AU2003200491A1 - Animal testing procedure - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): AgResearch Limited Invention Title: ANIMAL TESTING PROCEDURE The following statement is a full description of this invention, including the best method of performing it known to me/us: ANIMAL TESTING PROCEDURE TECHNICAL FIELD This invention relates to an animal testing procedure. Specifically, this invention relates to a method of determining the genetic value of an animal during animal breeding.

BACKGROUND ART Species that are under some form of artificial genetic selection provide the majority of the world's food and natural fibres, companion animals and racing animals.

While there are many methods and schemes to selectively breed and improve a species, these almost invariably require the objective measurement of specific traits related to the characteristic to be improved and accurate recordal of pedigrees or family relationships in order to distinguish genetic influences from environment.

In many species the need to record pedigree places serious restrictions on the management of breeding populations. For example, to be certain of pedigree in an extensively farmed species such as the sheep, ewes must be single sire mated and at lambing the birth must be observed and the lamb uniquely tagged.

These requirements of pedigree recording constrain management options at mating and lambing, require highly skilled stock managers to gather accurate records, and limit the size of breeding flocks and conditions under which breed flocks to manageable levels.

Further, single sire matings risk low pregnancy rates if the ram is not active or is infertile, while such extensive recording often causes stress to ewe flocks during lambing which may increase miss-mothering and lamb loss.

Only under extreme conditions, such as single pen lambing, can parentage be \PERTHO1 \home$ yasminp kccp %Speci\P48715 -Animal Testing Procedure.doc determined completely error-free.

Similar difficulties are experienced in virtually any breeding programme where animals or plants are in an extensive fannrming situation for at least part of their lifecycle.

One obvious alternative to such traditional recording of pedigree is to use DNA marker profiles to identify parents. It is well known that DNA can be used to determine paternity in humans.

Use of DNA markers to generate pedigrees on a large scale for animal breeding would enable management constraints of visual recording and confinement of animals to be relaxed.

DNA testing has been widely used in the breeding of high value animals such as horses to monitor the accuracy of pedigree records. Such parentage matching is typically very reliable and effective when there is a mother and progeny with two or more potential sires.

However, while DNA profiling has been available for nearly 20 years there are few examples of the widespread use of DNA generated pedigrees in breeding strategies.

One of the primary reasons for this is the cost and practicality of accurate DNA matching in large populations. If recording or management is relaxed at mating and parturition in an extensive breeding situation, then potentially there can be a very large number of parents for an offspring. For example, in sheep where ten sires may be mated with a thousand ewes, a lamb could have one of 10,000 possible combinations of parents.

An additional problem when trying to match a progeny to a unique pair of parents arises when allowing for genotyping errors. A common way to overcome this is to require more than one exclusion among the marker tests. Although this works \PERTHQ1 \home\yaminp \keep Speci\P48715 Animal Testing Procedure.doc 4 reasonably well in situations where many markers are typed, it is somewhat arbitrary, and is less useful for lower numbers of markers being scored.

The exclusion method tries to exclude all possibilities but the correct one, by excluding relationships with incompatible genotypes. A common way to cope with genotyping errors is to allow a discrepancy at one marker. This means that there must be at least two discrepant markers to exclude possible parents (as it is unlikely that two of the genotypes in the comparison will be in error). When a small set of markers is used, it is difficult to exclude all incorrect relationships, and if there need to be two out of a small number of markers showing an inconsistency this will exacerbate the problem.

As progeny produced in extensive breeding systems potentially have a very large number of parents, genetic evaluation of such animals are thus complicated by the uncertainty of the parentage. A number of statistical methods have been developed to attempt to assign pedigree to such offspring.

Such methods typically only aim to identify the sire. As multiple sires are often found to be genetically compatible with each offspring, the use of genetic markers may be used to identify the most likely male with a certain level of likelihood.

These methods are useful in tracing pedigrees through natural populations where there is no record or information on matings. This yields information on population parameters, such as the distribution of mating success, but the methods have not been widely used in extensive breeding situations due to the large amount of genetic analysis required.

