separate chromosomes then independent segregation would provide us with an adequate description of heredity. We now know, however, that chromosomes are made up of linear sequences of large numbers of genes all linked together. Linked genes don’t behave independently of one another in their inheritance, with neighbouring genes being inherited together. Accordingly we can define linkage as being the association of genes in their inheritance due to them being located on the same chromosome. The same term may also be used to describe the characters determined by the linked genes (i.e. the characters may be said to show linkage). When we look at the inheritance of characters determined by linked genes we do not see the Mendelian ratios typical for two genes inherited independently of one another.

Remember
Linked genes don’t behave independently of one another in their inheritance. Neighbouring genes are inherited together.
Linkage is the association of genes in their inheritance due to them being located on the same chromosome.

Night Owl Education and Equestrian Linkage and the gene for equine combined immune deficiency disorder

I will describe linkage through the example of the gene for equine combined immune deficiency disorder (equine CID). The recessive allele of this gene causes equine CID and its characterisation was one of the first success stories of equine molecular genetics. Equine CID results in a deficiency of the immune system and foals born with the condition usually die within 3 months of birth. It’s thought that about ¼ of all Arabs carry the allele for equine CID (McGuire and Poppie, 1973), presumably due to inbreeding within the breed.

The process of finding and characterising the gene involved identifying “marker” genes linked to the equine CID gene. (Looking for an unknown gene is a bit like looking for a needle in a haystack when you’re not quite sure what the needle looks like, but you know it’s attached to a very long piece of brightly coloured wool – you find the wool first! Briefly the idea is to start a search by first finding out what the gene is close to that you already know about, i.e. which known genes it’s linked to).

If 2 loci (the CID gene and a “marker”) are closely linked only 2 types of gametes would be expected. This was seen to be the case for the genetic “marker gene” called HTG8.

Suppose the equine CID gene is denoted D and the marker M, with d being the disease allele and m the other marker allele. If M and D were close together on one chromosome and d and m were on the other we would expect gametes with genotypes DM and dm in roughly equal proportions. If a carrier stallion was mated with a non carrier mares we’d see the following genetic ratios:

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Genetic contributions carrier stallion normal mares ↓ 50% DM 50% dm

100% DM DD MM Dd Mm 50% Normal foals 50% Carrier foals

If a carrier stallion was mated with a carrier mare we’d see the following genetic ratios:

Genetic contributions carrier stallion

carrier mare ↓ 50% DM 50% dm

50% DM DD MM Dd Mm Normal foal Carrier foal

50% dm Dd Mm dd mm Carrier foal Equine CID foal

Although two genes are involved we see a ratio like that for a monohybrid cross since the linked genes are inherited as a single unit . There are two phenotypes since carriers are phenotypically indistinguishable from normal foals unless a molecular genetics test is done to identify the equine CID allele, or breeding reveals carrier status.

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If the two genes were independently assorting they would not be genetically linked at all. Then 4 types of gametes would be expected in equal proportions. The d allele would then be as likely to segregate with either the M or the m allele during gamete formation, the same is true for the D allele. In that case a typical 9:3:3:1 dihybrid ratio would be expected.

Finding that a particular marker allele was always inherited with the disease allele was very helpful. The marker acted like a little flag that said the disease gene lives near here, this is where you should look for it. It’s like a detective being told that a murderer lives in a particular street when all he had previously known was that he lived in the town somewhere. This analogy is apt since the allele being searched for in this example is a killer!

Remember:
Genes whose position in the genome is unknown can be found by their linkage to genes whose positions are already known, or marker genes whose position can be easily found.
If 2 genes are closely linked there are only 2 genotypes among the gametes.

Partial Linkage

Many genes are partially linked. In such cases we get 4 gamete genotypes, but not in equal proportions. Once again the genotype and phenotype ratios do not conform to those for dihybrid crosses.

Partial linkage occurs because the maternal and paternal chromosomes come together at meiosis (gamete formation) and swap some genes in a process called crossing over. This gives rise to new combinations of characters and so is a source of genetic variation. The farther apart two genes are on a chromosome the more likely there is to be crossing over between them. This is

Night Owl Education and Equestrian because only one or a few crossing over events occur per pair of chromosomes.

The diagram below represents what might happen during gamete formation. Each chromosome is made up of two identical strands, each called a chromatid . One chromatid of each of the maternal and paternal chromosomes breaks, the break being in the same place on each chromatid. Each broken end of the paternal chromatid then rejoins with a broken end of the maternal chromatid so that the chromosomes exchange parts . It’s like swapping the lids of two cooking pans that are identical in all but colour. Both pans work as well with their swapped lids, they just look a bit different.

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The chromatids of each chromosome separate and are distributed into four newly forming gametes, each with one of the four chromatids (2 from each of the pair of chromosomes). Later each chromatid is replicated so that each gamete has a complete new chromosome.

The proportion of recombinant genotypes (Dm and dM) depends on the amount of crossing over between the loci, which increases with increasing distance between them. Given a limited number of cross-overs per chromosome (often only one) the greater the distance between two genes the more likely crossing- over is to occur there, rather than else-where in the chromosome.

If there is always crossing over between two genes (because they’re far apart on the chromosome) the genes aren’t considered to be genetically linked and the ratios expected for a di-hybrid cross are the same as those for independent segregation.

The genotype and phenotype ratios for partial linkage are therefore somewhere in between those for complete linkage (with only parental genotypes and no recombinant ones) and independent segregation (when recombinant and parental genotypes are equally likely).

Scientists use partial linkage as well as complete linkage in their searches for genetic disorders (such as the equine CID already discussed). This makes it easier to find, isolate and characterise those genes, and can lead and to improvements in the understanding and treatment of genetic disorders, and also to genetic tests useful to breeders.

Remember
If 2 genes are partially linked there are 4 genotypes among the gametes, but the parental genotypes are more frequent than the recombinant ones.

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The proportion of recombinant genotypes depends on the amount of crossing over between the genes, which increases with increasing distance between them.

example of partial linkage

The extension coat colour gene (E locus) is linked to the genes for roan (RN) and tobiano (To). The dominant extension allele is E + and horses with this allele produce black eumelanin pigment, resulting in the horses of black, brown or bay and their derivatives, depending on other genes. Allele e is recessive and horses of genotype ee are chestnut (including sorrel) or some derivative of that colour (such as palomino). The dominant allele at the tobiano locus is To T, with horses of genotype To +To + being solid coloured rather than white spotted.

We will consider what happens in di-hybrid crosses between two black tobiano horses (i.e. between horses of genotype E +e To TTo +). Because of crossing over any particular animal may have chromosomes with the genotypes E + To T, E + To +, e To T or e To + – and this will affect the expected ratios of the different phenotypes and genotypes. We will assume that both our horses have chromosomes of genotype E + To T and e To +. We will further assume that both horses are of genotype A aAa at the agouti locus so that only black or chestnut foals can result from the cross. Lets say for arguments sake that 20% of gametes had recombinant genotypes. The following shows what we expect from such a mating:

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Genetic contributions from sire from dam 40% To T E+ 40% To + e 10% To T e 10% To + Parental Parental Recombina E+ type type nt type Recombin ant type 40% To T E+ 16% To T To T 16% To T To + 4% To T To T 4% To T To + Parental type E+E+ E+e E+e E+E+ black tobiano black tobiano black tobiano black tobiano

40% To + e 16% To T To + 16% To + To + 4% To T To + 4% To + To + Parental type E+e ee ee E+e black tobiano chestnut chestnut black tobiano 10% To T e 4% To T To T 4% To T To + 1% To T To T 1% To T To + Recombinant type E+e ee ee E+e black tobiano chestnut chestnut black tobiano tobiano tobiano

10% To + E + 4% To T To + 4% To +To + 1% To T To + 1% To + To + Recombinant type E+E+ E+e E+e E+E+ black tobiano black black tobiano black

Black tobiano: 16%x3 + 4%x4 + 1%x2 = 66% Black: 4%x2 + 1% = 9% Chestnut tobiano: 4%x2 + 1% = 9% Chestnut: 16% (Total 100%)

(for those without much maths the multiplication is done before the addition in the above calculations)

From this you could see that, on average, about 2/3 of the foals would be expected to be black tobiano. Put another way any particular foal has a 66% chance of being black tobiano. The chances would be different if more or less recombination occurred.

The alleles may be linked differently in different horses, and particularly in different

Night Owl Education and Equestrian breeds of horses, so that, for example, To T e and To + E + might be the parental types rather than To TE+ and To +e. This is the case in the example that follows. There are also assumed to be fewer recombinant chromosome types to show how this can affect ratios:

Genetic contributions from sire mare 2% To T E+ 2% To + e 48% To T e 48% To + E + Recombina Recombina Parental Parental type nt type nt type type 2% To T E+ 0.04% To T 0.04% To T 0.96% To T 0.96% To T To + T + + + + T + + + Recombinant type To E E To E e To E e E E black tobiano black tobiano black tobiano black tobiano 2% To + e 0.04% To T 0.04% To + 0.96% To T 0.96% To + To + + + + + + Recombinant type To E e To ee To ee E e black tobiano chestnut chestnut black tobiano 48% To T e 0.96% To T 0.96% To T 23.04% To T 23.04% To T To + T + + T + Parental type To E e To ee To ee E e black tobiano chestnut chestnut black tobiano tobiano tobiano 48% To + E + 0.96% To T 0.96% To +To + 23.04% To T 23.04% To + To + + + + + + + + + Parental type To E E E e To E e E E black tobiano black black tobiano black

Black tobiano: 23.04%x2 + 0.96%x4 + 3%x0.04 = 50.04% Black: 0.96%x2 + 23.04% = 24.96% Chestnut tobiano: 0.96%x2 + 23.04% = 24.96% Chestnut: 0.04% (4 in 10,000) (Total 100%)

In this case you can see that about half the foals (on average) are expected to be black tobiano and half either black or chestnut tobiano. Another way of putting this is to say that for any particular foal the chances of it being black tobiano are about 50%. Very few foals would be chestnut – or to put it another way the chances of getting a

Night Owl Education and Equestrian non-tobiano chestnut foal are small.

Remember: The extension coat colour gene (E locus) is linked to the genes for roan (RN) and tobiano (To).

Sex Linkage

Sex linked genes are on the sex chromosomes, that is to say the X and Y chromosomes (non sex chromosomes are called autosomes). In actual fact most sex linked genes are on X chromosomes. The Y chromosomes are much smaller than X chromosomes and contain very few genes. Females have two X chromosomes while males have one X chromosome and one Y chromosome.

Since the X and Y chromosomes are largely non-homologous males have only one allele for almost all of the sex-linked traits. They are said to be hemizygous for these traits. This results in recessive alleles being expressed without them needing to be homozygous (which they would have to be in females). Especially when alleles are rare, such as those for genetic disorders, they tend to be in the heterozygous form in females (since the other allele is statistically more likely to be a common allele than a rare one – the same is true for autosomal traits). This leads to recessive sex-linked traits being more common in males than females. It also leads to altered genetic ratios compared with those for autosomal traits.

The inheritance of the sex chromosomes is shown below, and we can see that – on average – we expect a 50:50 ratio of males to females, even though in nature (and in domestication) breeding stallions tend to have harems of mares.

Genetic contributions stallions mares X Y

X XX XY Filly foals Colt foals

Night Owl Education and Equestrian

An example of a sex-linked gene is that causing haemophilia A (factor VIII deficiency). Although the recessive haemophilia A allele isn’t unique to horses it has rarely been reported to occur in Thoroughbreds, Quarter Horses and Standardbreds (Archer, 1961, Henninger, 1988, Hutchins et al , 1967). Sufferers do not make the blood clotting protein called factor VIII, or they make a defective form of it. This causes recurrent subcutaneous hematoma, hemathrosis and internal haemorrhaging with anaemia that often leads to death. In humans the disorder can be managed by providing factor VIII replacement therapy, but this is expensive and not used on stallions who suffer from haemophilia A (who couldn’t be used for performance or breeding even if they survived long enough).