Whilst other methods have been proposed for use in breeding situations, they often seek to account for multiple possibilities of sire pedigrees during the genetic evaluation process. The use of genetic markers has not been exemplified in such techniques, but rather is mentioned only as a possibility in assigning probabilities to possible parentages.

PERTH101\home$\yasminp Nkeep\Speci P48715 -Animal Testing Procedure.doc All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or'comprising' is used in relation to one or more steps in a method or process.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided a method of determining a breeding value for progeny, including the steps of identifying possible parents of progeny, and \PERTHOI \omrne$ \yasminp \keep \SpeciP48715 Animal Testing Proeedure.doc 6 (ii) genotyping of possible parents and progeny, and (iii) calculating parentage likelihoods and converting them to statistical probabilities, and (iv) estimating the breeding value of the progeny the method characterised by the step of using a selected number of genetic markers in the genotyping.

In preferred embodiments of the present invention this method is used to determine the parentage of progeny produced in large scale animal breeding.

In preferred embodiments of the present invention the procedure is used to determine the parentage in sheep.

However, this should not be seen as a limitation on the present invention in any way for this procedure may find use in any breeding programme where animals or plants are in an extensive farming situation for at least part of their lifecycle, such as other livestock (cattle, camels, camelids, goats), farmed fish (salmonids), poultry, and sexually reproducing plants.

The term "breeding value" should be taken to mean a measure of the genetic merit of an individual according to the mean of its progeny (and adjusted for the contributions of the other parent(s), for the trait or combination of traits of interest.

Identification of possible parents preferably involves the accurate recording of the mating mob (groups of sires and mothers which have the opportunity to mate) and the parturition mob (groups of mothers in defined areas and the animals which are potentially their offspring). Such recordal is standard practice in conventional pedigree recording.

\PERTH01\ honme\yasminp\keep\Spei\P48715 Animal Testing Prucedure.dc 7 Possible parents may also be identified by some form of objective measurement of specific traits or through other fann management practices.

Identifying possible parents allows animals obviously not the parents to be eliminated before beginning any statistical analysis, thus reducing the number of calculations required.

In addition, allowance can be made for unknown parents, such as when there are incomplete mob records. These will have higher relative likelihoods for cases where none of the other possibilities provides a good match.

The term "genotyping" should be taken to mean the analysis of the distribution of a number of polymorphic genetic markers. Preferably the markers are analysed simultaneously using standard molecular biology techniques known in the art, such as multiplex or parallel analysis systems.

Such markers will herein be referred to as DNA markers, though this should not be seen as limiting.

The term "selected markers" should be taken to mean a set of markers which together enable a cost-effective solution to parentage testing and genetic evaluation using the methods proposed here. Using current technology this would normally constitute four to ten microsatellite markers chosen to be highly polymorphic and able to be multiplexed analysed together), but the proposed technology will make -use of any marker information no matter how good or poor that information is.

Alternatively, a larger set (20-30) of markers with lower probability of pedigree exclusion (eg SNP markers). However this is not limiting. The key feature of the selected markers is that less markers are used than one would reasonably expect from simulation or know from experience to provide a perfect solution to parentage problem. Ideally between 50 and 90% of progeny would be matched to a single parent \PERTHO1 \home$\yasintnp\keep\ Speci\P4$715i Animal Tesing Froceduredoc 8 with high probability while the remainder would have multiple possible parents.

In the present invention the use of a selected number of DNA markers to generate DNA profiles does not enable the identification of the parents of all progeny. Instead, the partial parentage information provided by selected DNA marker information can be used to calculate the statistical probability for each possible parentage.

According to another aspect of the present invention there is provided software programmes to calculate the statistical probability for each possible parentage.

The identification of possible parents creates a database of likely parents for each offspring. Having a finite number of potential parents then allows the statistical probabilities of each being the true parents to be calculated.

The set of possibilities are more likely to contain relatives of the true parents than unrelated animals. In some cases incorrectly allowing these as possible parents may still be better than not assigning parents to a progeny.