Remember
Since the X and Y chromosomes are largely non-homologous males have only one allele for almost all of the sex-linked traits, and are said to be hemizygous for these traits.
Sex linked recessive alleles are expressed in males without them needing to be homozygous.
recessive sex-linked traits are more common in males than females.
Sex linked traits show altered genetic ratios compared with those for autosomal traits.

Summary

Linkage is the association of genes in their inheritance due to them being located on the same chromosome. Neighbouring genes on a chromosome are inherited together, so don’t behave independently of one another in their inheritance.

Partially linked genes are on the same chromosome but far enough apart that some crossing over occurs between them during meiosis. There will be 4 genotypes among

Night Owl Education and Equestrian the gametes, but the parental genotypes will be more frequent than the recombinant ones. The proportion of recombinant genotypes depends on the amount of crossing over between the genes, which increases with increasing distance between them.

Since the X and Y chromosomes are largely non-homologous males have only one allele for almost all of the sex-linked traits, and are said to be hemizygous for these traits. Sex linked recessive alleles are expressed in males without them needing to be homozygous, making recessive sex linked traits more common in males than females. Sex linked traits show altered genetic ratios compared with those for autosomal traits.

References

Archer, R.K. 1961. True haemophilia (haemophilia A) in a Thoroughbred foal. Veterinary Record 73 , 338-340. Henninger, R.W. 1988. Hemophilia A in two related Quarter Horse colts. Journal of the American Veterinary Medical Association 193 , 91-94. Hutchins, D.R., Lepherd, E.E. and Crook, I.G. 1967. A case of equine haemophilia. Australian Veterinary Journal 43 , 83-87.

Night Owl Education and Equestrian

Lesson six: Complex Traits and Polygenic Inheritance

Introduction

So far we have been concerned with traits that exhibit distinct phenotypes, e.g. coat colour, coat curliness and inherited disorders. Such traits are called discontinuous traits. There is usually a reasonably straightforward relationship between the phenotypes and the genotypes that determine them. Each genotype typically produces a single phenotype, although one phenotype might be produced by more than one different genotypes, e.g. in the cases of dominance and di-hybrid inheritance. Genotypes can often be inferred by studying the phenotypes of individuals and their relatives, particularly parents and offspring.

We have seen that the relationship between phenotype and genotype can be complicated by the occurrence of pleiotropy and epistasis. Also the expression of particular genotypes may vary with the environment, during development in utero and after birth. The occurrence of variable penetrance and expressivity mean that single genotypes may give rise to a range of different possible phenotypes.

Many (perhaps most) traits exhibit a wide range of different phenotypes, there being a continuous spectrum of phenotypes within a certain range. Such traits are said to show a continuous distribution of phenotypes, and are therefore called complex or continuous traits. Examples of continuous traits include birth weight, height at maturity, the percentage of white present for various coat patterns and markings, and various traits of conformation, performance and temperament. Even some colours could be thought of as occurring in continuously varying shades. Palominos, for example, can vary from very pale “Isabella’s” to deep golden horses that almost look like chestnuts, while chestnuts can look any shade from almost palomino to a dark liver chestnut. Night Owl Education and Equestrian

Continuous Traits

Generally continuous traits are described, or at least measured, in quantitative terms. They are therefore often called quantitative traits. Height, for example, is usually measured in hands and inches, or in metres and centimetres. Race winning times and stud fee earnings may be measured in seconds or pounds sterling (or another currency) respectively. The study of the inheritance of quantitative traits is known as quantitative genetics. Biologists have been developing methods for studying quantitative traits since the late nineteenth century, even before they were aware of Mendel’s principles of heredity. They demonstrated relationships between the phenotype values for parents and offspring for several continuously varying traits, even though they did not understand the inheritance of continuous variation.

Remember ● traits that exhibit distinct phenotypes are called discontinuous traits ● complex traits display a continuous spectrum of phenotypes within a certain range ● continuous traits are measured in quantitative terms ● the inheritance of quantitative traits is known as quantitative genetics ●

Describing the variation for a quantitative trait

Continuous traits are usually described by using a frequency distribution of the phenotypes for the trait. The frequency distribution shows the number or proportion of individuals whose phenotype falls within particular classes, where each class represents a portion of the range spanned by all the phenotypes.

Many continuous traits exhibit a symmetrical bell-shaped distribution called a normal or Gaussian distribution. The normal distribution occurs when a large number of independent factors influence the trait, as we might expect for multi-factorial traits, which Night Owl Education and Equestrian are influenced by a large number of genes and environmental factors.

All normal distributions can be described by two statistics called the mean and the standard deviation. The mean is often called the average and describes where the centre of the distribution of the phenotypes occurs along the continuous range of possibilities. Half of the individuals will have a phenotype at or less than the mean, half a phenotype at or greater than the mean. The mean phenotype is easily calculated by adding up all the individual measurements (∑xi) and dividing this by the number of measurements (n). This provides a convenient way, for example, to compare the phenotypes of parents and their offspring.

The standard deviation is a measure of the extent of variability (or spread) about the mean. Two distributions may have identical means but different standard deviations. The distributions with the larger standard deviations will have more individuals with phenotypes more different from the mean than the distributions with smaller standard deviations.

Figure 1 shows several distributions of horse height, as measured in hands. Although in each population shown the horses have the same mean height the standard deviations range from a hand (4 inches or about 10cm) to just one inch (about 2.5cm). The Night Owl Education and Equestrian distribution with the small standard deviation is tall and “peaky” showing that most horses in the population are close to the mean height. That with the largest standard deviation is wide and short showing that the population contains horses of a wide range of heights.