The parentage probabilities are then preferably used to calculate breeding values for each progeny, taking into account available biological information such as mating and lambing dates, pregnancy scanning data, specific trait records and so forth to modify the likelihood for each possible parentage.

Incorporating biological factors into the calculations improves the accuracy of the procedure. For example, a ewe scanned pregnant with one lamb is unlikely to give birth to five lambs.

DNA testing has been widely used in the breeding of high value animals such as horses to monitor the accuracy of pedigree records. Typically, DNA parentage matching is very reliable and effective when matching a mother and progeny with two possible sires. However, in a less controlled animal breeding situation there may potentially be a very large number of possible parents for an offspring and as such the costs of \\PERTH 01 \houme$\yasniap\keep\Speci\P48715 -Animal Testing Procedure.dc genotyping pedigree records is prohibitive.

The present invention utilises a set of DNA markers that do not identify the pedigree of all offspring. In comparison, the number of DNA markers used will only offer partial solutions to the pedigree of the offspring. While initially counter-intuitive, this offers a cost effective option to determining pedigrees in large-scale animal breeding using current DNA marker technology.

The optimal number of markers for a given situation will vary with the number of animals involved and the accuracy with which breeding value are required. We have used simulation to determine the optimal number of markers for situations commonly encountered in sheep farming. For example, for a particular situation ("single sire/mixed lambing", lambing mob of 250 lambs of 100 dams) the proportion of progeny uniquely assigned (assuming no allowance for genotype errors) was up to 80% and 90% for 4, 5 and 6 markers respectively.

Unexpectedly, the partial parentage solution provides similar genetic progress to that achieved using traditional pedigree recording, but with greatly relaxed management constraints and half or less than half the number of DNA markers required for a near complete pedigree solution. As such, the process makes genetic progress using DNA pedigrees feasible and cost effective even in large-scale farmed species such as sheep.

Automation and refinement of the genotyping process is anticipated to further reduce the costs of genotype analysis and broaden the applicability of this technology.

Further, the present invention also unexpectedly provides additional benefits that arise from the technology, including robustness to error rates in genotyping data, in addition to options for incorporating marked QTL in the breeding DNA profile and breeding analysis.

By combining information on the possible parents (such as mob records) and the \%PERThO 1~bome$\yasminp~keep\Speeflp4s71s Animal Testing Proceduredoc limited amount of genotypic information obtained by the partial pedigree solution with biological information collected during standard farm management such as mating and lambing dates, pregnancy scanning data, specific trait records and so forth then the estimated breeding value for each offspring can be calculated.

This ability to use DNA to generate pedigrees on a large scale for extensive breeding situations enables management constraints of visual recording and confinement of animals to be greatly relaxed. This allows more efficient management of animals at mating and lambing, thus lowering costs and stress on animals which could result in increased survival of young; the size of flocks to be increased more easily (less capital and staff training required); and breeding flocks to be run under similar conditions to production flocks, thereby increasing the relevance of selection.

It is expected that the present invention will provide a better solution, or at least a cost and labour effective alternative to traditional pedigree recording or to applications of DNA technology which aim to identify the exact parents of offspring in a breeding situation.

It is anticipated that there are a number of points in this procedure which can be modified or extended depending on breeding situation in question. Use of different data collected in standard genetic evaluation and pedigree recordal, and the manner these variables are analysed, could be used.

It is anticipated such modifications to the current procedure would be obvious to a skilled addressee and as such the present invention should not be seen as limiting.

DETAILED DESCRIPTION OF THE INVENTION As defined above, the present invention is directed to determining a breeding value of progeny in large scale breeding situations.

The invention is based upon the inventors investigations into the ability to use a \PERTHOI \home$ \yasminp \keep\Speci P 48715 Animal Testing Prucduredoc 11 selected number of DNA markers in conjunction with currently relaxed management restraints to achieve similar results to that achieved using traditional pedigree recordal or full DNA profiling.

A simulation model has been developed to demonstrate how partial pedigree information can be used for the calculation of breeding values. The simulation used parameters that are typical for a prolific sheep breeding operation, and using DNA markers typical of those that could be used cost-effectively for this situation. Breeding values were calculated using an inverse additive relationship matrix incorporating the parental uncertainty in the genetic evaluation.

Simulation Methods.

I Pediree simulation A pedigree is simulated comprising three generations. This allowed some genetic similarities between the parents, as would normally be the case in practice.

Generation I (grandparents); o assumed to be unrelated animals.

o paternal and maternal lines were produced.

Paternal line included 5 grand-sires and 20 grand-mothers.

Maternal line included 10 grand-sires and 120 grand-mothers.

Generation 11 (parents); o Parents were generated from the separate paternal/maternal grandparent lines.

\\PERTHO1 \homet\yasminp\keep\Speid\P48715 Animal Testing Procedure.doe 12 o The number of progeny produced from each mating was based on a distribution of 0.147, 0.460 and 0.393 for singles, twins and triplets respectively.

o Animals in each group were randomly assigned a sex.

Consequently 20-30 potential sires and in excess of 100 potential mothers were generated.

Generation III progeny o 10 animals were randomly selected for use as sires from the set of potential sires from generation II.

o 100 animals were randomly selected for use as mothers from the set of potential mothers from generation II. These animals were randomly assigned a mate from the set of sires.

o The number of progeny per mother was determined as in generation II.

o Progeny were randomly removed (to mimic deaths) according to birth rank.

The proportions removed were 0.10, 0.15 and 0.30 for singles, twins and triplets respectively.

o Birth rank and rearing rank data were stored for all progeny.

o All progeny were randomly assigned a sex.

Generation I and II animals not directly related to progeny were removed from analysis.

2 Genotypes Genotypes were applied to all animals according to pedigree.

\PERTHOI \home$ \yasminp\keep \Speci\P48715 Animal Testing Procedure.doc 13 o The marker data was simulated for six markers, based on a set of markers currently being used by the applicants. The allele frequency data for these markers was calculated from a set of unrelated animals.

o Genotypes were assigned to generation I animals by randomly selecting alleles based on the allele frequency data.

o Generation II and III animals' alleles were randomly selected from each parental set of alleles.

o There were two simulations where 5% and 10% of genotypes were removed at random, to conservatively model the proportion of results from a commercial cost-effective genotyping laboratory.

o All genotypes were assigned without error.

3 Parentage assignment Progeny assigned to parents using partial pedigree software designed by the applicants, as described below.

In this example all relevant animals belong to a single mating or lambing group.

o The likelihood of the data given a putative parentage is calculated relative to the likelihood of the data given no relationship.

o The likelihood is L is the product over all markers of L(H 1

)/L(H

2 with L(H1) T(g, I g o) P(g 0 e(-e) 2 [T(gf I g) P(go +T(g I go) P(go)+P(gp)] e2(1-e) [P(go) +P(gf)] e 3 L(H2) P(go) PERTHO 1 \home\yamminp\keep\SpeciP48715 Animal Testing Procedure.doc 14 e(l-e) 2 +P(gm)P(gf)] 02(1 +P(gf) +P(gm)J +e3 e is the assumed rate of genotyping errors and other quantities are found from the following tables according to the genotypes (AI represents the ith allele andpi its frequency).

Offspring Parents Genotype Genotypes T(gp P(gP) 2 4 A4,j A,4,x A4 p7 AP AA x A4 2 PI PI 4p j 2 ~22 A,Aj x A4k 2 Pj PI 8 Pk AjAAjx A4 p/ 4p/pj 22 A4~Aj Ak Pi fk 42 P 12 A,Ak x AA 1 fpk 8 Pi2PjPk A,4k xAjAk Pk 2 8PU21 Pk 2 AA&kX AAJ Pk P 8 pip PkCPA \PERT{O1\bome$\yasminp~kecp Speci\P487lfl Anial Tetting Proceduredoc Offspring Parent T(gf I go) P(gf) Genotype Genotype or or (gf or gr) (go) T(g. I g) P(gm) AAi AAj pi P 2 A.Aj p 2ppj AAj A Ap p12 P2 AAj (pi+pj)1/2 2 ppj A Ak p12 2ppk o A probability is assigned to putative parentage in proportion to its likelihood, from the set of parentages which are more likely than a randomly chosen set of parents.

o The rate of genotype errors was assumed to be 1% when perfonnrming the likelihood calculations.