Figure 2 shows a normal distribution and the percentages of individuals within 1 and 2 standard deviations of the mean. For about two-thirds of a population with a normally distributed trait that trait lies within one standard deviation of the mean. So if the mean height is 14.5 hands and the standard deviation is 2 inches then 2/3 of horses are between 14 hands and 15 hands. Ninety-five percent of horses are within 2 standard deviations of the mean. So in the example 95% of horses would be between 13.5 hands and 15.5 hands (2 standard deviations is 2x2 inches = one hand).

Selection tends to move a mean to that being selected for (the desirable phenotype). It narrows the variation so that individuals too far away from the desirable phenotype are selected against (that is to say not used for breeding). Night Owl Education and Equestrian

Remember ● continuous traits are usually described using a frequency distribution of the phenotypes ● normal distributions occur when a large number of independent factors influence a trait, including genes and environmental factors ● normal distributions are described by a mean and standard deviation ● the mean describes where the centre of the distribution is ● the standard deviation measures the variability about the mean ● 67% of measurements occur within a standard deviation of the mean ● 95% of measurements occur within 2 standard deviations of the mean ● selection narrows the variation around the mean desirable phenotype

Continuous Traits are Polygenic

Continuous traits are encoded by many genes and are therefore polygenic. The greater the number of genes involved in influencing a trait the greater the number of possible genotypes that are possible. We know that for a single locus trait there are three genotypes. For a trait controlled by two genes each with two alleles there are nine (i.e. 32) possible genotypes, as follows.

If locus one has alleles A and a and locus two has alleles E and e then gametes may have haplotypes AE, Ae, aE and ae. From a Punnett square we see that there are 9 genotypes. We can also see that, because one allele of each locus is completely dominant, there are 4 phenotypes in this case. Night Owl Education and Equestrian

gametes Male

Female ↓ AE Ae aE ae

AE AAEE1 AAEe2 AaEE3 AaEe4 Ae AAEe2 AAee5 AaEe4 Aaee6 aE AaEE3 AaEe4 aaEE7 aaEe8

aaee9 ae AaEe4 Aaee6 aaEe8

In general if there are some number of loci n controlling a trait, each with 2 alleles, then there will be 3n possible genotypes. You can imagine this for three loci by adding an additional locus, say C, to the example above. Every genotype given in the Punnett square above will be possible in combination with each of three possible genotypes at the C locus (say C+C+, C+Ccr, CcrCcr). There will therefore be 9x3=33=27 genotypes. Because alleles at the C locus are co-dominant there will be 4x3=12 possible phenotypes in this case. If there are more alleles per locus then there will be more genotypes. Even with just a few di- allelic genes the number of genotypes rapidly becomes large (34=81, 35=243, 36=729).

For polygenic traits there are many genotypes specifying many (but usually fewer) phenotypes, each different from the others to some degree that may range from very slight to quite different. Because the difference between similar phenotypes is slight there is a continuous spectrum of phenotypes between extremes.

Mature horses, for example, generally range in height from a diminutive 9 hands to a gigantic 18 hands, with there being a continuous range of horses of in-between height. However the complete range is even more astonishing. A miniature brown mare known as Thumbelina is probably the World's smallest living horse at a mere 17 inches (4 hands 1 inch or 43cm)! A Belgian draught horse called Radar was the tallest, at just ½ inch under 20 hands high (202cm). However a five-year-old shire horse in Australia measures a colossal 20.1 hands (205.7cm) tall. At over 1.3 tonnes (1300kg) he weighs about the same Night Owl Education and Equestrian as a small car, and is still growing!

We could classify this variation, for example by grouping horses using one hand intervals. This though is an artificial grouping since horses of say, 14 hh, might be grouped with horses of 14.3 hh, even though they aren’t of the same phenotype for the trait of height. If we reduced the interval size of groupings, say down to within an inch, there would still be differences between horses within a group, but on a finer scale.

Polygenic traits are usually influenced by the environment, so that a particular genotype may give rise to a range of phenotypes according to the environment. Height is influenced by the nutrition a horse receives during its development, temperament and ability depend on, among other things, the training a horse has received. Most continuous traits are multifactorial, meaning that both multiple genotypes and environmental factors influence the phenotype.

The same principles of inheritance and gene function that apply to genes for discontinuous traits also apply to those for continuous traits. However, understanding the relationship between the genotypes and phenotypes of continuous traits is far from simple and is usually tackled using specially developed statistical procedures.

Remember: ● continuous traits are polygenic ● if there are n loci controlling a trait, each with 2 alleles, then there will be 3n possible genotypes ● even with just a few di-allelic genes the number of genotypes rapidly becomes large ● most continuous traits are multifactorial, so that both multiple genotypes and environmental factors influence the phenotype ● the same principles of inheritance and gene function apply to continuous traits as do those for discontinuous traits Night Owl Education and Equestrian

How can quantitative genetics be useful to horse-breeders?

Recently there has been some increased interest in quantitative genetics as the tools of molecular genetics are applied to address previously difficult or practically impossible lines of research.

Associations are made between particular extremes of a character (such as very short and very tall) and particular alleles of molecular genetic markers. The markers can be generated to cover all the linkage groups of the genome, so that particular areas of the genome can be associated with particular characters (which are said to be in the control of quantitative trait loci, or QTLs). Using computers and mathematical techniques it is worked out which marker alleles are linked to which particular alternative characteristics of the phenotype. The markers are like little flags that say “the character you‘re interested in lives somewhere in this area of this chromosome”. From there the discovery, isolation and characterisation of the genes involved is made easier.

In livestock QTL maps are made and used to guide the development for specific traits, such as high milk production: animals are tested for markers correlated with high milk production, and those with lots of “high-milk yield markers” are bred together.