4 Trait assignment Generation I animals were assigned genetic values for a trait with a known mean residual standard deviation and heritability Genetic values determined for generation II and III animals using known pedigree relationships and quantitative genetics theory. Phenotypes for generation III animals were produced from their genetic values, a component that depended on their birth and rearing rank, and a randomly sampled environmental component. Phenotypes were not modified according to sex. The birth/rearing rank effects used were 0, -7 for 1/1, 2/1, 2/2, 3/1, 3/2 and 3/3 respectively.

\PERTHl1 home t\yasminp\keep\SpeLi\P48715 Animal Testing Procedure.doc Breeding value (BV) estimation Partial pedigree results used in estimation of animal breeding values.

o BVs were estimated using ASREML (software that is able to produce estimated BV's by using a supplied inverse relationship matrix or by using a supplied pedigree) with known (generation III) phenotypic information.

The model included fixed effects of sex and birth/rearing rank combination.

o It was assumed that the litter size of each mother was known. In practice this may rely on pregnancy scanning information.

o Three different methods were used to estimate BVs: 1. True pedigrees: The true pedigrees and birth and rearing ranks (known from the simulation) were used.

2. Partial pedigrees: Pedigrees were assumed unknown and the relationship matrix was formed, using the partial pedigree probabilities, and inverted. Birth ranks and rearing ranks were derived as the weighted (by the probabilities) means of the assigned mothers' litter sizes and rearing group sizes. These were rounded to integer values, with a maximum of three, for the purposes of the genetic evaluation.

3. Best pedigree: Pedigrees were assumed unknown and the set of parents with the highest probability were used as the parents in the genetic evaluation. Birth ranks and rearing ranks were taken as the mother's litter size, and as the number of live progeny assigned to that mother respectively.

\PERTHO01 \homes \yasminp kCcp\Speei P48715 Animal Testing Procedure.duc 17 In other words, the best pedigree method uses the parents with the highest probability as if they are the true parents, i.e. only one set of parents is assigned for each probability. Partial pedigree allows a number of different parent possibilities.

o The true genetic values as produced in the trait assignment were also available for comparison.

o Correlations were found between breeding values estimated by each of the methods and between these and the true genetic values. Results were partitioned into generation II males and females and generation III animals.

These results were then summarised over multiple iterations of the simulation.

Results from simulation.

Simulation results are presented as the means (over replicates of the simulation) of the correlations between breeding values calculated using the true pedigree and by the other two methods (partial pedigrees or best pedigrees). Results are presented for three different groups of animals: sires, mothers and progeny. Each of these has substantially different amounts of information (number and type of close relatives with trait information) for estimating breeding values.

\PRTHOl1\hme$\yasminp\keep\Speci\P48715 Animal Testing Procedure.doc missing, V2% error rate: Mean Correlation Pedigree Group replicates correlation Standard Error partial Sires 20 0.944 0.010 partial Mothers 20 0.778 0.011 partial Progeny 20 0.944 0.005 best Sires 20 0.906 0.013 best Mothers 20 0.730 0.012 best Progeny 20 0.913 0.005 missing, 1% error rate: Mean Correlation Pedigree Group replicates correlation Standard Error partial Sires 50 0.905 0.014 partial Mothers 50 0.693 0.008 partial Progeny 50 0.934 0.003 best Sires 50 0.860 0.018 best Mothers 50 0.652 0.009 best Progeny 50 0.895 0.003 The accuracy of estimated breeding values is the correlation between them and the true breeding value (which are known within the simulations) Accuracies are shown for the three groups of animals and for known and partial pedigrees below: \\PERTHOI \home$ \yasminp\keep Sped\ P48715- Animal Testing Procedure.duc missing, error rate: Accuracy Pedigree Group Accuracy Standard Error true Sires 0.741 0.057 true Mothers 0.372 0.017 true Progeny 0.611 0.019 partial Sires 0.716 0.053 partial Mothers 0.315 0.019 partial Progeny 0.585 0.019 best Sires 0.702 0.052 best Mothers 0.287 0.019 best Progeny 0.567 0.016 missing, 1% error rate: Accuracy Pedigree Group Accuracy Standard Error true Sires 0.692 0.027 true Mothers 0.358 0.013 true Progeny 0.590 0.011 partial Sires 0.659 0.028 partial Mothers 0.268 0.015 partial Progeny 0.554 0.010 best Sires 0.639 0.031 best Mothers 0.243 0.014 best Progeny 0.527 0.009 \\PERTH01\ home$\ynsminp\kccp\ Speci\P48715 Animal Testing Procedure.doc Genetic progress is proportional to the square of the accuracy of the estimated breeding value. The ratio of the squared accuracies (pedigree unknown to pedigree known) gives the proportion of genetic progress that can be made with unknown pedigrees compared to that with known pedigrees.

5% missing, error rate: Relative genetic gain Group partial best Sires 93% Mothers 72% Progeny 92% 86% missing, 1% error rate: Relative genetic gain Group partial best Sires 91% Mothers 56% 46% Progeny 88% These results show that the "partial pedigree" method we have described for estimating breeding values allows much of the genetic progress that could have been made had the true parents been known.

While the process has been modeled and used for one trait, the same process can be applied to any trait which can be measured objectively in organism and has a hereditary component. Genetic progress is made by selectively breeding from animals identified as having higher or more desirable breeding value. In this way the next \PERTHO1 khonme$\yasminp\keep\ Spei\P48715 Animal Testing Proedure.dcc 21 generation will be on average improved for the trait in question. The more accurate the estimated breeding value the faster the genetic progress all other things being equal.

The progress is especially high for the groups of animals which are usually most intensively selected, i.e. sires and progeny. The results also show that a simplified method ('"best pedigrees") could also be useful in this context, but at the cost of lower genetic gain than with the partial pedigree method.

The results presented compare the gain against a "gold standard", i.e. perfect pedigree recording. Pedigree recording errors in practical farming systems mean that this "gold standard" is seldom, if ever, achieved. A number of reports have investigated the level of pedigree errors, and some of these are listed below.

There have also been a number of reports investigating the reduction in genetic gain due to errors in pedigree recording. For example, Israel and Weller (2000, J Dairy Science 83:181-187) predicted a loss of 4% gain with 10% incorrect sire identification when modeling selection in dairy cattle, while Banos et al. (2001, J Dairy Science 84:2523-2529) estimated a reduction of 11 to 15% in a similar situation, but incorporating international comparisons.

While the loss in genetic gains due to pedigree errors will depend on the precise nature of genetic selection, it would appear that the gains with the proposed marker-based methods relative to what can practically (due to pedigree errors) be achieved would be up to 10% higher than those tabulated above.

The genetic progress that could be made if there was no pedigree recording at all has not been simulated. This is expected to be 0% for sires and mothers, and <80% for progeny (would be 80% if fixed effects did not need to be estimated in the process).

\PERTHo1 \huwe$\yaminp\ keepSpeci\P48715 -Animal Testing Procedure.doc Type Estimated Reference pedigree errors Sheep, New Zealand 1-15% Crawford et at. (1993) Proc NZ Society of Animal Production 53: 363-366.

Sheep, New Zealand 10% Welch Kilgour (1971) Proc VZ Society ofAnimal Production 31:41.

Sheep, Australia 12% Alexander et at. (1983) Aust J Experimental Agriculture Animal Husbandry 23: 36 1-368.

Sheep, USA 14% Wang Foot (1990) Theriogenology 34: 1079-1085.

UK dairy cattle -sire 10% Visscher et at. (2002) J Dairy assignment Science 85:2368-2375.

Dairy cattle sire 11% Banos et at, (2001) J Dairy Science assignment. Average of 84:2523-2529.

surveyed reports.