Such scientific breeding techniques aren’t yet used in horses. However a recent development means there is now a marker screening test for Cerebellar Abiotrophy (CA), which is a recessively inherited neurological condition found in Arabian horses. Although the specific mutation that causes CA is not yet known, it is known to be near a group of particular markers which are usually inherited with CA. These are used as a diagnostic tool to identify affected foals and potential carriers of the disease. Arab breeders can test their horses before breeding in order to avoid breeding two suspected carriers together. CA may not be polygenic, but it does illustrate that so called marker assisted selection (MAS) is now occurring in horses, so that the day when polygenic traits are similarly selected seems a little closer than it did. A wide range of markers are now available that specifically flag up particular parts of the horse chromosomes (there is a Night Owl Education and Equestrian

“map” of markers). The horse genome has been sequenced and this is driving research to new levels and at a pace previously impossible. Scientifically based selection procedures for quantitative traits in horses seems an ever more likely future development.

Although molecular genetic breeding of horses would be an exciting development classical quantitative genetics can and is currently being used to address various issues of horse breeding. For example we might want to know how much continuous traits are due to genotype and environment. If there is a significant genetic component then we should expect to be able to select for particular phenotypes. Quantitative genetics is the branch of science concerned with addressing the “nature versus nurture” issue.

The genetic component of a phenotypic trait is called heritability. Heritability varies between zero and one, with one indicating high heritability. It represents the proportion of variation for a character that is due to genetic influences rather than environmental ones. If you don’t think well in proportions then multiply by a hundred and think of it as the percentage of variation due to genetics. For any particular trait the heritability may vary between populations, including between breeds. When the heritability is high there’s genetic variation and low environmental influence on the trait. If there is genetic variation we can change a trait by selecting for particular phenotypes.

When heritability is low there’s little genetic variation. This may mean that only one allele exists for some, several or most of the genes involved with the trait. When there is only one allele for a gene that allele is said to be fixed in the population. Alleles are usually fixed if they are more strongly selected for than other alleles, although they can be fixed for other reasons, such as the founder effect (which is discussed in the lesson on evolution). When heritability is low the affects of the environment will be great, including training and nutrition: they will have more of an effect on the phenotype.

Most analyses of performance and temperament traits show that they have from low to moderate values of heritability. For your reference the table shows some published levels of heritability for different traits, along with the associated references. Night Owl Education and Equestrian

trait and reference estimated heritability pulling ability in Finnish draft horses 0.23-0.27 Hintz, R.L. 1980. Genetics of performance in the horse. Journal of Animal Science 51: 582-594.

traits averaged between studies for French, German and Swiss warm blood stallions, judged by log earnings (Hintz, 1980.):

jumping 0.18 3 day eventing 0.19 dressage 0.17 traits averaged between studies for Thoroughbreds, trotters and pacers:

log of earnings 0.49 earnings 0.09 handicap weight 0.49 best handicap weight 0.33 time 0.15 best time 0.23 log of earnings (trotters) 0.41 earnings (trotters) 0.20 time (trotters) 0.32 best time (trotters) 0.25 best time (pacers) 0.23 traits among German stallion warm bloods: Bruns et al, 1985. cited in Klemetsdal, G. 1990. Breeding for performance traits in horses - a review. In Proceedings of the 4th Congress of Genetics Applied to Livestock Production 16, 184-193.

riding ability 0.36 gaits 0.50 jumping ability 0.72 cross country 0.33 racing time 0.53 character/temperament 0.25 Night Owl Education and Equestrian

cutting ability among elite quarter horse sires 0.19

Ellersieck, M.R., Lock, W.E., Vogt, D.W. and Aipperspach, R. 1985. Genetic evaluation of cutting scores in horses. Equine Veterinary Science 5: 287-289.

White facial markings in Arabian horses 0.69

Woolf, C.M. 1989. Multifactorial inheritance of white facial markings in the Arabian Horse. Journal of Heredity 80, 173-178.

White leg markings in Arabian horses 0.68

Woolf, C.M. 1990.Multifactorial inheritance of common white markings in the Arabian Horse. Journal of Heredity 81, 250-256.

Appaloosa markings Unknown, http://www.appaloosaproject.info/index.php?&MMN_position=6:6 2008. high?

To look at some of the issues around quantitative traits it is helpful to consider some examples, which we’ll now do.

Remember ● by associating quantitative trait loci (QTLs) with those of already mapped marker genes the whereabouts of QTLs can be determined ● by associating particular marker alleles with QTLs for a particular phenotype breeding stock can be selected with high number of genes for that phenotype ● classical quantitative genetics can be used for horse breeding ● now that the horse genome has been sequenced and mapped molecular breeding might one day be used Night Owl Education and Equestrian

Running Speed in Race Horses

One example of a quantitative trait is that of running speed. Race horse breeders would like there to be a single gene that confers superior racing performance. If such a gene ever existed then our ancestors have undoubtedly already selected race horses that have “that gene”. More likely there are many genes affecting racing performance. They will include those that enhance muscle performance, respiration, heart function, conformation and musculoskeletal integrity, as well as the mental ability to race. Genetic studies of humans have lead to the discovery of over 100 genes affecting performance, and these same genes are now being studied in horses.

Thoroughbred horses were developed over 350 years ago from a group of about 100 founder horses. Since then selective breeding has produced the modern race horse. Horses of all sizes and types win races. Secretariat was a large horse of 16.2 hands while Northern Dancer, another noteworthy race champion of the 20th Century, was only 15.2 hands tall. Some horses, like Man O War, had long stride lengths (reported to be about 28 feet) while others, like the recent Kentucky Derby winner, Smarty Jones, have a much shorter stride. Not all horses win races in the same way, each has some superior qualities, but not necessarily all the same ones as the others. Clearly there are many aspects of the phenotype that contribute to racing quality.

Despite 350 years of selection recent studies indicate that perhaps about 30% of racing performance is determined by genetics. The rest is down to management and training (and probably opportunity too). Thirty percent still represents a considerable and significant chunk of variation and indicates that we can use genetics to breed better race horses.

The Thoroughbred race-horse Secretariat is probably one of the most well known among horses. In 1973 the 3 year old colt won the American triple crown. Many people consider him to be the best Thoroughbred race horse of all time. Some have suggested cloning such outstanding horses. Night Owl Education and Equestrian

A clone of Secretariat may or may not turn out to be as outstanding as Secretariat himself. Although Secretariat was probably a near perfect a genetic example of his type we’ve seen that only about 30% of winning races is down to genetics. Not only would the clone not be raised in exactly the same environment, or by the same trainers, but aspects of his development would also be different, both before and after birth. Things such as the number of times a cell divides or how far it migrates in a developing embryo are not under strict genetic control. It may be that clones of Secretariat would not develop to be exact replicas of him. Even the conformation, brain development and physiological development may be different in the clones. Such small differences which could be significant to the chances of the clone be a superior winning race horse. A clone wouldn’t be the same horse!

Remember ● running speed is a multifactorial trait ● there are many genes affecting racing performance ● genetic variation accounts for about 30% of racing performance ● cloning great winning horses is not a guaranteed way of getting more horses of the same greatness ● most, probably all, performance traits are similarly multifactorial ● the genetic component of a phenotypic trait is called heritability

Personality traits, and their possible connections with colour and pattern

To illustrate this we consider whether it may be possible to breed for personality traits, such as relative calmness and trainability. There is no doubt that personality traits are at least partly inherited. However the inheritance of behaviour is very complex. There are Night Owl Education and Equestrian probably lots of genes involved - with the brain being the most complex organ of the body, and this working in conjunction with the senses and nervous system. The expression of particular characters also depends a lot on the environment. In domesticated horses the nature and expertise of the handlers and riders plays a big part in the nature of a horse, including its manners and the way it controls its natural fear responses. You often see horses that can’t be properly controlled by one person, but then go on to do good things for another owner or trainer. In addition the personality of a mare will influence a foal more than that of its sire, because the mare makes up a bigger part of the foals environment. Personality traits are clearly multi-factorial.

Horses are naturally nervous animals. They have evolved this way to ensure their survival in the wild. Flight is the chief response to danger: horses try to escape from dangerous situations by running away. In evolutionary terms those horses that successfully escaped were the ones that survived to breed. They passed their survival abilities on to their offspring. Horses tend to only fight if they think they have no other choice. Occasionally stallions will fight over mares, but usually the weaker stallion will give in before fatal damage is done.

The flight response of horses is under biochemical control, and therefore there is likely to be genetic variation for it. It is fairly likely though that horses have little or no variation at some of the genes that control the flight response. Alleles that make horses more prone to run away would‘ve been strongly selected for, because natural selection favours successful runners. This may mean that only one allele exists for some of those genes, in which case those alleles are fixed in the population. It may be that some genes are di- allelic, but that both alleles have the same, or almost the same, affect on the phenotype. Against this there may be some selection in favour of horses that don’t run away unnecessarily, which is a waste of precious energy, especially when nutritious food is more difficult to come by. This alternative selection pressure probably provides some genetic variation in favour of quieter horses.

Humans have bred horses for a long time, applying their own selection pressures according to what they require from a horse. It is for this reason that there is variation in temperament between breeds, and probably between colours too, especially in “hotness” Night Owl Education and Equestrian or nervousness. Some breeders do specifically choose breeding stock according to the calm nature of their forbearers, and this is a great idea. Breeds in which the stallions are commonly used for riding, such as Quarter Horses, tend to be calmer because they have been selected to be that way: even the stallions have to be relatively docile! In some breeds, the stallions are shown in-hand and little else is done with them. Some very beautiful stallions with good show records may nevertheless have nervous temperaments. I’ve seen pony and cob stallions at local shows sometimes act appallingly badly - but because they are show winners some mare owners will choose them anyway, perhaps unaware of their temperament. They may pass aspects of their temperaments on to offspring that consequently often don‘t make good children’s or ridden ponies.

Animal domestication, including horse domestication, has inevitably been associated with selecting for docility and tameness. It has been shown in foxes that selection for tameness leads to foxes with areas of de-pigmentation (Belyaev, 1979, Trut, 1999). In other animals too piebaldness (coloured and white, as in paints) is thought to be associated with docility, including in cats, dogs, hamsters, rats, cows, birds and horses.

The connection between colour and temperament in animals is interesting, and illustrates how genetics and selection can affect behaviour. Some of the evidence seems to point to “coloured” horses being, on average, more docile than others. Various genes cause different variants of piebaldness. Most forms in horses are caused by pigment cells (melanocytes) not being found in the white areas. The overo gene, for example, is involved with both the migration of pigment and nerve cells during development (Metalinos et al, 1998). Pigment cells also migrate to the brain. They occur in various (and possibly all) parts of the brain, and in the membranes surrounding the brain (the leptomeninges). This includes, for example, the substantia nigra, a part of the midbrain that regulates mood and produces dopamine (which is also produced in pigment metabolism). Mutations that prevent melanocytes from reaching the brain are known in various animals and can have a range of affects on behaviour, including stress response.

Variation associated with pigmentation can also cause a range of affects in non-piebald animals, and be selected for by breeding for colour and pattern. For example, hooded rats homozygous for a red-eyed dilution gene have numerous associated disorders including Night Owl Education and Equestrian hypertension leading to kidney damage and altered behaviours, such as high anxiety, low aggression and a predisposition to alcoholism! Because the red-eyed dilution gene affects different cell components in different types of cells there are several (i.e. pleiotropic) effects of the mutation. This is rather a dramatic example of how selecting for colour and pattern can cause selection for particular behavioural traits at the same time. I believe that some colours and patterns in horses may well be associated with behavioural characteristics, though perhaps not usually in such an extreme way as in this example. There might even be some truth in the old tale about red horses being more fiery than others!

The extension locus is responsible for the switch from black to red coat pigment production. It encodes a melanocortin receptor (MC1R). In other mammals, including rats and humans, the agouti signalling protein also binds to the melanocortin receptors in the brain (Lu et al, 1994, Willard et al, 1995), which act as potent neuromodulators, with various affects on behaviour and physiology. It has long been realised that non agouti rats are calmer and tamer than agouti rats (Keeler, 1942, Cottle and Price, 1987). More recently it has been realised that this is probably to do with the effect of the agouti signalling protein on neural melanocortin receptors, and the consequent effects on the brain. This is another example of how colour is associated with behaviour: selection for docile laboratory rats has led to over 80% of them being non agouti.

There may be an association between colour and docility in horses. Perhaps black horses – which don’t produce agouti signalling protein - are more docile than bays. Maybe there’s a gradient with redder horses being friskier than darker ones. There are, however, bound to be complicating factors so that a simple relationship might be hard to detect. In lab rats there are no mutant forms of the extension locus known. However in horses the chestnut mutation occurs and perhaps this also has an effect on the biochemistry of brain, and ultimately on behaviour. Interestingly the chestnut mutation occurs in the same gene that causes red hair in humans. It is well known that both chestnuts and “red heads” are associated with “old wives tales” about them being more fiery!

Overall there’s very good evidence for genetic variation for behavioural characters in horses. This means it’s possible to breed more docile horses. It is well known that some Night Owl Education and Equestrian breeds are more fiery, and the evidence suggests that probably some colours are too. Nevertheless the skill of a horses handlers and riders may hugely influence the final temperament of any particular horse.

Remember ● personality traits are multifactorial ● there may be little or no variation at some of the genes controlling certain types of behaviour that are essential for survival ● selection for temperament has affected breed development, with some breeds being, in general, more docile than others ● horse domestication has been associated with selecting for docility and tameness ● there is some evidence that pattern genes may also affect temperament, with white patterned horses (paints, piebalds etc.) possibly being more docile on average ● the extension and agouti colour genes also affect brain function, so there might some relationship between temperament and colour, though this is unlikely to be a simple one ● the environment - especially training and handling - affect temperament to a large degree

Summary

Traits that exhibit distinct phenotypes are called discontinuous traits. Complex traits display a continuous spectrum of phenotypes within a certain range that can be measured in quantitative terms. They are usually described using a frequency distribution of the phenotypes. A common distribution in genetics is normal distribution, which occurs when a large number of independent factors influence a trait, including genes and environmental factors. Normal distributions are described Night Owl Education and Equestrian by a mean, indicating the centre of the distribution, and a standard deviation, which measures the variability about the mean. Selection narrows the variation around the mean phenotype.

By associating quantitative trait loci (QTLs) with those of already mapped marker genes the whereabouts of QTLs can be determined. These marker alleles can then be used to breed animals for a particular phenotype. Now that the horse genome has been sequenced and mapped molecular breeding might one day be used.

Running speed, probably like all other performance and personality traits, is multifactorial. The genetic component is called heritability. Although there are many genes affecting such traits heritability levels are modest, with the environment - especially training and handling - affecting these traits to a large degree. For this reason cloning great winning horses is not a guaranteed way of getting more horses of the same greatness.

There may be little or no variation at some of the genes controlling certain types of behaviour that are essential for survival (such as running from danger). Nevertheless selection for temperament has affected breed development, with some breeds being, in general, more docile than others. There is some evidence that pattern and colour genes may also affect brain function, so that horses of some patterns and colours may be more docile, on average, though the relationship is unlikely to be a simple one. Night Owl Education and Equestrian

References

Belyaev DK, 1979. The Wilhelmine E. Key 1978 invitational lecture. Destabilizing selection as a factor in domestication. Journal of Heredity 70:301–308. Cottle, CA and Price, EO. 1987. Effects of the nonagouti pelage-color allele on the behavior of captive wild Norway rats (Rattus norvegicus). Journal of Comparitive Pyschology 101(4):390-4. Jung, Gi-Dong, Yang, Jeong-Yeh, Song, Eun-Sup and Park, Jin-Woo. 2001. Stimulation of melanogenesis by glycyrrhizin in B16 melanoma cells. Experimental and Molecular Medicine 33 (3), 131-135. Keeler, C.E. 1942. The association of the black (non-agouti) gene with behavior. The Journal of Heredity 33(11):371-384 Lu, D., Willard, D., Patel, IR, Kadwell, S., Overton, L., Kost, T., Luther, M., Chen, W., Woychik, R.P., Wilkison, W.O. and Cone, R.D., 1994. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature 371, 799 - 802. Metalinos, D.L., Bowling, A.T. and Rine, J. 1998. A misense mutation in the endotheline-B receptor gene is associated with Lethal White Foal syndrome: an equine version of Hirshsprung Disease. Mammalian Genetics 9, 426-431. Rieder, Stefan Taourit, Sead Mariat, Denis, Langlois, Bertrand and Guérin, Gérard. 2001. Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus). Mammalian Genome 12 (6), 450 – 455. Trut LN, 1999. Early canid domestication: the farm fox experiment. American Scientist 87:160–169. Willard DH, Bodnar W, Harris C, Kiefer L, Nichols JS, Blanchard S, Hoffman C, Moyer M, Burkhart W, Weiel J, Luther, M.A., Wilkinson, W.O. and Rocque, W.J. 1995. Agouti structure and function: characterization of a potent alpha-melanocyte stimulating hormone receptor antagonist. Biochemistry 34(38):12341-6. Zalfa, A. Abdel-Malek, M. Cathy Scott, Minao Furumura, M. Lynn Lamoreux, Michael Ollmann, Greg S. Barsh and Vincent J. Hearing. 2001. The melanocortin 1 receptor is the principal mediator of the effects of agouti signalling protein on mammalian melanocytes. Journal of Cell Science 114, 1019-1024. Where do rat colors come from? http://www.ratbehaviour.org/CoatColorMutations.htm. Night Owl Education and Equestrian

Module two assignment

Q1. What would be the ratio of the different phenotypes expected from a di-hybrid cross between a mare and a stallion who are both palomino and overo? Illustrate your answer using a Punnett square. What’s the chance of a live overo foal? What’s the chance of a live non-overo foal? Imagine you had a friend who wanted to make such a cross described. What advice would you give her?

Q2. Give short answers to the following questions (a sentence or two should be sufficient). A. In the text I mention a lethal gene that occasionally causes death in Arabian foals. The foals are born a dilute “lavender” colour, have neurological problems and fail to stand and nurse. Do you think the gene responsible is dominant lethal or recessive lethal? Briefly give the reason for your answer. B. How might a dominant lethal gene occur? Do you think it could ever be passed on to the next generation? If so briefly explain how? (tip: this is not a trivial question, you need to think about when (I.e. at what age) such a gene might be expressed. It might help you to think about human conditions if you’re stuck.). C. What is the name given to the phenomenon where a single gene has more than one affect on the phenotype, as does the lavender gene in Arab horses and lethal white overo gene in coloured horses? (A single word is enough). D. Appaloosa spotting (discussed in detail later in the course) is controlled by several genes. One of the genes causes horses to have at least minimal phenotype characteristics. Depending on the genotype at other genes, and possibly some environmental factors, between one and three of these characters may show (mottled skin pigment on the face and/or genitals, hooves striped with narrow vertical bands and white sclera of the eye). Some horses may have one characteristic, others will have two or all three. What is the term that describes when the expression of the characteristics like this may or may not occur, even though the genotype for them is present? E. Appaloosas can show various other traits, such as blankets and various types of spotting, where the extent of the trait varies according to background genotype and environmental factors. What is the term used to describe this kind of gene expression? Night Owl Education and Equestrian

Q3. What would be the phenotype ratios expected from di-hybrid crosses between a tovero stallion and tovero mares? (Tovero is the combination of tobiano and overo). Classify the phenotypes into tovero, tobiano, overo, solid (i.e. with no pattern) and lethal white overo (LWO, which can be with or without tobiano, but all foals will have the LWO phenotype so that you couldn’t distinguish between the two by just looking). What proportion are expected to have the LWO phenotype? You can see from this that one has to be careful when crossing coloured horses together, breeding together overo horses is definitely not to be advised.

Q4. All foals homozygous for the overo allele will have the LWO phenotype whether or not they also have a tobiano allele, so that you couldn’t distinguish between the two genotypes by just looking. What kind of relationship is this between overo and tobiano?

Q5. Research by Sponenberg et al (1984) observed 57 foals of a bay roan Brabant Belgian stallion, out of eight chestnut American Belgian mares. Thirty foals were bay roan, 25 were chestnut, one was bay, and one was chestnut roan.

A. Chestnut is due to a homozygous recessive allele at the extension locus (of genotype ee), bays can be homozygous or heterozygous for the dominant extension allele E. What genotype was the stallion at the extension locus? Briefly state how you know that (one sentence should be sufficient). B. Roan is caused by a dominant allele. Was the stallion homozygous or heterozygous at the roan locus? Briefly state how you know that (one sentence should be sufficient). C. If the roan alleles are designated Rn (dominant, causing roan) and rn (non-roan) what are the genotypes of the mares? Write down their genotype at both genes. D. If the two genes were independently segregating what theoretical ratios of phenotypes would you expect among the foals? Do you think the foals occur in approximately the expected ratios? Night Owl Education and Equestrian

E. If the two genes were completely linked together what theoretical ratios of phenotypes would you expect among the foals? (Hint: you need to look at the data to decide if E is more likely to be linked to Rn or rn, consider the dams first). F. Briefly state what conclusion you reach about the linkage of the alleles at the roan and extension loci. Are they linked? If they are linked are they completely or closely linked, or probably only distantly linked? If they are linked which of the roan alleles (dominant or recessive) is linked to the dominant extension allele?

Q6. A. If we use the symbols XH and Xh to denote the dominant normal and recessive haemophilia alleles of the gene that causes haemophilia A, and Y to denote Y chromosomes what genotype would haemophiliac colt foals be? What’s the special name for this kind of genotype? B. Who would a colt foal inherit a recessive allele for haemophilia A from and what would their genotype be? What would you suggest happens to this parent? C. Explain why haemophilia A only occurs in colt foals and not in fillies. (Please be brief and to the point).

Q7. Which of the following is not an example of a polygenic trait? A. running speed in race-horses B. Lethal white overo C. temperament D. jumping ability E. shading within a colour, such as from dark to light chestnut or palomino Night Owl Education and Equestrian

Q8. Among 1000 horses running time for completing a particular course is normally distributed with a mean completion time of 1 minute and a standard deviation of 8 seconds.

A. How many horses complete the course with a time between 52 seconds and 1 minute 8 seconds? B. What is completion time above which the slowest 2.5% of horses run the course? C. What is completion time below which the fastest 2.5% of horses run the course?

Q9. How many possible genotypes are there for a trait controlled by 5 di-allelic genes?

Q10. For some breeds the heritability of some performance traits is low. Does this mean the traits are not under genetic control? Briefly say why, or why not?

Q11. A foal inherits 50% of his genes from the dam and 50% from the sire. Nevertheless a foals dam generally has more influence on his or her behavioural traits than the sire. Briefly explain why this is.

Q12. The heritability for dressage traits is low in many Warmblood breeds. Despite this many Warmbloods seem to be very well suited to dressage and excel in the discipline. Briefly say why you think this is. What influences the success of such Warmbloods in this case?

Reference Sponenberg, D.P., Harper, H.T. and Harper, A.L. 1984. Direct evidence for linkage of roan and extension loci in Belgian horses. The Journal of Heredity 75: 413-414.