Alternative methods There are several places where the proposed procedures could be modified or extended. In the simulations we have rounded birth ranks and rearing ranks for use in the genetic evaluation. A similar method could also be used for other effects used in genetic evaluation and depending on pedigree records, for example date of birth and age of mother. There are other ways that any of these could be used in the genetic evaluation. Rather than rounding, the estimated values could be used directly by fitting polynomials or splines to these values.

The best pedigree method described here has made no use of measures of confidence (eg ratio of probability of the best to the second best pair of parents) in the parentage chosen. It could be possible to improve this simple method by excluding cases where there is another possible parentage with a similar probability.

\FERTHO I1\bcmeS \yasminlp~ktep\Spevi\P48716 Animal Testing Proceduredne 23 Another embodiment of the present invention is described below. The first three steps of the parentage procedure are identical between Examples 1 and 2. The differences between the two examples involve the manner in which the breeding values are calculated.

Summary While it is anticipated other statistical approaches could also be used to calculate breeding values, the current protocol has the advantage that it is more useful and versatile for smaller herds of animals more common in many farming situations. With advances in computing it is anticipated that the inverse matrix approach could also be used for flocks larger than 3000 and it is anticipated that alternative systems would also likely make use of the inverse matrix approach of the present invention.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

\PERTIIOI01ome$ \yasmiinp \keeBp\Spii\P487 Animal Testing Piocedure.doc

Claims (19)

1. A method of determining a breeding value for progeny, including the steps of identifying possible parents of progeny, and (ii) genotyping of possible parents and progeny, and (iii) calculating parentage likelihoods and converting them to statistical probabilities, and (iv) estimating the breeding value of the progeny the method characterised by the step of using a selected number of genetic markers in the genotyping.

2. A method as claimed in claim 1, wherein there are four to ten highly polymorphic genetic markers selected.

3. A method as claimed in claim 1 wherein there are 20 to 30 SNP genetic markers selected.

4. A method as claimed in any one of claims 1 to 3 wherein the genetic markers are analysed simultaneously in a multiplex analysis system.

A method as claimed in any one of claims I to 3 wherein the genetic markers are analysed simultaneously in a parallel analysis system.

6. A method as claimed in any one of claims 1 to 5 wherein the selected genetic markers do not identify a single set of parents for the progeny.

7. A method as claimed in any one of claims 1 to 6 wherein the progeny is sheep.

8. A method as claimed in any one of claims 1 to 7 wherein the step of estimating breeding value incorporates biological information.

9. A method as claimed in any one of claims 1 to 8 wherein the biological information is progeny birth rank.

A method as claimed in any one of claims 1 to 8 wherein the biological information is progeny rearing rank.

11. A method as claimed in any one of claims I to 8 wherein the biological information is progeny birth date.

12. A method as claimed in any one of claims 1 to 8 wherein the biological information is age of mother.

13. A method as claimed in any one of claims 1 to 12 wherein the selected genetic markers provide a unique pedigree solution for between 50 to 90% of progeny.

14. A method as claimed in any one of claims 1 to 13 wherein the breeding value is calculated using an inverse additive relationship matrix.

A method as claimed in any one of claims 1 to 14 wherein the breeding value is calculated using partial pedigree information.

16. A method as claimed in any one of claims I to 14 wherein the breeding value is calculated using best pedigree information.

17. Software configured to undertake a method of determining a breeding value for progeny, including the steps of inputting information on possible parents of progeny, and 26 (ii) inputting genotyping information of possible parents and progeny collected using a selected number of genetic markers, and (iii) calculating parentage likelihoods and converting them to statistical probabilities, and (iv) estimating the breeding value of the progeny using an inverse additive relationship matrix.

18. A method of determining a breeding value for progeny substantially as described herein with reference to the accompanying description.

19. Software configured to undertake a method of determining a breeding value for progeny substantially as described herein with reference to the accompanying description. Dated this 14 th day of February 2003 AGRESEARCH LIMITED By Its Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia.