WO2006090136A2

WO2006090136A2 - Genetic screening of animals

Info

Publication number: WO2006090136A2
Application number: PCT/GB2006/000604
Authority: WO
Inventors: Peter Keightley; Ian Hastings; Karyn Megy; Nader Deeb; John Bastiaansen; Graham Plastow; Paul Denny; Steve Brown
Original assignee: University Court Of The University Of Edinburgh
Priority date: 2005-02-22
Filing date: 2006-02-22
Publication date: 2006-08-31
Also published as: WO2006090136A3

Abstract

The present invention relates to methods for genetically screening animals to identify those possessing commercially important traits or those possessing polymorphisms associated with commercially important traits. In addition the invention relates to improved methods for breeding animals with commercially important traits.

Description

GENETIC SCREENING OF ANIMALS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Understanding the mechanisms which underlie phenotypic variation within species is crucial to addressing fundamental issues in medicine, agriculture and evolutionary biology. However, identifying the genes that contribute to variation in traits affected by multiple genetic and environmental factors has proven extremely difficult and consequently the genetic basis of variation in complex traits remains poorly understood and few genes have been identified.

Variation in body size is an archetypal model for complex trait variation, but with the exception of a few Mendelian mutations (1), no gene contributing to quantitative variation in this trait has been identified in animals.

The availability of modern genetic analysis resources has encouraged the study of the genetics of complex traits in many species with the ultimate goal of being able to positionally clone and characterise quantitative trait loci (QTL). Such resources typically include coarse genome-wide linkage mapping and fine-mapping of specific regions. The widespread availability of molecular genetic markers has led to a proliferation of QTL mapping projects in diverse species. In many of these, attention is now directed at fine- mapping in those regions of the genome that have shown significant associations in genome-wide scans. However to date, the ultimate goal of positionally cloning QTLs has remained difficult. Recently however researchers were able to positionally clone the QTL fw2.2 which influences fruit weight in tomato (Frary et al, 2000). In addition two QTL regions, one associated with an impact on meat quality (Milan et al, 2000) and another associated with an impact on growth (Van Laere et al, 2003), have been positionally cloned in pigs.

In mice, several QTL mapping studies have identified loci that are scattered over the genome (Barsh et al, 2000, Pomp 1997) and attention is now focused on fine mapping of individual QTL.

The "P6" lines of mice have been divergently selected on body weight for more than 50 generations. Reciprocal crossing experiments between high and low lines provided evidence that an X-linked factor accounts for approximately 20% of the selection response. Further analysis by marker-based QTL mapping indicated that a single QTL located between the microsatellite markers DMXU50 and DMXH25 explains almost the entire X-linked effect. By backcrossing and selection of marker genotypes, the region containing the high-line QTL "allele" was introgressed into an inbred low selection line background. Efforts to refine the position of the QTL by high-resolution mapping indicate that the QTL maps to a short interval of the X-chromosome between the microsatellite markers DXMU226 and DXMU68, and that the QTL does not recombine into more than one factor. However knowledge of the phenotypic effects is rather sketchy, and is limited to body weight and fatness. SUMMARY OF THE INVENTION

The present invention is based upon the finding that genes involved in Mendelian diseases contribute to complex trait variation. In particular, the present inventors have determined that variation in the level of expression of two genes located within a large- effect QTL may have substantial effect upon the phenotype of animals. In addition the present inventors have determined that commercially important phenotypes such as increased size, increased birth weight, increased juvenile weight, increased lean meat content and/or increase in the size and/or quality and value of certain meat joints, may be linked to reduced expression of genes encoding members of the glypican family of membrane-bound heparin sulphate proteoglycans or the activity of these proteins. These proteins are involved in morphogenesis and growth regulation (8). Furthermore the inventors have established that polymorphisms within the QTL may be associated with the variation in gene expression and/or activity of the products of said genes.

Thus, in a first aspect of the present invention there is provided a method of screening non-human animals for potential inclusion in or exclusion from breeding programs said method comprising the steps of: a) providing a nucleic acid sample from a non-human animal; and either

(i) comparing the level of expression of the quantitative trait locus genes Gpc3 and/or Gpc4 with a reference nucleic acid sample; or (ii) detecting a polymorphism associated with modulated Gpc3 and/or Gpc4 expression and/or modulated activity of the products of the Gpc3 and/or GpcA genes; wherein modulation of the level of expression of the quantitative trait locus (QTL) genes, Gpcb and GpcA and/or modulation of the activity of the products of said genes, is associated with effects upon the phenotype of non-human animals.

For example, animals in which the level of Gpc3 and/or GpcA expression is modulated may possess commercially important traits. Additionally or alternatively, animals in which the activity of the products of the genes Gpc3 and/or Gpc4 is modulated may also possess commercially important traits. Furthermore, animals which harbour polymorphisms associated with modulated expression of Gpc3 and/or Gpc4 and/or polymorphisms associated with modulated activity of the products of the genes Gpc3 and/or GpcA, may be useful in breeding programs designed to generate progeny which may possess commercially important traits.

The present method may find particular use in, for example, agricultural livestock screening. By way of example, the present method may have wide use among animals such as cattle, pigs, sheep and other commercially important livestock. In addition, the present method may have application among poultry, for example chickens, turkeys and other farmed birds for example ostrich and pheasant. Alternatively, the present method may have applications in scientific research, m such instances it is envisaged that the present method may have uses in a range of other animals, for example rodents, particularly the guinea pig, rat and mouse.

Commercially important traits or phenotypes may be considered to be those which have an impact upon the value of a particular animal. Examples of commercially important traits/phenotypes may include animals which exhibit an increase in growth generally or more specifically animals which exhibit an increase in growth in certain areas of the body. Animals which grow more quickly or which reach maturity more quickly may also be considered to possess commercially important traits/phenotypes. Other commercially important traits/phenotypes may include animals with greater overall size, increased lean meat content, increase in the size and/or quality and value of certain meat joints and/or superior overall meat quality (drip loss, pH, tenderness, colour) .

Other commercially important traits or phenotypes may include increased birth weight and/or increased juvenile weight. Traits such as increased birth weight and/or increased juvenile weight may have an impact upon the ease with which a female non- human animal may give birth. For example traits such as birth/juvenile weight may be associated with conditions such as dystocia which is characterised (in humans) as a difficult birth or labour caused or contributed to by abnormalities of the cervix and/or uterus, the foetus, the maternal pelvis, or any combination of these factors. In addition a condition such as dystocia and juvenile growth may be associated with health and early mortality.

Dystocia is a serious problem in, for example cow-calf producers where the condition results in a birth which requires the assistance of a qualified attendant. The economic impacts upon the beef industry are high although difficult to evaluate precisely, the annual losses may be estimated at between 500 million and 750 million dollars. These losses can be attributed to several factors. First of all a high percentage of all calf deaths are a result of dystocia, for example, Montana studies show that 57% of all calf death losses are attributable to dystocia. In addition poor calf performance must be considered together with calf death losses as calves born during dystocia have lower weaning weights and are therefore likely to be more susceptible to disease. In addition to the above other commercially important traits may include increase in the size and/or quality and value of certain meat joints. Meat quality may be assessed in a number of ways. For example, pH of muscle/meat may be determined at different times post mortem using specialised pH meters for example the PH/LF-Cotrol System (Wurthinger, Pettenbach, Austria). The PH/LF-Cotrol System is equipped with a glass electrode standardised at pH value 4.64 and 7.00. Drip loss may be determined by, for example, using the bag method (Honikel, 1987) or EZ-DripLoss method (Rasmussen and Andersson, 1996; Otto et al 2004; Honikel, K. O., 1987).

In addition carcass traits may also be assessed by many means known in the art. For example, the carcass of pigs can be measured with the AutoFOM- System (SFK- Technologies) (Fat-O-meter - FOM) and colour measurements can be obtained with the Opto-Star (Matthaus, Pδttmes, Germany) and Chroma-Meter 200b (Minolta, Langenhagen, Germany) at 24 h post-mortem after cutting the carcass and cleaning the surface of the muscle/meat. The Opto-Star is calibrated against a yellow and a blue and the Chroma-Meter 200b against a white standard. For the Chroma-Meter 200b high L* values indicate paler meat, whereas for the Opto-Star a lower value indicates paler meat.

The Gpci and Gpc4 genes are located within a "large-effect" X-linked quantitative trait locus (QTL) in mice. This particular QTL has been identified as occupying approximately 600Kbp of the X-chromosome and comprises four genes, Gpc3, Gpc4, Q8C9S7, and Q9D9G4. Gpci and GpcA are known to encode members of the glypican family of membrane-bound heparin sulphate proteoglycans involved in morphogenesis and growth regulation. Specifically, animals found to exhibit a reduced level of expression of either of these genes may possess commercially important traits. Furthermore animals in which the activity of the products of the Gpc3 and/or Gpc4 genes are modulated may possess commercially important traits.

The present method requires a sample of nucleic acid from a non-human subject. The term nucleic acid may be taken to include both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In the latter case RNA may be taken to include all forms of RNA and in particular messenger RNA (mRNA).

Nucleic acid, for example DNA, may be obtained from almost any cell. For example, it may be possible to obtain a nucleic acid sample, for example DNA, from the skin or buccal cavity of a subject. Such samples may be obtained by means of a swab or other sampling device. Alternatively samples of hair or fur may be removed from the subject in a manner which ensures that at least a portion of the hair/fur follicles and/or skin surrounding the hair or fur is also removed.

The sample obtained may be subjected to a nucleic acid extracting protocol. Such protocols are well established and known to the skilled person (see for example Sambrook et al, 1989). hi addition a number of kits are available which facilitate the extraction of nucleic acid from a variety of sample types. Such kits are available from manufacturers such as, for example, Qiagen, Invitrogen LifeSciences and Amersham.

RNA may be extracted from a variety of sources and is useful in allowing levels of gene expression to be determined. RNA may be obtained, for example, from samples of tissue, for example muscle tissue (e.g. skeletal muscle, cardiac tissue), skin and/or tissues such as mammary tissue. In addition, samples of nucleic acid, particularly RNA, may be obtained from samples of organs such as the liver, kidney, pancreas, thymus, spleen, brain, intestine (small/large), testis, prostate gland, adrenal gland, uterus and lung. Furthermore body fluids, for example blood, may also be used to obtain a sample of RNA.

In order that the RNA may be extracted from a particular source, the sample must first be disrupted and or homogenised. Conveniently samples may be disrupted by means of the addition of lysis buffers together with, for example an enzyme capable of digesting molecules comprising the cell membrane or other essential cell structures. By way of an example, enzymes such as lysozyme may be used. Additionally a sample may be disrupted and/or homogenised by means of a mechanical device, for example a blender or vortex mixing machine or the sample may be subjected to disruption and/or homogenisation in a pestle and mortar. Tissue samples which comprise a significant amount of connective tissue, collagen and/or contractile proteins, may require additional treatment with an enzyme, for example a protease, in order to facilitate the disruption/homogenisation procedure.

Nucleic acid, specifically RNA, may be extracted from a sample using a variety of techniques all well known in the art (Sambrook et al, 1989). A number of kits are available (Qiagen, Invitogen LifeSciences, Amersham) which facilitate the purification procedure. By way of examples RNA may be extracted with the use of silica gel based membranes which selectively bind nucleic acid, for example RNA. In addition enzymes such as DNase may be used to further reduce the level of non-RNA contaminants. RNA may be isolated by direct elution from the silica gel based membrane.

In order to reliably analyse gene expression using, for example, RNA, it is necessary to ensure that the sample is not subject to specific/non specific degradation and/or transcriptional induction. There are a number of techniques, well known in the art, which ensure safe storage of RNA. RNA samples may be diluted in, for example, ultra pure water (Sigma) or other suitable buffers and stored for prolonged periods of time at, for example -20⁰C, -70⁰C or -8O⁰C. Preferably RNA samples are "snap" frozen in, for example, liquid nitrogen. In this way degradation of the RNA sample is prevented or minimised. In addition a number of specialised storage solutions are available which facilitate the storage of nucleic acid, specifically RNA. Such solutions include the RNA stabilisation solutions known as "RNAlater" available from QIAGEN or Ambion Inc.

Genes may be over expressed, constitutively expressed, under expressed or completely inhibited (i.e. no expression) and the term "modulated expression" may be taken to encompass each type of expression. The expression of a particular gene or genes may be modulated as a result of a number of factors which may in turn affect the transcription and/or translation of a gene. For example, a promoter associated with a gene may be repressed in some way such that the gene cannot be transcribed. Alternatively a promoter associated with a particular gene or genes may not function correctly due to the presence of a mutation or mutations. The expression of a gene or genes may be further modulated by the presence of mutations which affect the translation of mRNA. For example, mutations which introduce stop codons may prevent translation of a gene.

The level of expression of a particular gene may also vary as a result of the presence of polymorphisms at specific sites. Polymorphisms within regions associated with particular genes may affect the level of expression. For example polymorphisms within promoter regions and/or regions associated with mRNA stability may cause a gene to be over expressed, constitutively expressed, under expressed or not expressed at all. Specifically for example, polymorphisms within the 5' and 3' untranslated region (UTR) may affect the level gene expression.

The term "modulated activity" may be taken to encompass the activity of a particular gene or the product of a gene. In the present case therefore, the term "modulated activity" may relate to the activity of the genes Gpc3 and or Gpc4 and/or the activity of the products of these genes, glypican 3 and/or glypican 4.

It is to be understood that the term "modulated activity" may refer to genes or gene products which, when compared to a normally functioning gene or gene product, are shown to exhibit greater activity, lower activity or no activity. For example, in the case of an enzyme, the activity may be said to be modulated if, when compared to a normally functioning enzyme, the activity of the enzyme is increased or reduced. In some circumstances the activity of the enzyme may be modulated such that it no longer functions.

The activity of, for example, a protein may be modulated by the presence of, for example a polymorphism or mutation which affects the structure of the protein. Conformational changes may, for example, result in a reduced and /or increased ability to bind a certain ligand or catalyse the production of a certain compound.

It is to be understood that the present invention relates to the identification of non- human animals in which the level of expression of Gpc3 and/or Gpc4 and/or the activity of the products of the genes Gpc3 and/or Gpc4, is reduced rather than completely inhibited (i.e. not expressed or not active). Similarly the present invention relates to the detection of polymorphisms associated with reduced expression of Gpci and/or Gpc4 and/or polymorphisms associated with reduced activity of the products of the Gpci and/or GpcA, rather than polymorphisms associated with complete inhibition of expression or activity (i.e. not expressed or not active).

In order to screen non-human animals for reduced expression of Gpch and/or GpcA, it is necessary to compare the level of expression of these genes with a reference or standard nucleic acid sample. This may be achieved by quantitative and/or qualitative analysis. It is to be understood that the term "quantitative analysis" may refer to the comparison of the level of transcription of Gpci and/or GpcA with the level of nucleic acid present in a reference sample.

The term "reference" or "standard" nucleic acid sample refers to a sample of nucleic acid which may be applied to an assay system at a known concentration. Nucleic acid encoding constitutively expressed "house-keeping" genes in cells may be used to analyse the amount of nucleic acid in a particular sample. By way of an example it is possible to use nucleic acid encoding β-actin or the like as a reference nucleic acid sample.

There are many techniques which may be employed to compare the level of expression of a particular gene with a standard or reference nucleic acid sample. For example a reference or standard sample of known concentration may be resolved by agarose gel electrophoresis and the intensity of the bands visualised, compared with those of the test sample in order to determine the approximate concentration of the nucleic acid in the test sample. Alternatively a standard or reference nucleic acid sample may be used in an assay system such as real-time PCR wherein the amount of PCR product generated from the reference or standard nucleic acid sample may be directly compared to the amount of product generated from the test sample. Other techniques may include the use of high-performance liquid chromatography (HPLC).

Generally, techniques such as quantitative reverse transcriptase polymerase chain reaction (RT-PCR) may be used. Typically RT-PCR may be performed as a single step reaction in which the mRNA isolated as described above is first converted, by means of an enzymatic reaction involving reverse transcriptase, to complementary DNA (cDNA). Specific regions of the cDNA are then amplified by PCR. Alternatively RT-PCR may be performed in a two-step reaction in which the conversion of mRNA to cDNA and PCR stages are distinct.

The principles and methods involved in PCR are well established and may be found, for example, in Sambrook et al, 1989. Briefly, PCR relies on the provision of oligonucleotide primers which specifically bind to regions of DNA known to harbour genes or sequences of interest. Oligonucleotide primers are designed to be complementary to these sequences such that under the appropriate conditions, they bind. Conveniently, oligonucleotide primers are designed to flank the genes or sequence of interest and preferably comprise a sequence which causes each primer to bind at a location proximal to the gene or sequence of interest. Preferably a PCR may comprise a pair of primers in which the first primer binds at the, for example 3' end of a gene or sequence of interest and the second primer binds at the 5' end of the gene or sequence of interest. In the case of multiplex PCR a number of primer pairs may be added to a single reaction such that a number of genes or sequences of interest may be amplified for analysis. In the present case oligonucleotide primers may be designed to permit the amplification of target sequences within the QTL. The target sequences may be the genes which comprise the QTL. Additionally or alternatively the target sequences may represent portions or fragments of the genes that comprise the QTL. In the present case the QTL is known to comprise the genes Gpc3, GpcA, Q8C9S7 and Q9D9G4. Specifically, the present invention requires analysis of the expression and/or sequence of the Gpci and GpcA genes and optionally upstream/downstream regions.

Conveniently the PCR reaction should be optimised such that it does not reach saturation. This may be achieved by determining the concentration of RNA, by for example spectophotometry and/or denaturing agarose gel electrophoresis analysis. Preferably the RNA concentration is determined by spectrophotometry analysis of a sample at 260nm. Typically the PCR reaction should proceed such that the amount of product produced varies linearly with the concentration of RNA.

Generally the amount of PCR product generated within a specific period of time is proportional to the amount of nucleic acid template present. For example genes which are constitutively expressed or those whose expression has been induced or increased as a result of an up regulation event or the like, will likely yield more mRNA than those whose expression is suppressed or reduced. As such, by quantitatively analysing the products of RT-PCR, it is possible to establish the level of expression of a particular gene.

There are many methods by which the levels of RT-PCR product may be quantified. For example the products of such a reaction may be resolved by agarose gel electrophoresis and the intensity of the bands compared with a known concentration of a reference sample. Preferably RT-PCR is conducted in real-time allowing detailed analysis of the kinetics of the reaction in the early phases. All PCR reactions comprise three stages. The first stage is known as the exponential phase where, (assuming 100% reaction efficiency), the amount of product exactly doubles after each complete cycle. The second stage, or linear phase marks the point at which the reaction components are being consumed and the reaction begins to slow. Finally PCR enters the plateau stage where the reaction has stopped and no more product is produced. Crucially, depending upon the amount of template nucleic acid present, the time taken to enter each phase varies. By plotting, in real time, the progression of a PCR reaction, it is possible to accurately determine the amount of starting material.

Alternatively RT-PCR products may be quantified by chromatography systems, for example dHPLC.

The present invention further describes the identification of polymorphisms associated with reduced expression of Gpci and/or GpcA and/or polymorphisms associated with modulated, preferable reduced, activity of the products of the genes Gpc3 and/or GpcA. Polymorphisms may be detected in a number of ways, for example nucleic acid samples, or amplified target genes, may be sequenced and aligned with a reference sequence such that regions and/or nucleotides which vary from the reference sequence may be easily identified. Additionally or alternatively polymorphisms may be detected by, for example, restriction fragment length polymorphism analysis, heteroduplex analysis, single strand conformation polymorphism analysis, denaturing gradient electrophoresis or temperature gradient electrophoresis. Other ways in which polymorphisms, particularly single nucleotide polymorphisms (SNP) may be detected include, single base extension in which primers are designed to anneal immediately upstream of the SNP to be typed, pyrosequencing (Biotage) and/or minisequencing in which multiple oligonucleotide primers are immobilised on, for example, a solid support and extended in the presence of fluorescent ddNTPs with and enzyme, for example DNA polymerase. Finally it may be possible to use mass-spectrometry techniques, for example matrix-assisted laser desorption time-of- flight (MALDI-TOF) analysis, to type/analyse SNP(s).

Preferably the amplified nucleic acid fragments are sequenced and aligned with a reference sequence. In this way polymorphisms and the like may easily be detected. Frequently a polymorphism will disrupt or introduce a restriction site, therefore certain polymorphisms may be detected using restriction endonuclease enzymes.

Briefly, a reference nucleic acid sample is digested by a single or a number of restriction endonuclease enzymes. After treatment and enzyme denaturation, the resulting fragments are resolved by agarose gel electrophoresis. By comparing the band profile of the test sequence with that of a reference nucleic acid digested with the same restriction enzyme(s) it is possible to detect polymorphisms which have either created additional restriction sites and/or destroyed existing restriction sites.

A number of methods, all well known to the skilled artisan, may be used to obtain a nucleic acid sequence. Generally nucleic acid fragments may be cloned into vectors and subsequently sequenced with the aid of oligonucleotide primers designed to flank the cloning site on the vector. Typically, especially where the nucleic acid fragment to be sequenced is more than about 700bp long, both forward and reverse oligonucleotide primers are used to obtain the maximum amount of sequence data possible. Missing sequence may be obtained by designing further primers which "walk" along the nucleic acid fragment. Conveniently there are a number of kits available, for example TOPO " PCR4 cloning kits from Invitorgen LifeSciences, which provide a means of rapidly cloning nucleic acid fragments and obtaining sequence information.

It may also be possible to determine the level of expression of the genes Gpci and/or Gpc4 by detecting the levels of the products of these genes, glypican 3 and/or glypican 4. Techniques such as, for example, enzyme liked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radial diffusion assays and other immunological techniques, all well known in the art, may allow the detection of the products of the genes Gpci and/or Gpc4.

By way of an example it may be possible to coat the surface of, for example, a microtitre plate with an antibody specific for either glypican 3 and/or glypican 4 such that when a sample provided from a non-human animal is contacted to said antibody, any glypican 3 and/or glypican 4 in the sample results in the formation of an antibody/antigen complex. Antibody antigen interactions may be detected with the use of an antibody conjugated to an enzyme capable of reporting a level via a colourmetric chemiluminescent reaction. Such conjugated enzymes may include but are not limited to Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (AIkP). Other types of conjugated molecule may include fluorescent or radiolabeled antibodies.

By "sample" is meant any sample provided/obtained from a non-human animal from which proteins, for example antibodies may be derived. Suitable samples may include whole blood, serum, saliva, faecal isolates and/or mucus. In the case of Western blot analysis samples of glypican 3 and/or glypican 4 may first be resolved by agarose electrophoresis or other similar techniques. The resolved proteins may then be transferred to a suitable substrate, for example nitrocellulose and probed with an antibody capable of interacting with the resolved glypican 3 and/or glypican 4. Said antibody may be conjugated to a molecule for reporting a level of bound antibody as described above. Alternatively a secondary antibody, conjugated as described above may be used to detect antibody/antigen interactions.

In each case the level of antibody/antigen complex detected in the sample provided from the non-human animal may be compared to the level of a reference sample which may be applied to the assay at a known concentration. In this way a quantitative assessment of the level of glypican 3 and/or glypican 4 in the sample may be obtained.

The methods described herein may aid in the selection of animals with reduced expression of the genes Gpc3 and/or GpcA and/or animals harbouring polymorphisms associated with reduced Gpc3 and/or GpcA expression. Similarly, animals in which activity of the products of the genes Gpc3 and/or GpcA is modulated, or animals which harbour polymorphisms associated with modulated activity of the products of the genes Gpc3 and/or GpcA, may also be identified by the methods described herein. As such the present invention may provide a method by which potential breeding partners are identified. In this way it may be possible to improve the current breeding procedures such that the progeny are more likely to exhibit reduced Gpci and/or GpcA expression. In addition the present invention may provide a method of improving current breeding procedures such that the activity of the products of the genes Gpc3 and/or GpcA in the progeny, is modulated. Animals generated by such a method may be more likely to possess a particular commercially important trait or traits. Such a method may involve using the method described in the first aspect of the present invention to select either male or female non-human animals which exhibit reduced expression of Gpcb and/or GpcA and using such animals in breeding programs such that the progeny of animals selected in this manner are more likely to also exhibit reduced expression of Gpc3 and/or GpcA. Such animals may also be more likely to possess commercially important traits.

Thus in a second aspect of the present invention there is provided use of non- human animals, selected in accordance with the first aspect of this invention, in breeding programs.

Current breeding programs may rely on the selection of animals with desirable traits. For example, animals which are larger than normal, particularly animals with a greater amount of muscle tissue, may be selected for use in breeding programs. Accordingly it is envisaged that the methods described herein may be used in conjunction with current breeding selection methods in order to maximise the likelihood of the progeny possessing commercially important traits.

In a third aspect of the present invention there is provided a kit for detecting animals exhibiting reduced expression of Gpcb and/or GpcA and/or animals possessing polymorphisms associated with reduced expression of Gpc3 and/or GpcA, said kit comprising oligonucleotide primers designed to amplify a region of the QTL comprising the Gp c3 and/or GpcA genes. hi addition said kit may also aid in the detection of animals in which the level of activity of the Gpc3 and/or GpcA genes and /or the products of said genes is modulated. Thus there is provided a kit which, for example, may allow the user to identify non-human animals in which the expression of Gpci and/or GpcA is reduced and/or the activity of the products of the Gpci and/or GpcA genes is modulated. In addition the kits may allow the user to identify animals which possess polymorphisms associated with reduced expression of Gpci and/or GpcA or reduced activity of the products of the genes Gpci and/or GpcA. Animals identified by the kits described herein may possess a commercially important trait or traits or may be used in breeding programs to generate progeny with an increased chance of possessing commercially important traits.

The kit may further comprise the reagents required to execute a PCR reaction. For example the reagents may comprise ultra-pure H₂O, DNA polymerase (e.g. Taq- polymerase), buffers (PCR buffer), magnesium chloride and/or nucleotides (dNTPs). In addition the kit may comprise tools to facilitate the collection and storage of samples from which nucleic acid is to be obtained.

The kit may further comprise a reference nucleic acid sample derived from a non- human subject in which the genes Gpci and GpcA are normally expressed. By normally expressed it is meant that the genes Gpci and GpcA are not over or under expressed. Furthermore the kit may comprise the sequence of the reference nucleic acid sample such that polymorphisms within the PCR product may be easily identified. Alternatively the reference nucleic acid may be added to an assay system such that the level of expression of the region of the QTL comprising the Gpci and GpcA genes may be determined.

The techniques used to amplify and sequence genes are well known to one of skill in the art and are substantially described above. In addition, the techniques used to investigate the level of expression of particular genes are also known to the skilled man and are substantially described above.

In a further embodiment there may also be provided a kit for detecting the level of expression of the genes Gpci and or GpcA and/or the level of activity of the products of the genes Gpci and/or Gpc4, said kit comprising a substrate upon (or in) which an antibody specific for, for example, glypican 3 and/or glypican 4 is immobilised and a secondary antibody for detecting antigen/antibody complexes. Said secondary antibody may be conjugated to a molecule capable of reporting a level of bound antibody or alternatively said kit may comprise a further antibody, specific to the secondary antibody, and which further comprises a conjugated molecule for reporting the level of bound antibody.

Such conjugated molecules may include, but are not limited, to enzymes such as Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (AIkP). Other types of conjugated molecule may include fluorescent or radiolabeled antibodies.

Substrates suitable for the immobilisation of for example proteins may include polymeric substances such as plastics, agarose, glass, nitrocellulose or the like. Said kit may further comprise a reference protein, for example glypican 3 and/or glypican 4 at a known concentration to permit quantification of the level of, for example glypican 3 and/or glypican 4 detected in a sample provided from a non-human animal. In this way the level of antibody detected in a sample obtained from a non-human animal may be representative of, for example, the level of glypican 3 and/or glypican 4 expressed and/or active in the non-human animal. By sample is meant any sample provided/obtained from a non-human animal from which antibodies may be derived. Suitable samples may include whole blood, serum, saliva, faecal isolates and/or mucus.

In addition to screening the potential breeding partners, the abovementioned kit may also be used to screen the resulting progeny for those animals most likely to develop commercially important traits.

In a fifth aspect of the present invention there is provided a method of identifying and storing reproductive material from a non-human animal, wherein said non-human animal exhibits modulated expression of the genes Gpc3 and GpcA and/or the activity of the products of the genes Gpc3 and/or Gpc4 is modulated, comprising the steps of: a) providing a nucleic acid sample from a non-human animal; and either

(i) comparing the level of expression of the quantitative trait locus genes Gpci and/or GpcA with a reference nucleic acid sample; or

(ii) detecting a polymorphism associated with modulated Gpc3 and/or GpcA expression and/or modulated activity of the products of the genes Gpc3 and/or GpcA; b) identifying suitable animals and obtaining semen or egg samples from said animal for breeding purposes.

In a sixth aspect of the present invention there is provided a method of conducting an animal breeding business comprising the steps of identifying and storing reproductive material from a non-human animal according to the fifth aspect and providing samples to farmers for breeding purposes. The above method may farther comprise the step of screening a number of female animals and determining their genetic identity with regards the QTL described herein and providing male semen to inseminate said female animals based on their genetic identity, in order to seek to breed off-spring with desired commercial traits.

In a further aspect of the present invention there is provided a method of obtaining non-human animals for inclusion in breeding programs, said method comprising the steps of; a) screening animals for inclusion in breeding programs by the method of claim 1 b) identifying animals with either; i. modulated Gpc3 and/or GpcA expression; and/or ii. modulated activity of the products of the genes Gpci and/or GpcA c) providing said animals for breeding purposes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described by way of example and with reference to the figures which show: Figure 1

QTL region. Top panel: The extent of chr X segregating in three recombinant families, and the QTL effect size in males (i.e., difference in body mass between hemizygotes ± SE). The horizontal grey bars indicate the region known to segregate, while the error bars show the uncertainty in the location of recombination. Families 103 and 105 segregate for the QTL whereas family 101 does not. The differences in effect size between families 103 and 101 and between families 105 and 101 are highly significant (P<0.0001 in both cases), whereas the difference between families 103 and 105 is not significant (P=0.75) Second panel: Genes within the QTL region according to the Ensembl database (7). Third panel: LOD score plot for body mass at 6 weeks. Triangles indicate the locationof markers, lower panel: Recombination rates in the intervals delimited by diamonds (the chr X average is 0.40 cM/Mb (26)). Within ~600Kbp interval containing the QTL, no recombination events were detected.

Figure 2.

Transcript levels in newborn liver and kidney in homozygous low-allele females and hemizygous low-allele males (black bars) and in heterozygous females and hemizygous high-allele males (grey bars). Data are from 23 low allele mice and 24 heterozygous females. Values are least squares means (± 2 x SE) from a general linear model fitting effects of genotype, sex and litter; * = P <0.05. (a) Raw transcript levels of Gpc3, Gpc4

and β-actin; (b) Transcript levels of Gpc3 and Gpc4 divided by β-actin levels; correcting

for β-actin by including it as a covariate in the model yielded qualitatively similar results.

Figure 3.

Polymorphisms between high and low line derived chr X (a) 80 bp from the stop codon of Gpc3 in the 3' UTR, (b) 332 bp from the stop codon of Gpc3 in the 3' UTR and

(c) 1455 bp downstream of Gpc3. Sequence in common with reference mouse sequence (http://www.ensembl.org/Mus_musculus) is denoted by '.'.

Figure 4.

Human gene structure for GPC3 and link with the pig sequence. Pig sequences obtained by concatenation of public ESTs. Figure not to scale. The exon and intron sizes are indicated.

Figure 5. Human gene structure for GPC4 and link with the pig sequence. Pig sequences obtained by concatenation of public ESTs. Figure not to scale. The exon and intron sizes are indicated.

Figure 6.

Human gene structure for GPC3 and link with the cattle sequence. Cattle sequences obtained by concatenation of public ESTs. Figure not to scale. The exon and intron sizes are indicated.

Figure 7.

Human gene structure for GPC4 and link with the cattle sequence. Cattle sequences obtained by concatenation of public ESTs. Figure not to scale. The exon and intron sizes are indicated.

Figure 8.

SNPs were identified in the chicken homologue of the candidate gene(s) by searching public databases.These SNPs were then set up as genotyping assays and used to obtain the frequency of the polymorphisms within a broiler dataset using high vs low pools for either fat or breast muscle yield. The SNPs are ordered 5' to 3' and interestingly the largest frequency difference was found with a polymorphism 3' downstream of the gene

(the yellow bars) for both traits.

Figure 9

The difference in allele frequency (allele "1") between the High and the Low pools for the five polymorphic markers. Materials and Methods Experimental mice

The inbred low line and a line congenic for a high line segment of chr. X were described previously (1). We continued marker assisted backcrossing to the low line to produce an interval specific congenic strain (ISCS) with a contribution from high line autosomes <0.1%. The mice used in this study were at backcross generation 10-12. All experiments were carried out in accordance with U.K. Home Office regulations. Progeny testing

Heterozygous ISCS females were crossed with low line males and mice recombinant between DXMir226 and DXMit68 were used for progeny testing. Recombinant males and females were crossed with low line mice to produce families that segregated for the recombinant segment. Body weights at 6 weeks of age from the progeny were recorded and flanking markers genotyped. Further genotyping using a range of microsatellite markers established the recombinant breakpoints; microsatellite primer sequences are available in table S4. PCR genotyping was carried out on DNA extracted from ear clip or tail clip samples (1). Maximum likelihood analysis

The marker allelic states and phenotypes of the progeny test data set were analysed by maximum likelihood interval mapping (1). Briefly, each recombinant event was assumed to have been replicated across litters, and the phenotypic and flanking marker data at a given chromosomal position were used to estimate a hemizygous effect in males, homozygous and heterozygous effects in females, normally distributed litter effects, and effects for litter size, parity and sex. Likelihood ratio for the model with a QTL relative to that for the reduced model with no QTL was calculated every O.lcM in the region of interest, and converted to a LOD score. There were 937 males and 972 females in the data set.

Post mortem and histological analysis

34 mice, matched for genotype and sex, were sacrificed between 8 and 16 weeks of age and immediately underwent a comprehensive post mortem and histological investigation.

Tissue samples were fixed in 10% phosphate buffered formalin and processed. Sections were cut at 4μm and stained with haematoxylin and eosin for histological analysis.

DNA sequencing

Sequencing was carried out in forward and reverse directions using DYEnamic ET terminator Cycle Sequencing Kits (Amersham) on an ABI Prism 3730 DNA Analyzer according to manufacturers instructions. Sequencing primer sequences are shown in table

S5.

RT-PCR

Transcript levels were examined in kidney and liver from 47 newborn mice from 7 litters which were segregating for the QTL region (23 low allele mice 24 high allele males or heterozygous females). Tissue smples were collected into RNAlater solution (Qiagen) and stored at -20°C until required. Total RNA was isolated from tissue using Qiashredder homogenisers (Qiagen) and RNAEasy Extraction kits (Qiagen) according to manufacturer's instructions. We performed RT-PCR using One Step RT-PCR kits

(Qiagen) with the addition of RNAsin RNAse inhibitor (Promega). Reaction conditions were optimised for each gene tested and for each tissue type to ensure the PCR reactions did not reach saturation. Specifically, we determined the number of PCR cycles and starting RNA concentration such that the amount of product varied linearly with RNA concentration. RT-PCR primer sequences are provided in Table S6, and RT-PCR conditions (total RNA per reaction, number of cycles, etc) are listed in Table S7. 5μl of each RT-PCR product was run on 1% agarose gel to check for DNA contamination. RT-PCR product quantification by DHPLC

RT-PCR products were quantified using a WAVE dHPLC instrument at an oven temperature of 50°C. We sampled 5μl of each RT-PCR product on a DNASep column. Samples were eluted from the column using an acetonitrile gradient in a 0.1M triethylamine acetate buffer, pH7, at a constant flow rate of 0.9ml min^'1. The gradient was created by mixing eluent A (0.1 M triethylamine acetate, 0.1 M Na₄EDTA) and eluent B (25% acetonitrile in 0.1 M triethylamine acetate) according to manufacturer's specifications (Transgenomic). Each litter of mice was measured for all three genes in one assay to eliminate variation due to differences between runs. Transcript levels of Gpc3 and GpcA were expressed relative to that of β-actin by dividing the amount of Gpcb

or Gpc4 product by that of β-actin. Because the RT-PCR and quantification provided only an index of transcript levels, these are arbitrary units. AU samples were analysed in triplicate and the average within-assay coefficient of variation was less than 5%.

Examination of expression of genes of unknown function.

The tissue distribution patterns of expression of Q8C9S7 and Q9D9G4 were surveyed using mouse Rapid-Scan Gene Expression panels (OriGene Technologies; MSCBlOl) which included cDNA fom brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, muscle, lung, testis, skin, adrenal gland, pancreas, uterus, prostate gland, b reast (virgin, pregnant, lactating and involuting) and embryo (e8.5, e9.5, el2.5 and el9). The kits were used in accordance with manufacturer's instructions using an initial activation of 3min @ 94°C followed by 35 cycles of 30 sees @ 94°C, 30 sees @ 55°C and 2 min @

72⁰C then a final extension of 5 min @ 72°C (primer sequences are provided in Table S6)

Supporting material reference (1)

Supporting Table 1. Body mass at 6 weeks of age in families 103 and 105, which segregate for the QTL (see Fig. 1). Values are least squares means from a general linear model including litter, sex, genotype and sex*genotype interaction.

Least squares mean Standard Sample (g) eixor size

Females Homozygous 13.36 0.15 75 low

Heterozygous 14.80 0.13 103

Males Hemizygous 16.09 0.13 99 low

Hemizygous 18.85 0.13 100 high _EET

Supporting Table 2. Sequence polymorphisms between the high-growth line, low-growth line, and reference mouse sequence (http://www.ensembl.org/Mus_musculus).

Location High Low Ensembl 5¹ flanking sequence Polymorphism 3' flanking sequence Strand

Line line shown

Q9D9G4 C G C GGGAGACAAGAGACCTGGAGGCAGG [C/G] GAAGAAGAGGCCAGCACCTCAGGAG 1

22 22 25 GAACCACACAAAGTAAATGTGTGCA [TG] AATTTAGGCACTTGATCCTGATTCC repeats repeats repeats

Gpc3 intron 13 14 12 TTGGTTGGAACACACACATAGATGT [G] ATAGAACTCTATTTAGTAAACCTTT m 1 repeats repeats repeats

Gpc3 3'UTR, 13 17 17 AGGGAGCCACCTTCTTTTTTTTTTC [T] ATCTTTTATGCCTCCTCCCACCACC -1

80 bp from repeats repeats repeats stop codon

Supporting Table 3. Transcript levels (expressed as ratio of beta-actin levels) in newborn mice from litters segregating for the QTL. Values are least squares means (LSM) and standard errors (SE) from a general linear model including litter, sex, genotype and sex*genotype interaction.

Sample Liver Kidney size

CO

C CD Gpc3 Gpc4 Gpc3 Gpc4 CO

LSM SE LSM SE LSM SE LSM SE m

CO UJ Females Homozygous 13 0.70 0.04 0.55 0.02 1.61 0.08 1.23 0.08

I m m low

73 ι- Heterozygous 12 0.71 0.04 0.61 0.02 1.39 0.08 1.07 0.09 m

IO

Males Hemizygous 10 0.73 0.05 0.60 0.03 1.45 0.09 1.04 0.10 low

Hemizygous 12 0.49 0.04 0.63 0.02 1.25 0.08 1.13 0.09 high

Supporting Table 4. Microsatellite primer sequences. Physical positions are from Ensembl Build 24.33.1 (http://www.ensembl.org/Mus_musculus).

Position Position Primer pair Left sequence Right Sequence Approx.

(cM) (Mb) name size

0 39636328 DXMit226 CTACTCCCTGGGACAAGCAC CACAAAGGTGAGTAGAAGATGCA j24

0.658 41935562 DXIcpl3 GACTTAGGGATGTTGTGCAT TTGTCCCTAGGATCAAATTC 182

1.217 43168194 DXIcp20 TTCAACTACCAAAGGGCACA TGAGGACACAACACACACATT 275

1.405 44062558 DXIcp30 TTΓTCTTTATGTCTTTGTΠTG ACGTACATCTTCACTTATGG 427

1.405 44147989 DXIcp35 GGCAGCTGTTAGAAATGGTT AAAACCTCTCAGTAAAAGTGAAAA 300

1.405 44175340 DXIcp36 GGCTTTCGATΠTCTTCCTT AACCAAACATTTTCAAGACCA 331

1.611 44291635 DXIcp31 GCACAACATTGATTTGGTAGA GGAGAGCAGAATGGAACCT 240

1.611 44949790 DXIcp49 GAGCAAAATGCCACGATATT TACACATGGAGAGGGGAAGA 209

1.704 44973023 DXIcp34 TATAGGCCACCCAAGCAGT TCAATCCTCTCTCCCTCTCA 268

1.843 44996671 DXIcpl2 GCAAGTTCCAGGACAGTCT TGTTCTGTGTTGAGCCTAGA 199

2.169 45092842 DXIcplό GAAGGGATCACATGCTTTTc AGGGCATΠTAACACACAAAG 212

2.260 45095530 mtr20 TACCAAGGTGGGGGTAAAGG TGGAGTGGGAAAAGAACACA 194

2.624 45329989 DXIcpl5 ACAACATGAGCCCAGAGAAA CACCCTCATTCCCATACTGA 195

2.985 45438513 DXIcp22 CCAAGAAATGTGTGAATGCTT GTACCCCGCTCCATTCTATC 235

4.901 45758525 DXIcp51 CTCCCCAAAATCTAAACTCAAG TGCCACAGTTCTGTTTTCTTC 199

4.918 45762708 DXIcp29 CCTCCCCCAATGATTTTTAT GCCCCACTAAAGAGTTTGTT 242

4.936 45843712 DXIcp23 AAGTGCAGATGTCGCTCACT GAGAGCAGAGACAGGGATGA 236

4.956 45847566 DXMit68 TCCTTTGGCCTCCTGCATAT TGTTCTTACAATGAGCCTCATAGG 128 Supporting Table 5. Sequencing primer sequences. Primers for Gpc3 and Gpc4 were designed to span introns and were used to sequence cDNA. All other primers were designed for direct sequencing of genomic DNA.

Region Primer pair Forward primer Reverse primer name

Gρc3 Gpc3-1 GCTCCTCTCTTGCTCTGTC CTGCCAAATACTTTCAGGTC

Gpc3 Gpc3-2 GCCTCTTTCCAGTCATCTAC TTCTGGCTGTATCTCTCCAC

Gpc3 Gpc3-3 GAAAATGATACCCTGTGCTG TGGCTCCCTTTCTGTAGG

Gpc4 Gpc4-1 GGTCTCATTCTGTCCCCTAA AAAATGGTACTGGGAGTTCA

Gpc4 Gpc4-2 ATGTGAACCTGGAAGAAATG GCTCTGGATGATAAGGTCTG

Gpc4 Gpc4-3 AAGGTTTTCCAAGGCTGT CATTGTTGTCCATTCATTT

Gpc3 upstream Gpc3upO CTGGAAGAAAGAACGGACCT CCACCTTGGTAAACCCAATC

Gpc3 upstream Gpc3upl TCAGCAAGTCTGGCAGTAG TCCCAGAGCCAGTCAGAG

Gpc3 upstream Gpc3up2 CAATCCTGCCCTGACACTC TGGAACGTGACTATTTGTGG

Gpc3 upstream Gpc3up23 AGAGCCCACACAAGGTTTC ATTGAATGAGACCCAGCAAG

Gpc3 upstream Gpc3up3 GGTTTCTTGGGAACCCTTT AGGGCTCTACTCCAGGTATG

Gpc3 upstream Gpc3up4 TAAAGCAAAGTGTAGCTGGTG TAAATTGCTCAGTTGCCTTC

Gpc3 upstream Gpc3up5 GGGGCTATGAAGAAAGAGAAA GGGGTGACTAAGCCCTCTG

Gpc3 upstream Gpc3up56 AACTAAGGCAGGAGGGAGAG TATCAAACTCACCAGCCAAA

Gpc3 upstream Gpc3up6 GGACAGAGGGCTTAGTCACC CCTCATATTTTGCTGAACTGGA

Gpc3 downstream Gpc3down2 CAAACCTCAAATTCAGAAGCA CTGTACGTGGCTTTTCTACCA

Gρc3 downstream Gpc3down3 ATTTCCCAGAATCAAAGAGAA CTAAGCCGCAGCCTCAGT

Gpc3 downstream Gpc3down4 CAGCTCCATGAGGGTAGTGA CACACGAATTTCAAAGAGAAAA Gpc3 downstream Gpc3down5 AGAGCAAGAGAGAGCTGAGAGA CCTTAGGTΓGGATTGTCCTG

Gpc3 downstream Gpc3down6 AAAGAGAGGTCATGGTGGAAC TCTCTGCATGGATTTCTTTGA

Gpc3 downstream Gpc3down7 GAAGGCCAGATAATAAGAGCAA GGAAATCAGAAGGACAACGA

Gpc3 downstream Gpc3down8 TCCAACAGTCAAATGTAAGAAA CCATCTATGTGGCGTGCT

Gpc3 intron 1 Gpc3inl CTCTCCATCTTAGGGGAGTTG TCCAATCGCATGTTTCATTT

Gpc3 intron 1 Gpc3in2 CCCCTTCTTTAAGCACCATT CGTTCACTGGCAGGAGGT

Gpc3 intron 1 Gpc3in3 GAGATCAGAGTAAAAGGAAGCA CCTTATGGGCTGGTAGATTGT

Gpc3 intron 1 Gpc3in4 CTCAAGTGTCTGCCTGCTTT ATTCCTTTGCACTTTGCTTG

Gpc3 intron 1 Gpc3in6 AAGCTGGGCAGTAATGAAAG ACCATTTGGGTTTCTTTTGG

Gpc3 intron 1 Gpc3in7 TGGGATCTGAAGACATGGAG CATGATATAGGCATTGCTTTC

Gpc3 intron 1 Gpc3in9 GGAAGGAGAGCCTGGAAG CCAAATTCATCTGCCTTGAC

Gpc3 intron 1 Gpc3inlO TGCCCCTACAAAATGATCTG GACTGTGACTCGGGAATTTATC

Gpc3 intron 1 Gρc3inl 1 GGGACACACGGTGGATAAA CCGTTCTTTCTTCCAGAGAC

Gpc3 intron 1 Gρc3inl2 AATCCCAATGCCTCCTTCT TAGATGCAGTGTGTGTGCTG

Gpc3 intron 1 Gpc3inl3 TGCGGCCTCTATTATATCTTTC CCTTTCTCCAACACTTTCTCC

Q9D9G4 Q9Dexl GCCTCTGCTTCACCATCTAC TGGACTATCCTATGCTAAAACC

Q9D9G4 Q9Dex2 AGTCTGGAAGAGGTGTGTGG GTCTTGACATCCCTGCTΠT

Q9D9G4 O9Dex3 CCTGCTTCTGGTCTGTGAG CCTGTGCATTCCTCCTCTT

Q9D9G4 Q9Dex41 CACATGCTGGAACTTTGTCC CTTTGGTACGCCTCGAAAAC

Q9D9G4 O9Dex42 CCGTGTCCACTCCTACTCTC ATGCCATGTTCTTTCCCATT

Q8C9S7 Q8Cexl GCAAAATACAGGGAGTCTGG CCTCTAAGCTGGAAGTGGTG

Q8C9S7 Q8Cex2 GATCACAAAGATCGGAAAGG ATGTGTGAGTTGAGGGGTTC Q8C9S7 Q8Cex3 TGGGATCTGAAGACATGGAG TGCTTTCCCAAGGAAGTAAA

Q8C9S7 Q8Cex4 ACGCTCATCTGCACAAACT AGGACATGGGGAGTCAAAA

Q8C9S7 O8Cex51 CAGGAAGGAAGGAAACTCA CAGCCATGCTATCTAAACACTT

Q8C9S7 Q8Cex512 ATTCGAGACAAGCTCCCTTC CATGGAACCAGACTCTCCTC

Q8C9S7 Q8Cex52 AATCATGGCTCTGTGCTCTC CCTCATTGCCCCTCAGTT

Q8C9S7 O8Cex523 GCTGACAAACTCATCCTATCTC GCAGCAAGCAGCCTAACA

Q8C9S7 O8Cex53 AAGTATTTCAACAGGGATCAA CCTATGAAGACACAAAATGCT

Q8C9S7 Q8Cex54 GCACACTGATCCCAATTCTC CCTATGAAGACACAAAATGCT

Supporting Table 6. RT-PCR primer sequences.

Gene Forward primer Reverse primer Spanning intron number

Gpc3 GATCTATATTGGCGTTGCTG GTGGTCATGCAAGGCTGTA 3

Gpc4 GAATGGTCAGTGGCGTTGTA CCAGAAGTCCAGGTTGACAC 8 β-actin TCTGGCACCACACCTTCTAC AGGATCTTCATGAGGTAGTCTG 3

Q8C9S7 CGGTGCCTATTTATGAAGGA TGAGATTCTTCCCACCTCTG 1

Q9D9G4 TCCCTTTCAGAATGAGTGCT CGCTCCCCAAACAGAAGA 2 and 3

Supporting Table 7. RT-PCR conditions. In all cases, the RT-PCR consisted of an initial reverse transcription of 30 minutes @ 50°C, initial activation of 15 minutes @ 95°C followed by a variable number of cycles of 1 minute @ 94°C, lminute @ 55°C and 1 minute @72°C, then a final extension of 10 minutes @ 72°C.

Tissue Gene Total RNA per reaction Number of cycles

(ng)

Liver Gpc3 100 22

Liver Gpc4 100 25

Liver β-actin 50 20

Kidney GpcS 20 25

Kidney Gpc4 20 25

Kidney β-actin 20 25

Sequence concatenation

The pig and cattle ESTs {Expressed Sequence Tags) corresponding to the GPC3 and GPC4 genes were extracted from the public databases (see below). For each species and for each gene, the sequences were concatenated in contigs, using the usual public software cap3. Primer design

The boundaries exons/introns were identified on the contigs using the similarity with the human genes GPC3 and GPC4. Primers were designed within the exons, or between several exons if the final product (intron included) is still in the limits set. In the case where no exon exists for a pig or cattle sequence, the human sequence, or a consensus mouse/human, was used. Primers were designed using the public software

Primer 3. (see below for more detailed information).

Sequencing

Using the primers previously designed, the sequences were amplified. For the pig, several pools of individuals were used — for the cattle, Angus and Holstein individuals were used. SNPs were identified within these sequences using proprietary software.

SNP validation

SNPs were validated using standard methods e.g. resequencing of several individuals or confirmation of polymorphism by analysis of amplicons from individuals of different SNP genotypes (eg PCR-RFLP, TaqMan, Snapshot etc). Confirmed SNPs are indicated in f black* in the sequences.

Results

We previously showed that much of the selection response between lines of mice divergently selected for body size is due to a large-effect X-linked quantitative trait locus (QTL) with an approximately 20% difference in growth rate between homozygotes (2). To determine the molecular basis of this QTL, we progeny-tested recombinant offspring of females that were heterozygous for a segment of chr X containing the QTL, but otherwise congenic for the low-growth inbred background (3); this narrowed the plausible interval containing the QTL to a ~660kb region (Fig.

I)-

The estimated effect of the QTL on body mass at 6 weeks is 2.82 g (standard error of the mean, SEM 0.11 g) between low- and high-allele hemizygous males, and 1.48 g (SEM 0.1 Ig) between homozygous low-allele and heterozygous females (see also Table Sl). The entire effect of the QTL is attributable to the 660kb region, as demonstrated by three recombinant families (Fig. 1). Thus, in contrast to previous studies that have either found QTL to be composed of multiple QTL (4, 5), or have lacked the statistical power to dissect a single QTL, this large-effect QTL is caused entirely by one small chromosomal region. Further fine-mapping of the QTL has not been possible because the target region is located in a recombination cold-spot (Fig. 1), There is substantial heterogeneity in the recombination rate within the region, roughly similar in magnitude to variation observed in humans (6).

The QTL region contains four genes according to the Ensembl database (7), and function is known for only two of these, Gpci and GpcA (Fig. 1). Both of these genes encode members of the glypican family of membrane-bound heparin sulphate proteoglycans that are involved in morphogenesis and growth regulation (8). Loss-of- function mutations in Gpci lead to Simpson-Golabi-Behmel syndrome (SGBS) in humans, a disorder with numerous phenotypic effects, including overgrowth, skeletal and renal developmental abnormalities, an increased frequency of embryonic cancers, and neonatal mortality (9, 10, 11). Gpci knock-out mice show similar phenotypes, including increased body mass, renal dysplasias, and increased prenatal mortality (12). hi contrast, no obvious phenotypes are seen in GpcA knock-out mice (13).

DNA sequencing (3) revealed no differences in coding sequence between the high and low line QTL alleles at Gpci, GpcA, or Q8C9S7, one of the genes of unknown function. In the other gene of unknown function, Q9D9G4, there was one synonymous SNP (Table S2). The lack of nonsynonymous differences indicates that the QTL must be associated with regulatory variation. We therefore measured mRNA transcript levels in tissues from congenic mice from litters in which the QTL segregated (3). We focused our attention on newborns, since the effect of the QTL on neonatal body weight is as large as that in adults (14).

To investigate the transcript levels of Gpci and GpcA, we examined kidney and liver since both genes are expressed in these tissues in mice (15), and kidney abnormalities are often observed in Gpc3 loss-of-function mutations in humans and mice (9, 10, 12). Quantitative RT-PCR of Gpc3 and GpcA (with expression scaled

relative to that of β-actin) points to Gpc3 as the causal gene underlying the QTL (Fig.

2) (3). Mice with the high line allele show 15% lower expression of Gpc3 in liver and kidney (P = 0.017 and P = 0.012, respectively), whereas the differences in transcript levels for GpcA are non-significant (P = 0.08 and P = 0.74, respectively) and the trends vary in direction between tissues (Fig. 2; Table S3). The lower level of expression of Gpc3 in mice with the high body mass QTL allele is consistent with the overgrowth seen in Gpc3 knock-out mice (12). These results underscore the potential significance of relatively small changes in expression levels on phenotype. While a number of studies have used microarrays to identify genes underlying QTL (16-19), such an approach would have been unlikely to have been successful here because of its low power to detect subtle differences in expression levels.

To examine whether the two genes of unknown function might contribute to the effect of the QTL, we examined their expression using a 24-tissue gene expression panel (3). Q9D9G4 was originally identified in adult male testis cDNA (7), and we observed clear expression in this tissue, as well as very low levels of expression in muscle, lung and small intestine; no expression was detected in embryos. Q8C9S7 was originally identified in 3 -day neonate thymus cDNA (7) and while we were able to detect very low levels of expression in the thymus of 3-day old mice, we were unable to detect its expression in any adult tissue or embryonic stage using the commercially-available expression panel. Q8C9S7 appears to be homologous to an annotated human pseudogene, and there are premature stop codons in the mouse sequence and the homologous sequence in rat (7). Because genes of unknown function with restricted patterns of expression did not present strong candidates for the causative factor underlying a QTL with a general effect on growth rate in both sexes, we did not pursue these two genes further.

To identify candidate polymorphisms that might be responsible for the difference in Gpc3 transcript levels, we sequenced the 5' and 3' untranslated regions (UTR), 2876 bp upstream from the 5' UTR of Gpc3 (including its promoter region (20)), 1724 bp downstream of the 3' UTR, the first 1048 bp of intron I, as well as 3377 bp of other regions of intron 1 that were identified as having high conservation with human. We focused on these regions because they are the regions near genes that show the highest levels of sequence conservation in rodents (21), The only sequence differences between the high and low line derived regions were three mononucleotide repeat polymorphisms (one in the first intron of Gpc3 and two in the 3' UTR), two dinucleotide repeat polymorphisms downstream from Gpc3, and a SNP 1455 bp downstream of the 3'UTR (Table S2). This low level of polymorphism is consistent with previous findings (2) and a low frequency of microsatellite polymorphism between the lines. The 3'UTR polymorphisms present strong candidate polymorphisms for the differential expression of Gpc3, since 3'UTRs are known to play a role in mRNA stability (22, 23). Furthermore, the polymorphic segments show high conservation across mammals (Fig. 3 a, b). A BLAST search of a 450 bp region surrounding the downstream SNP yielded hits in the region of Gpc3 in both human and rat, and indicated that this base pair is also conserved across these species (Fig. 3c). Whatever the sequence polymorphism(s) responsible for the phenotypic effect, the QTL is not due to mutation that occurred during the selection process because a large X-linked effect was observed in replicate selection lines derived independently from the same base population (24). Since knock-out mutations of Gpc3 generate a range of pathological phenotypes, it might be expected that QTL-associated regulatory variation at Gpc3 would generate milder forms of these pleiotropic effects. We therefore conducted post mortem and histological analyses on a sample of 34 age and sex-matched individuals (3). Some of the most prominent pathological conditions of Gpc3 -deficient mice are cystic and dysplastic kidneys, imperforate vaginas leading to swelling of the perineum and fluid filled uteri, and susceptibility to respiratory infections (12). However, there was no evidence of cystic medullary dysplasia resembling that seen in Gpc3 -deficient phenotype in mice of either genotype and no other significant pathological phenotypes were observed in other organs in either group. Since Gpc3 -deficient mice have a reduced survival probability to weaning (12) we compared the numbers of high and low genotype mice surviving to weaning age in segregating litters. There is no evidence of an effect of genotype on numbers of high and low allele mice at weaning

(512 and 554, respectively; %²ι= 1.65; P = 0.2). For litter size, congenic females

homozygous for the high-line QTL allele have somewhat higher performance than females homozygous for the low-line allele (5.38 [SEM 0.22] versus 4.78 [SEM 0.18], respectively; t₁₉₈ = 2.13; P =0.03).

In this study we have fine-mapped a growth QTL to a region containing only two genes of known function, found no coding sequence variation in these two genes, and demonstrated significant variation in the transcript levels of Gpc3. The phenotypic and expression differences between QTL genotypes are consistent with known loss-of-function mutations and knock-out phenotypes, i.e., reduced/absent Gpc3 expression leads to increased body size. Our results therefore show that a gene underlying a Mendelian disease in humans can contribute to quantitative variation in mice. Unlike loss-of-function mutations, allelic variation in Gpc3 has no pathological side-effects that we were able to detect; it affected growth rate only, and did so at all ages and in all tissues that we have studied (14). This work provides further evidence that the glypicans are involved in normal growth processes in addition to their role in SBGS and a variety of cancers (25).

Candidate gene for QTL Glypican 3 (Gpc3)

We carried out a study of differences in organ weights and cell numbers between the mouse lines congenic for a section of the X chromosome containing the Gpc3 gene; this has been shown in our previous work to change growth rate by about 20%. Our new studies confirmed the effect on growth, and showed differences in organ weights that are consistent with the presence of a major QTL at the Gpc3 gene.

High resolution mapping placed the X-linked QTL within an ~660kb region of the X chromosome, containing only 2 genes of known function, Gpc3 and Gpc4. Relative reductions of 15 % in Gpc3 mRNA expression levels were found between High and Low line kidney and liver. Sequence differences between High and Low line mice were found in the 3' end of the Gpc3 gene. No significant histological differences were found between the two lines. (Oliver et al PLoS Biol 2005 May;3(5):el35)

Gpc3 is a cell surface heparan sulphate proteoglycan. The effects of Gpc3 are varied and appear to be developmental stage and tissue specific. Its effects are associated with both cell proliferation and apoptosis. Gpc3 promotes growth by affecting canonical/ non-canonical Wnt signalling, and therefore cell proliferation, in liver carcinomas. (Capurro et al, Cancer Res. 2005 JuI 15;65(14):6245-54) Gpc3 modulates Bmp2-Smad signalling which in turn inhibits renal branching morphogenesis. A reduction in Gpc3 increases cell proliferation in uteric buds. (Hartwig et al, Mech Dev. 2005 Jul;122(7-8):928-38 ) Expression of Gρc3 within lung tumours is associated with an increase in the rate of apoptosis and is therefore suggested as a candidate lung cancer suppressor gene. (Kim et al, Am J Respir Cell MoI Biol. 2003 Dec;29 (6):694-701)

SGBS

Simpson Golabi Behmel Syndrome, a human overgrowth syndrome, caused by loss of function mutations of the Glypican 3 gene is characterised by several factors including, increased body size, craniofacial abnormalities, kidney dysplasia and Polydactyly. SGBS patients can also exhibit liver and kidney carcinoma (usually in infancy), hepatomegaly and splenomegaly as well as polycythemia (large increase in circulatory red blood cells).

Gpc3 Knockout Mice

Gpc3 knockout mice show many of the symptoms of SGBS including increased body mass and cystic kidneys. They also show a reduction in spleen mass of as much as 60%. Gpc3 deficient mice show morphological differences in kidney, spleen and placenta.

Current Work

Aim of Current work

The QTL is known to be associated with an -20 % increase in body mass. This work will determine whether the increase in body mass is associated with proportional increases in organ masses. It will also determine whether the effects of the QTL are associated with changes in cell numbers. Muscle composition has been found to be altered with increases in body mass (Rehfeldt et al J Muscle Res Cell Motil. 2005;26(2):103-12 ). This work will also investigate whether the QTL has an effect on skeletal muscle fibre composition.

Methods

Weights and organ collection

Body weights were recorded at either 3 or 10 weeks. Mice were sacrificed by continued exposure to CO₂. Spleen, kidney, liver and heart were removed and weights recorded before being snap frozen in liquid nitrogen. The right quadriceps femoris muscle group was dissected from the bone, orientated in 1% agar, mounted on cork discs and frozen in liquid nitrogen cooled isopentane (-70 ⁰C). All samples were stored at -85 ⁰C. 10 μm Transverse sections of muscle were prepared and slides stored at -85⁰C until required.

Single cell suspensions

Weighed tissue was placed between two layers of 100 μm gauge nylon gauze in 3ml of medium (Dulbecco's Modified Eagle Medium, 2 μl/ml Proteinase Inhibitor Cocktail, 400μM L-glutamine). Sterile forceps were used to gently tease the tissue through the gauze and the medium containing the cells was centrifuged at 1400rpm for 3 minutes at 4 ⁰C. Red blood cells were removed using RBC Lysis Buffer and the resulting pellet was washed twice in medium before being resuspended and diluted with 0.4 % Trypan Blue. Samples were counted in duplicate using an Improved Neubauer Haemocytometer. Resulting counts were averaged and converted to cells/ mg tissue. NADH-TR staining

Slides were incubated in NBT solution (2mg/ml Nitro-Blue Tetrazolium, 1.6mg/ml NADH, 0.05M Tris HCl) for 30 minutes at 37 ⁰C in a wet box then washed in distilled water. Unbound NBT was removed with 3 changes each of 30, 60 and 90% acetone. Slides were washed in distilled water and mounted in aqueous mounting medium. Micrographs were obtained and counts of the different fibre types, (aerobic, anaerobic and intermediate), were performed in duplicate. Counts were averaged and converted to proportions.

Results

Organ weight study

Body weights, Liver, Heart, Kidney and Spleen masses were measured in newborn, three and ten week old mice (Table 1). As expected, overall body mass showed a statistically significant relative increase in body mass of 20-25% in the High line mice at both 3 and 10 weeks of age compared to the Low line. The relative increased body mass in the High line was ~14 % in newborn mice. Newborn mice showed no significant differences in organ weights.

Table 1

Cell counts

Cell counts were carried out on liver, spleen and kidney cells from both lines of mice at 3 and 10 week of age. At 3 weeks all three tissues showed a relative decrease in the number of cells per mg of tissue in High line mice but only the 19% decrease in kidney cells was statistically significant (p =0.021, n = 44). At 10 weeks Kidney and Spleen Cell counts showed no significant differences between the two lines. An 32% relative increase was found in Liver cells per mg in the High line (p=0.014, n=20)

Muscle fibre composition

Muscle fibre composition was calculated for the quadriceps femoris muscle group of 44 sex and genotype matched 3 week old mice. No significant differences were found in either the total count or the fibre composition with both mouse lines giving proportions of 0.23, 0.24 and 0.53 for aerobic, intermediate and anaerobic muscle fibres respectively. Growth Rate

Reanalysis of the data collected in 1999 by Xiaujun Lui, (Lui et al Heredity 2001:92(4) 356-257), shows differences in the growth rate between the two mouse lines. An increase of ~10% in growth rate, in tandem with an increase in body mass of ~30% is found in the High line relative to the Low line at 4 weeks of age. This is equivalent to the differences found in newborns and corresponds to a second peak of relative increase in growth rate between the high and Low mouse lines making 4 weeks a good age to use for further study. The 1999 data also shows a relative increase of only ~3 % in the carcass weights of 10 week High line mice in comparison to Low, (0.7g and 0.5g increase in males and females respectively), (ρ= 0.012, n=166).

Conclusions

The QTL s effect varies at different stages of development and in different organs. The QTL does not affect skeletal muscle composition at the age examined. The overall increase in body mass caused by the QTL does not lead to proportional increases in all organ masses. A decrease in the relative mass of kidney at 3 weeks coupled with a reduction in cell numbers at that age are consistent with findings from Gpc3 knockout mice. A reduction in kidney mass is also found in GPC3 knockout mice, as are changes in kidney cell composition. The reduced spleen mass is also consistent with findings from Gpc3 mice where reductions in spleen size can be up to 60% of expected size. This provides further evidence linking our QTL to the Glypican 3 gene and further confirms that Gpc3 has a varied role in the development of different organs. The reductions in expected liver mass at 3 and 10 weeks, coupled with increased cell counts at 10 weeks is unexpected and requires further investigation. Gpc3 promotes growth in liver cancers possibly the decrease is connected with the reduction in Gpc3 mRNA expression previously found in High line liver. Candidate genes GPC3 and GPC4: Sus scrofa and Bos taurus

I. General rules to design primers

When available, pig (or bovine) sequence was used to design primers for sequencing. If not, a consensus between the human and the mouse (and sometimes the rat) genes was used.

Primers were designed in exons. When possible, the primers were designed in two different exons (forward primer in one exon, reverse primer in the following) — however, this was not possible in most cases due to the large introns in these genes. The amplicon size was designed to be as large as possible, but not more than 600bp.

II. Pig

The SNPs were tested initially on three lines with growth data to be used in the association analysis.

a. GPC3 (Fig. 4 - Table 2) Table 2: Primers for the pig gene GPC3

The pig gene sequence was obtained from the concatenation of ESTs available from public databases (dbEST: http://www.ncbi.nlm.nih.gov/dbEST/) and two contigs were obtained. In order to match them to the human gene structure (Ensembl: http://www.ensembl.org/), both contigs were then concatenated to one sequence by adding a stretch of "N" between them. Result of the primer design:

Primers were designed using "Primers3" software, si and s2 are subjective scores for "degree of confidence" of the SNP: si = high confidence and s2 = lower confidence. Hs, Mm, Rn, Ssc and Bt are the species used to design primers - respectively Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Sus scrofa (pig) and Bos taurus (cattle).

b. GPC4 (Fig. 5 - Table 3) Table 3: Primers for the pig gene GPC4

The pig sequence was obtained from the concatenation of ESTs as above and two contigs were obtained. For primer design, and to match the human gene structure, they were concatenated into a single contig separated by a stretch of "N". (see above). Result of the primer design:

The 5'UTR was too different between the human, mouse and rat and was not considered. Three primer pairs were designed in the 3 'UTR in order to cover as much as possible the UTR length - the overlap between two amplicons is around 150bp.

III. Cattle

The SNPs were tested on samples of sires from a breed to be used in the association analysis. a. GPC3 (Fig. 6 - Table 4) Table 4: Primers for the cattle gene GPC3

Bovine sequence was obtained by the concatenation of ESTs from public databases (IBISS: http://www.livestockgenomics.csiro.au/ibiss/) and one contig only was generated. The primers designed for pig were tested on the bovine sequence and only the ones not working were specifically redesigned for bovine. Result of the primer design:

a. GPC4 (Fig.7 -Table 5)

Table 5: Primers for the cattle gene GPC4

Few ESTs were found in the databases to characterize the bovine sequences. They were concatenated in 2 short contigs and a consensus human/mouse sequence was used to generate primers in the missing parts (Gene sequences from Ensembl: http://www.ensembl.org/ and concatenation using cap3: http://genome.cs.mtu.edu/cap/cap3.html). Result of the primer design:

IV. Comparison of GPC3 3'UTR between mouse, pig and cattle

The mouse, the pig and the cattle sequences were aligned in the 3'UTR regions of the GPC3 gene. The three regions are highly similar except for an insertion of 8 Ts in the mouse sequence.

Table 6: Comparison of GPC3 3'UTR between mouse, pig and cattle.

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I. Allele Frequency

a) Pig

Two SNPs were identified and confirmed in the GPC3 gene. These were located in exons 3 and 8. A single SNP was identified and confirmed in the 3' untranslated region of GPC4.

Allele frequencies were determined for the SNPs identified in exon 8 of GPC3 and GPC4 using samples from herd sires of 20 pig populations. Allele frequencies for the SNP in GPC3 exon 3 was determined in sires from four populations (I, M, N and R) used for trait association analysis and for females from five other lines (B, E, H, O and U, see below: results for these 8 lines are also provided for the other SNPs). Results are presented in Table 7. Table 7: Allele frequencies in PIC populations

b) Cattle

A single SNP was identified and confirmed for both of the genes, in exons 4 and 2 in the GPC3 and GPC4 genes respectively. Allele frequencies were determined for these SNPs in sires from two cattle populations. The GPC3 SNP was only found at an extremely low frequency of 0.003 in one population and it was fixed in the second population, whilst the GPC4 SNP was present at a frequency of 0.65 (n= 350) and 0.31 (n=48).

II. Association Analysis a. Porcine GPC3/4

Five datasets were used for the porcine association analysis. The first four were performance test data for four populations (lines I, M, N and R in Table 7). These datasets included phenotypic measurement of test daily gain (TDG), lifetime daily gain (LDG), P2 backfat thickness (P2), ultrasonic muscle depth (MD) and feed intake (FI) (the last only for males). The fifth dataset nominally consisted of 100 animals from each of lines B, E, H, O and U in Table 7). These animals have a large number of traits recorded including carcass and meat quality measurements as well as performance test data.

Results for the performance datasets were analysed across (Model: Trait = line + genotype) and within lines (Model: Trait = farm+genotype) and separately for each sex. Potentially interesting results for each of the markers are presented in Tables 8- 10. Table 8: Association Analysis for GPC3 exon3 in males

1 LS Means significance levels: a- b p<0.3, c-d p<0.1, e-fp<0.05

The only significant difference for LSMeans for GPC3 exon3 in males (Table

8) is for muscle depth in line I, however, there is a tendency across lines for the 10 genotype to grow faster and to have more muscle depth. This trend is also found for line I for growth and there is also a tendency for 10 animals to be leaner in lines I, N and R. Table 9: Association Analysis for GPC3 exon3 in females

1 LS Means significance levels: a — b p<0.3

No significant difference was detected between genotypes in the females with the GPC3 exon3 SNP (Table 9), although there was a suggestion that growth and muscle depth were different across lines and in line I, with allele 2 being associated with faster growth and more muscle. Allele 2 tended to be associated with greater backfat in line I. These results are opposite to those found in males for growth and muscle depth (Table 8). Table 10: Association Analysis for GPC3 exon 8 in males

1 LS Means significance levels: a- b p<0.3, c-d p<0.1, e-fp<0.05

Although there is a tendency for GPC3 exon 8 genotype 10 to have greater growth across lines (and in lines M and R) the only significant result was found for backfat across lines and in line I where genotype 10 was associated with greater backfat thickness (Table 10). Table 11: Association Analysis for GPC3 exon 8 in females

1 LS Means significance levels: a - b p<0.3, c-d p<0.1, e-f p<0.05, g-h p<0.01, i-

There appears to be a significant association between the GPC3 exon 8 genotype and performance traits in the females (Table 11). Highly significant effects were found for growth in lines N and R and for muscle depth in line R. There is a suggestion that the effect is dominant in line N, and in line R (where only 1 observation was available for 11) the heterozygote is also associated with more growth and more muscle. Note that allele 1 was somewhat associated with more growth in the males (Table 10). Table 12: Association Analysis for GPC4 3'UTR in males

1 LS Means significance levels a - b p<0.3, c-d p<0.1 , e-f p<0.05

There appears to be a significant association between the GPC4 3'UTR genotype and P2 in the males (Table 12) especially in line I with genotype 10 being associated with greater fat. Significant effects were found for FI in lines I, M and R although the effect differs with genotype 10 being associated with greater FI in lines M and R and genotype 20 in line I. Table 13: Association Analysis for GPC4 3'UTR in females

1 LS Means significance levels: a - b p<0.3, c-d p<0.1, e-f p<0.05, g-h pO.Ol

For the females, in contrast to the males, there did not appear to be a significant association between the GPC4 3'UTR genotype and P2 (Table 13). However, significant effects were found for TDG and muscle depth across lines and in line R. The heterozygote appears to be associated with more growth and more muscle (although there were no observations for genotype 11 in line R). The results presented in Tables 8-13 suggest that there may be an effect of GPC3/4 genotype on performance traits in the four lines tested, although the results are not completely consistent across lines. However, in most cases the frequency of allele 1 is relatively low (see Table 7) reducing the power of the analysis. The most "favourable" frequencies are for GPC3 exon 3 in line R and for the other two polymorphisms in line N. In the case of the first of these there is little suggestion of an effect on any of the traits analysed (Table 8 and 9). In the case of the exon 8 polymorphism in line N, a significant effect was found for growth in females, especially between genotypes 12 and 22 (Table 11). The effect appears to be over- dominant, although genotype 11 has greater growth than genotype 22. In males of line N, significant effects were not found, although lifetime daily gain (but not TDG) tended to be greater in 10 animals than 20 (646g/d versus 633g/d).

The final porcine dataset utilised in the study consisted of a detailed phenotypic characterisation of carcass and meat quality traits for females of five diverse genotypes. This dataset was included as four of the lines had relatively balanced genotype frequencies for the GPC3- exon 8 and GPC4-3'UTR polymorphisms (lines B, E, 0 and U, see Table 7) and growth had also been determined for these animals. However, relatively small numbers of animals were available per line (100 per line were genotyped, but results were not obtained for all samples).

Results for growth were presented in Table 14 as analysed by line. Additional analysis was undertaken combining data from all Lines using the models:

Ml : Y = Batch+Line+Add+Add*Line

M2: Y = Batch+Line+Add (Add = additive effect) and Table 14 updated. Again the analyses do not indicate a significant effect on growth, although there is a significant difference between 11 and 12 LSMs for the first marker (note the interaction Add* Line was not significant for any of the markers). Table 14: Association of GPC3 and 4 polymorphisms with growth

As significant effects were seen for fatness and muscle depth in some of the previous analyses other traits were considered in this dataset. This review was limited initially to those line/marker combinations where the allele frequency was between 0.3 and 0.5 (see Table 7) in order to concentrate on analyses where the number of observations per genotype class would be more favourable for comparison. Lines B (GPC3 exon3 and GPC4 3'UTR) and O (all three markers) showed the most interesting results. There appears to be a significant effect of the GPC3 exon3 genotype on the weight of bone in three of the joints measured (ham, loin and shoulder) in line B (Table 15). Allele 2 appears to be associated with more bone in these joints. In addition there is a suggestion of an effect on the meat quality traits, ultimate pH (24h after slaughter) and drip loss (which are positively correlated) in Line O, however, the effect on ultimate pH tends to be in the opposite direction in line B. This tendency for opposite alleles to be associated with the trait mean is also shown for the weight of belly and the weight of lean in the belly.

Table 15: Effect of GPC3 polymorphism on the yield of primal cuts and pH and drip loss

The most significant results were obtained for the GPC3 exon 8 polymorphism in line O (Table 16). Significant effects were observed for fat depths, weight of some joints and the weight of lean in the joints, with a trend to effects on the amount of lean in some of the other joints (e.g. ham and loin). Similar effects were also observed for the GPC4 3'UTR polymorphism, although the association was in the opposite direction (e.g. allele 1 for GPC3 is associated with more lean in belly, ham and loin, whereas allele 2 for GPC4 is associated with a higher LSmean - not shown).

Table 16: Table 10 GPC3 exon 8 effects in line O

= e signi icance eve o e a ve e ec = .

Again an "across line" analysis was undertaken using models Ml and M2.

For GPC3 exon3 effects were seen for several traits e.g. Table 15 in Line B (and Line O). Table 16B shows the results for these traits using models Ml and M2. As would be expected based on the initial results, the Add*Line interaction was significant for many cases in which case data should be evaluated by line (see above). However, for drip loss, this interaction was not significant (P=0.81) and a very significant additive effect was observed.

Table 16B: Association of GPC3 Exon 3 polymorphism using models Ml and M2

Table 16C shows significant results obtained for this marker for the combined Lines as the interaction Line* Add (Ml) was found to be not significant (>.3). In this case the combined lines can be treated as a single population in order to improve the power of the analysis, as larger numbers of observations are available for each genotypic class.

Table 16C: GPC3 exon 3 Association analysis for combined data (where Ml is not significant)

Genotype 11 is associated with less fat at the shoulder, higher intermuscular fat and skin plus subcutaneous fat in ham and shoulder and less lean meat in ham and loin (and a similar trend in the shoulder).

In the case of GPC3 exon 8 most traits showing an effect showed significant Add*Line interaction (Ml) including for the traits significant for line O (Table 16) (results not shown). Interestingly, Add*Line interaction was not significant and the additive effect for drip loss of the combined Lines was found to be significant, which is similar to the effect found with the other marker in GPC3 (Tables 16B, C). The LSmeans were 3.14, 2.96 and 2.61 for genotypes 11, 12 and 22 respectively (contrasts ll/12 ρ=.36; 12/22 .04; 11/22 .02).

In the case of GPC4 most traits showing an effect showed significant Add*Line interaction (Ml) however, in this case the marker (GPC4) had no significant effect on drip loss. Further Association Analysis Pig:

Markers were analyzed with residual phenotypes which have been adjusted for all relevant fixed effects. A mixed model including sire as random effect was used to estimate LSmeans for genotype classes within sex. A second analysis, again mixed model with random sire effect, was done to estimate additive effects and for the female group also dominance effect. Table 17: Marker genotype frequencies, contrasts in bold have a minimum of 50 observations.

Results for pre-wean piglet survival, average daily gain and loin depth are summarised in the Tables 18 and 19 respectively.

Table 18: Summary of pre-wean piglet survival association analysis. Marker EDSOOl

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I m Table 19: Summary of average daily gain and loin depth association analysis. m Marker ID EDS002

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II. Association Analysis a. Cattle GPC4

Sires from a breed with estimates of growth breeding values were genotyped for the bovine GPC4 polymorphsim and "Expected progeny differences (EPD)" used to analyse for a genotype effect. EPD is the estimate of how future progeny of each sire are expected to perform for the traits listed. The model used was TRAIT = EPDsource+genotype. Significant effects were observed for birth weight and yearling weight and there was a suggestion of a genotype effect on weaning and carcass weight (Table 20). Note: as sires only have one X chromosome genotypes are defined as 1, 0 (10, see table 16) or 2,0 (20) when allele 1 or allele 2 is present respectively. The estimated contrast of 10 and 20 breeding values will be Vi the additive effect of the marker or VA of the expected difference between 11 and 22 females (assuming additivity) or Vi the expected difference between 10 and 20 males (assuming effect is equal in both sexes etc.).

Table 20

1 LSmeans significance levels: a-b p<.3, c-d p<.l, e-f ρ<0.05

As birth weight is related to calving ease (dystocia) variation in these genes may explain variation in this important welfare and economic trait Further Association Analysis Cattle:

The estimated progeny differences (EPD's) were obtained from the Angus Association webpage for sires born between 1990 and 2002. The EPDs were used as dependent variables in a regression on genotypes to estimate LSmeans for the 10 and 20 genotype classes. The additive effect is also shown in the tables in the appendix. GPC4 SNP has a good intermediate frequency.

Growth traits come up significant for the GPC4 SNP. This fits with the candidate status of the genes which are expected to affect growth. The GPC4 SNP shows associations of interest where yearling weight and feedlot_value have favourable results with the 20 genotype and the %_retail product and grid_value have favourable results with the 10 genotype. The 1 allele appears favourable for the packers where the 2 allele would be favourable for the feedlot operation.

Table 21 : GPC4

Significant effects of this marker on several growth related traits in Aberdeen Angus:

Aberdeen Angus:

Table 23: GPC 4 across both breeds

Variation of GPC3 and GPC4 in the chicken. a) putative orthologues were identified for GPC3 and GPC4 in the chicken by searching [Ensembl database at: http://www.ensembl.org/Homo_sapiens/, the Orthologue Prediction] using [reciprocal BLAST analysis].

Both of GPC3 and GPC4 are on GGA4 (i.e. not the sex chromosome); GPC3 (ENSGALT00000009820) at 3659761-3772143 Mb and GPC4 (ENSGALT00000009812) at 3586738-3597158 Mb.

b) Information on chicken SNPs in these genes was obtained from the Chicken Variation Database (ChickVD) at: http://chicken.genomics.org.cn/index.jsp.

c) 7 SNPs were selected from GPC4 and the adjacent genomic region.

d) The sequences Cl 154 and Cl 155 are SNPs between GPC4 and GPC3, near the 5' end of GPC4.

Table 24

LabED SNP (BGI ID) PosChr Flanking sequence and SNP Gene SNP location

CO Cl 150 snp.l l.502.30285.S.l 3597520 CTCAGAAATGACTCACGGTC[A/G]TTCATGTTTGATTCACTCCC GPC4 5' upstream

C CD CO C1151 snp.l l.502.34591.S.l 3593214 GGAAAGTAATAATTGCCTTT[A/G]GAGAATGCGTTCGACTGTAG GPC4 iαtron

Cl 152 snp.l l.502.36868.S.l 3590937 GTGTCACTTCTGACCAGCACCA/GICACCTCCAGGAAACATTCTG GPC4 intron m Cl 153 snp.l l.502.39575.S.l 3588230 AACAAAGCCCACCAAGAATA[T/C]GTCTCCTGGAACAGGTATTT GPC4 iαtron

CO

I Cl 154 snp.l l.502.22677.S.l 3605128 GCTTTTAAACCCACGTATAG[AZC]ATTTCCCAATTGTGTCCTAC m m C1155 snp.l l.502.17849.S.l 3609956 TCTGAAGGAACCCAACGCAA[TZC]GTTTTAAGCCAAGAAAGTTA

73 C1156 snp.l l.502.41712.S.l 3586093 ACATCCCGTAAAGAAAGCAC[AZG]ACCTGCTCCTGGCATTACCA GPC4 3' downstream ι- m

IO

e) primers used for amplification of chicken DNA and SNP genotyping:

f) genotype frequencies were estimated for each SNP in sample pools selected for high and low amounts of breast meat and fat. Frequencies were estimated from the peak heights using SnaPshot genotyping.

One of the SNPs (Cl 155) was fixed in this population of chickens. AU of the other six SNPs were found to be segregating. The allele frequency for Cl 156 appears to be more different between the selected pools of animals, having a higher frequency in the high pools for both breast meat % and amount of fat. The allele frequency differences for Cl 150 are also different, but in this case the frequency is higher in the low breast meat pool and the high fat pool. A relatively large difference is also observed for Cl 153 in the breast meat pools. Smaller differences are also observed for the other markers, g) 8 SNPs were selected from GPC3 and the adjacent genomic region Table 25

LabE) SNP (BGI ID) PosChr Flanking sequence and SNP Gene SNP location

Cl 157 snp.l 1.494.5054.S.3 3774143CTTCACTTAACAGCTCCCAG[A/T]GATTGATCAACTGGAGATGC GPC3 5' upstream

Cl 158 snp.l 1.494.9061.S.1 3770136 CCAATGGCCGAGAGGAGAGC[TZA]CAGCCCTGAGCTGCAGAACC GPC3 intron

Cl 159 snp.11.494.21591.S.I 3757606GTTCAACTGCTTGGAAGTTG[A/G]ACACAACTTCTCCAGAGCCT GPC3 intron

C1160 snp.l l.494.38690.S.l 3740507CCCCACCAAAAACCTCCCCA[G/A]TGCGTTTGCCTCTGAGAATC GPC3 intron

Cl 161 snp.l 1.494.56125.S.1 3723072GGACCCCAGAGGATGTCAGC[TZC]GTGTGACACCCAGACCTACT GPC3 intron

CO Cl 162 snp.11.494.76751.S.I 3702446 ACTTCGTGCCACCGTGCCAA[TZC]CTGAGGAACGCATCCGGCGG GPC3 intron

C CD CO Cl 163 snp.l 1.495.22445.S.1 3666793TGGGCTAGGAACTGCCCTAT[AZG]GGCTCAGTCCACCAGCATGG GPC3 intron

Cl 164 snp.l 1.495.30100.S.1 3659138TTCTTCCTTGACCTTATATC[CZT]CATCTTCCCAGTCCCGCCAG GPC3 3' downstream m

CO

I Table 26 m m

LabID PrimerJL Primer_R Primer_I (for SNaPshot)

73 ι- m Cl 157 ATTTGTAGGCACTGAGAAGTGT GTATGGAGTCATGCAGTCGTA CTTCACTTAACAGCTCCCAG

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Cl 158 CAAAGAGAACAGCCAAAAAG GTGCTGTTTCATGACTGGTA CCAATGGCCGAGAGGAGAGC

Cl 159 CGGAGTTGAAATATATTATTGTTG ATTGTGAATATTTCCCTTTCATT GTTCAACTGCTTGGAAGTTG

Cl 160 CAGAGATTGTTTCTTTGGAATAA GAAATGTCAAGTTAATGTGCTCT gattctcagaggcaaacgca

C 1161 CTGAGTTACAGGAAGGAGAAAA CTTTTGGTATGGTTCTTATTCAG GGACCCCAGAGGATGTCAGC

Cl 162 ACAGATACACCCAAGACACAATA TGTTTCTAAATTCAGTTCACAGC ACTTCGTGCCACCGTGCCAA

C 1163 ATTTTTCCTTTGTGACTCACTG TTCAGGAGTAGGAAAGATGTGT TGGGCTAGGAACTGCCCTAT

C 1164 CTTTGTCTGGAGCTGTGTTT ATACAGACTTTTTCTGTGGTTGA ctggcgggactgggaagatg

Association between polymorphism in chicken GIypicans GPC3 and GPC4 and variation in commercially interesting traits.

Summary: Significant associations have been found between two polymorphisms in chicken GPC4 and commercially important traits such as weight gain (to market weight) and breast meat yield. In addition, at least one polymorphism in chicken

GPC3 appears to be a good candidate for a marker associated with variation in lean breast meat yield.

Introduction:

This work makes use of the polymorphisms described in the US patent application serial number 60/655099. Individual samples or high and low pools from a growth trial of a commercial broiler cross (i.e. broilers — chickens produced for meat - produced from parent stocks purchased from poultry breeding stock companies) were genotyped using the following markers Cl 150, Cl 151, Cl 152, Cl 153 and Cl 156 (all in GPC4) and Cl 158, Cl 160, Cl 161, Cl 163 and Cl 164 (in GPC3).

Results:

1) GPC4

Table 27: Cl 150 males (frequency of allele 1 = 0.47)

Although Sex by Genotype interaction was not significant for any of the traits, the marker effect was more significant in males than in females (not shown). Cl 150 had a significant effect (or an effect approaching significance) on growth (BW20, BW42, Gain and CW) in males (Table 1) but not in females. The favourable allele (allele 1) appears to be recessive for body weight and breast yield. Heavier (faster growing) birds tend to have a higher yield of breast meat. When expressed as % of BW, BRST appeared to show an over-dominance effect in males (although this did not reach statistical significance).

Cl 156: Significant dominance effects were found for all traits that are not expressed as percentages in both males and females, however the "12" genotype had the highest values in females while the "12" animals had the lowest values in males leading to significant Sex by Genotype interaction for Body Weight 42 days and GAIN. A significant additive effect was found for %BRST in males only.

Table 28; Cl 156 (frequency of allele 1 = 0.57)

2) GPC3

Screening for association between polymorphism in the chicken GPC3 locus and breast meat and fat content. Eight markers (SNPs) were tested in high and low pools based on trait scores. Three markers were found to be monomorphic. Figure 9 shows the difference in allele frequency (allele "1") between the High and the Low pools for the five polymorphic markers. The frequency difference for marker Cl 161 appears to be interesting for both traits with the results in opposite directions so that it appears this marker is associated with increased breast meat and reduced fat. The frequency difference is similar to that observed using Cl 150 in GPC4 (using pools). Effect of GPC3 and GPC4 genes on growth and meat yield in broilers from a second cross in a different farm. (Batches A, B, C and D)

Summary:

Based on the preliminary results from the initial study (see above), three SNPs were selected to be tested in a second dataset collected from four batches of broilers for association with growth and yield.

Batches A, B and C were based on the same cross of commercial broiler parents, whereas Batch D used the same male as Batches A-C crossed with a commercial female parent of a different genotype. Analysis and result evaluation was done separately for the two crosses.

Cl 150 had no significant effect in all tested traits in both crosses.

C1156

Effects observed with this marker were significant or approached statistical significance (p<0.05) for a number of traits. These were as follows:

Cross 1 (Batches A-C)

Additive effect of 0.055% on %wings (P=0.07J)

Additive effect (P=0.043) of 0.255% on %front_half, where the '22' genotype was associated with the highest values. The '22' animals had also the highest growth and %Breast, but results were not significant.

Cross 2

An additive effect of 1.05 g/d (P=0.093) with the '22' animals having the highest mean. C1161

Cross 1 a significant (P=0.001) additive effect of -0.85 g/d on growth with the ' 11 ' genotype having the highest mean.

The ' 11 ' genotype was also associated with lower %Front_Half (P=O.032), lower %Breast (not significant) and lower %wings (not significant).

This marker had no effect in Cross 2.

When compared to the initial results (effects on Gain and %Breast), Cl 156 showed similar effects and direction as in Cross 1 in this study.

Overall:

Cl 161 (GPC3) has a large effect on growth only in Cross 1 (additive effect of 0.85 g/d is equivalent to -0.85 generation of selection or ~1.7 generation between the ' 11 ' and '22' genotypes), and a moderate frequency of -0.6 in both crosses. Although not significant, Cl 161 had a negative trend with respect to meat yield.

Details:

(See also Table 30 for more details).

Batch: We collected four batches. In the current analysis we analyze separately batches (A. B and C) and Batch D.

Cross: Association analysis was done separately for the two cross types (see above).

Sex: We selected only Males. Sex was confirmed by macroscopic inspection of sex organs after slaughter. However there were few animals that had no sex organs after the evisceration - those 'unconfirmed males' were genotyped with sex-specific SNPs and all were confirmed to be Males (n = 100). Test Age: Animals were slaughter at a relatively late age (~8 wks) than accepted in the industry (~6 wks). Deboners: 4 to 5 deboners worked in each batch. The deboner technique has a strong effect on meat yield. Traits: Table 29.

Table 30. Batches

Feed Breeder Flock

Batch Date Farm Weight Conversion Flock age age Mortality Condemnation

11/1/2005 6.4629 2.0012 26 56 6.1726 0.1412

11/15/2005 6.9718 2.1084 44 56 7.8247 0.0953

11/16/2005 6.4353 2.1099 33 57 6.5012 0.4958

11/29/2005 7.357 2.1301 41 57 3.7436 0.2382

Table 31. Cross and number of animals tested by Batch.

Genes and SNPs: Table 32. SNPs within gene.

Table 33. Linkage disequilibrium between the three SNPs

Table 34. Number of genotyped animal for each SNP

* There are 26 animals that were not genotyped for any of the markers.

Table 35. counts of genotyped animals by Cross

Table 36. Polymorphism and recoding to 1/2.

— Batches A, B and C (Cross 1)~

Statistical Models:

Y = Batch <N> <Random>

Deboner (Batch) <N> <Random> Genotype <N>

Results:

Table 37. Legends

unites

' genotype (LSM11 )

(10) LSM(11 )-LSM(12)

(11) LSM(12)-LSM(22)

(12) LSM(11)-LSM(22)

(13) P value of the contrast '11 ' vs i 2'

(14) P value of the contrast '12' vs '22'

(15) P value of the contrast '11 ' vs '22'

(16) Number of '11' animals (N11) (17) N12

(18) N22

(19) Total number of animals

(20) Frequency of allele '1 '

Table 38

Table 39

Table 40

Table 41

Table 42 Results from previous trial.

* * We discovered that gender was not accurately recorded in this dataset. Batch D (Cross 2) -

Statistical Models:

Y = Deboner <N> <Random>

Genotype <N>

Table 43. Legends

unites

' genotype (LSMl 1)

(10) LSM(11)-LSM(12) (11) LSM(12)-LSM(22)

(12) LSM(11)-LSM(22)

(13) P value of the contrast ' 11' vs ' 12'

(14) P value of the contrast '12' vs '22'

(15) P value of the contrast '11 ' vs '22'

(16) Number of '11 ' animals (Nl 1)

(17) N12 (18) N22

(19) Total number of animals

(20) Frequency of allele '1'

Table 44. Detailed results

GIypicans GPC3 and GPC4 Sequences details

Sus scrofa

• GPC3

0 ESTs

The following ESTs (Expressed Sequences Tags) were extracted from the public databases: 40424230, 40428661, 46177097, 4043860, 46175690, 37984877, 34166466 and 34169307.

0 Contigs

>Contigl

GAGGTAGCTGCGAGGAAACTTTTGCAGCGGCTGGGTAGCGGCAGGTCTCCTGCTCCTCGG

GGCCACTGCCAGGCTTGCCGAGTCCTGGGACGGCTCTCGCCCCCGCCGCTACTCTCCCGC

GCTCTCCTCGCTCCCCGCGAAGCAGGATGGCAGGGACCGTGCGCACCGCGTGCTTGGTGG

TGGCGATGCTGCTTAGCTTGGACTGCCCCGGACAGGCGCAGCCCCCGCCGCCGCCGGACG

CCACCTGTCATCAGGTCCGCTCCTTCTTCCAGAGACTGCAGCCCGGACTCAAGTGGGTGC

CAGAAACCCCGGTGCCAGGATCAGATTTACAAGTATGTCTCCCCAAGGGCCCGACATGCT

GCTCAAGAAAGATGGAAGAAAAATACCAACTAACAGCGCGATTGAACATGGAACAACTGC

TTCAGTCTGCAΆGTATGGAGCTCAAGTTCTTAATTATTCAGAATGCTGCGGTTTTCCAAG

AGGCCTTTGAAATCGTTGTTCGCCATGCCAAGAACTACACCAACGCCATGTTCAAGAACA

ACTATCCGAGCCTGACCCCACAAGCTTTTGAGTTCGTGGGTGAATTTTTCACCGATGTGT

CTCTCTACATCCTGGGTTCGGACATCAACGTGGATGACATGGTCAATGAACTGTTTGACA

GCCTGTTCCCAGTCATCTATACCCAGCTGATGAACCCAGGCCTGCCCGAGTCGGCCTTGG

ACATCAATGAGTGCCTCCGAGGGGCGAGGCGTGACCTGAAAGTGTTTGGGAATTTCCCCA

AGCTTATTATGACCCAGGTTTCCAAGTCACTCCAAGTCACGAGAATCT

>Contig2

GCGGCTCGAGCAACACAACAGATCACCTGAAGTTCAGTAAGGACTGCGGCCGAATGCTTA

CCCGAATGTGGTATTGCTCGTACTGCCAGGGGCTGATGATGGTTAAGCCTTGTGGTGGCT

ACTGCAACGTGGTCATGCAAGGCTGTATGGCGGGCGTGGTGGAGATTGACAAGTACTGGA

GAGAATACATTCTGTCTCTGGAAGAACTGGTGAATGGCATGTACAGAATCTATGACATGG

AGAATGTACTGCTTGGGCTCTTTTCAACCATCCACGACTCCATCCAGTACGTCCAGAAGA

ATGGAGGAAAGCTGGCCACCACTATTGGCAAGTTATGCGCCCATTCTCAGCAACGTCAGT

ATAGATCTGCTTATTATCCTGAAGATCTGTTTATTGACAAGAAGGTATTAΆAAGTTGCTC

ATGTAGAACATGAAGAAACCTTATCCAGCCGGAGAAGGGAACTAATTCAGAAGTTGAAAT

CTTTCATCAGCTTCTACAGTGCTTTGCCTGGCTACATCTGCAGCCACAGCCCTGTGGCCG

AAAATGACACTCTCTGCTGGAATGGACAAGAACTCGTGGAGAGGTACAGCCAAAAGGCAG

CCAGGAATGGAATGAAAAACCAGTTCAACCTCCATGAGCTGAAAATGAAGGGCCCTGAGC

CAGTGGTCAGTCAAATTATTGACAAACTGAΆGCACATTAACCAGCTCCTGAGAACCATGT

CTGTACCAAAAGGCAGAGTGCCGGACAAΆAACCTCGACGAGGAAGGGCTGGAAAGCGGAG

ACTGCGGCGACGATGAAGATGAGTGCATCGGAGGCTCTGGGGATGGAATGATGAAAGTGA

AGAACCAGCTCCGCTTCCTGGCAGAACTGGCCTATGACCTGCACATGGACGATGCTCCTG

GAAGCAAGCAACATGTGAATCAGAAGGACAATGAGATAGTTGCCTCTCACAACCTTGGGA

ATGGCCATCCTCCCCTGAAGCTTCTCACCAGCTTGGCAATCTCTGTGCTGTGCTTTTTCC

TCCTGGTGCACTGACTGCCAAGCTCACAGGATATGTGCTGCCCTTCAGCACCCTGTGGTC

TCCCTCTATAAAGGGAACCACCTTTTTCTTTTTCCCTCTTTTTTTTATCTTCTATACCTC

CTTCAGCCATTAAGTAGAAAACTAACCCTGCGTTACGTTTTCGAAAATCACGTGGCATCT

CCATGAGGAGGGCAAATTTTAGTGGTAGTATAGATTGTCTTTTGCAATACAAAAAATCAA

ATTGTGCCAAATTATTTTCCTATGTTTGGCTGCTAGAACATGGTTGCCATGTCTTTCTCT

CTGTCTTTCCCTTTGCATGGATTTCTTTGAAAAAAAAATAATAAACACTCAAAAAAA

G

0 Primers

- Exon 1

EX1_F: TGGCGATGCTGCTTAGCTTG EXl R: GGGTTTCTGGCACCCACTTG - Exon 2

EX2_F: CCCGACATGCTGCTCAAGAA EX2_R: GGAAAACCGCAGCATTCTGAATAA

- Exon 3

EX3_F: GCGAGGCGTGACCTGAAAGT EX3_R: TGGCCAGCTTTCCTCCATTC

- Exon 4

EX4_F: TGGCCACCACTATTGGCAAGT

EX4JR: CATGAGCAACTTTTAATACCTTCTTGTC

- Exon 4-redesigned

EX4_F_redes: TGCGCCCATTCTCAGCAA EX4_R_redes: CATGAGCAACTTTTAATACCTTCTTGTCA

- Exon 4-redesigned2

EX4_F_redes2: TGGCCACCACTATTGGCAAG EX4_R_redes2: TCTTGTCAATAAACAGATCTTCAGGA

- Exon 5

EX5_F: GCCGGAGAAGGGAACTAATTCAGA EX5_R: GTGTCATTTTCGGCCACAGG

- Exon 5 -redesigned

EX5_F_redes: CAGCCGGAGAAGGGAACT EX5_R_redes: CTTGTCCATTCCAGCAGAGAGTGT

- Exon 6

EX6_F: AGCCAAAAGGCAGCCAGGAA

EX6_R: GTTTGTCAATAATTTGACTGACCACTG

- Exon 7

EX7_F: AGGCAGAGTGCCGGACAAAA EX7_R: TCATCATTCCATCCCCAGAGC

-Exon 8-3'UTR

EX8_utr_F: GACCTGCACATGGACGATGC EX8_utr_R: TCCATGCAAAGGGAAAGACAGA

0 Sequences

- Exon 1

>P00L2_C

MTCAYMTCGCTRTCTCACTCASACYCAATACGACACTCAGAATACTATTCCTTTCACTCG CACTTATTGCAAGCATACRTTAΆGTRATCTCTCTTRCCGACGGGACCACCTTATGTTATT YCAYCATGTCGCGSMYYAYATTAYGCGAGCGGAMWAACCGSMAAAGCTCCCKCGSCCGAC CCCTGWCCACCTGAGAMCGGMGMCCCAGCCTRGGAAACAGCTATGACCGGGTTTCTGGCA CCCACTTGAGTCCGGGCTGCAGTCTCTGGAAGAAGGAGCGGACCTGATGACAGGTGGCGT CCGGCGGCGGCGGGGGCTGCGCCYGTCCGGGGCAGTCCAAGCTAAGCAGCATCGCCACTG GCCGTCGT

>P00L3_C GGAAACAGCTATGACCGGGTTTCTGGCACCCACTTGAGTCCGGGCYGCAGTCTCTGGAAG AAGGAGCSGACCTGATGACAGGTGGCGTCCGGCGGCGGCGGGGGCYGCGCTGTCCGGGGC

AGTCCAAGCTAAGCAGCATCGCCACTGGCCGTCGT

>POOL15_C

AACAGCTATGACCGGGTTTCTGGCACCCACTTGAGTCCGGGCTGCAGTCTCTGGAAGAAG

GAGCGGACCTGATGACAGGTGGCGTCCGGCGGCGGCGGGGGCTGCGCCTGTCCGGGGCAG

TCCAAGCTAAGCAGCATCGCCACTGGCCGTCGTTTT

>POOL27_C

ACGAYGGCCAGTGGCGATGCTGCTTAGCTTGGACTGCCCCGGACAGGCGCAGCCCCCGCC

GCCGCCGGACGCCACCTGTCATCAGGTCCGCTCCTTCTTCCAGAGACTGCAGCCCGGACT

CAAGTGGGAGCCAGAAACCCGGTCA

>POOL62_F

CATGYCCCCTCCTTCTTCAGAGACTGCACCCCAGWCTCGKGKGGGTGCCAGAAACCCGGT

CATAGCTGTTTCCTGGGGGCGTTGTGTAGGGGTTGGGGWAGTRAAAWAAKGTGGWAAAGT

AGGGGAGAGACATCGACTTAATGTWTGATTKCGATWAGTG

>POOL62_R

GGAGCGGACCTGATGACAGGTGGCGTCCGGCGGCGGCGGGGGCCGCGTCTGTCCGGGGCA

GTCCAAGCTAAGCAGCATCGCCACTGGCCGTC

>POOL65_C

ACAGCTATGACCGGGTTTCTGGCACCCACTTGAGTCCGGGCTGCAGTCTCTGGAAGAAGG

AGCGGACCTGATGACAGGTGGCGTCCGGCGGCGGCGGGGGCTGCGCCTGTCCGGGGCAGT

CCAAGCTAAGCAGCATCGCCACTGGCARTWRTTW

- Exon 2

>POOL2_C

GAAWTWGCTATGACCGGAAAACCGCAGCATTCTGAATAATTAAGAACTTGAGCTCCATAC

TTGCAGACTGAAGCAGTTGTTCCATGTTCAATCGCGCTGTTAGTTGGTATTTTTCTTCCA

TCTTTCTTGAGCAGCATGTCGGGACTGGCARWAGTT

>POOL3_C

GAAACAGCTATGACCGGAAAACCGCAGCATTCTGAATAATTAAGAACTTGAGCTCCATAC

TTGCAGACTGAAGCAGTTGTTCCATGTTCAATCGCGCTGTTAGTTGGTATTTTTCTTCCA

TCTTTCTTGAGCAGCATGTCGGCACTGGCCGTARTT

>POOL15_C

GAAACAGCTATGACCGGAAAACCGCACCATTCTGAATAATTAAGAACTTGAGCTCCATAC

TTGCAGACTGAAGCAGTTGTTCCATGTTCAATCGCGCTGTTAGTTGGTATTTTTCTTCCA

TCTTTCTTGAGCAGCATGTCGGGACTGGCTGWAGTTTT

>POOL27_C

CAGCTATGACCGGAAAACCGCAGCATTCTGAATATTAAGAACTTGAGCTCCATACTTGCA

GACTGAAGCAGTTGTTCCATGTTCAATCGCGCTGTTAGTTGGTATTTTTCTTCCATCTTT

CTTGAGCAGCATGTCGGGACTGGCCRWCGTT

>POOL62__F

CTAACAGCGCGATTGAACATGGAACAACTGCTTCAGTCTGCAAGTATGGAGCTCAAGTTC

TTAATTATTCAGAATACTGCGGTTTTCCGGTCATAGCTGTTT

>POOL62_R

TGGAYGGAGTCGTKGCATGGTTGAAGCGAKCCCAWSCAGTACMTTCTCCAYGTCATAGAT KCTGTAYATGCCMTTCACCAGTKCTTCCAGAGACAGRATGTATTCTCTCCAGTACTTGTC AATCTCCACCACGCCCGCCATACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGG CTTAACCATCATCAGCCCCTGGCAGTACGAGCAATACCACΆTTCGGGTAAGCATTCGGCC GCAGTCCTTACTGAACTTCAGGTGATCTGTTGTGTTGATCACTTCGATTCCGAGATTCAG GGCCTGAAGGAAGATTCTCGTGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTT GGGGAAATTCCCAAACACTTTCAGGTCACGCCTCGCACTGGCCGTCGTTTTA

>POOL65_C

GCTATGACCGGAAAACCGCAGCATTCTGAATAATTAAGAACTTGAGCTCCATACTTGCAG ACTGAAGCAGTTGTTCCATGTTCAATCGCGCTGTTAGTTGTATTTTCTTCCATCTTTCTT GAGCAGCATGTCGGGACTGGCCRWCGTTTT

- Exon 3

>POOL2_C

AGGAAACAGGYTATGACCTGGCCAGCTTTCCTCCATTCTTCTGGACGTACTGGATGGAGT CGTGGATGGTTGAAAAGAGCCCAAGCAGTACATTCTCCATGTCATAGATTCTGTACATGC CATTCACCAGTTCTTCCAGAGACAGAATGTATTCTCTCCAGTACTTGTCAATCTCCACCA CGCCCGCCATACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGGCTTAACCATCA

TCAGCCCCTGGCAGTACGAGCAATACCACATTCGGGTAAGCATTCGGCCGCAGTCCTTAC

TGAACTTCAGGTGATCTGTTGTGTTGATCACTTC*R*)VΓTCCGAGATTCAGGGCCTGAAGGA

AGATTCTCGTGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTTGGGGAAATTCC

CAAACACTTTCAGGTCACGCCTCGCACTGGCCGTCRTTT

>POOL3_C

AGGAAACAGGCTATGACCTGGCCAGCTTTCCTCCATTCTTCTGGACGTACTGGATGGAGT

CGTGGATGGTTGAAAAGAGCCCAAGCAGTACATTCTCCATGTCATAGATTCTGTACATGC

CATTCACCAGTTCTTCCAGAGACAGAATGTATTCTCTCCAGTACTTGTCAATCTCCACCA

CGCCCGCCATACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGGCTTAACCATCA

TCAGCCCCTGGCAGTACGAGCAATACCACATTCGGGTAAGCATTCGGCCGCAGTCCTTAC

TGAACTTCAGGTGATCTGTTGTGTTGATCACTTQ*R*)YTTCCGAGATTCAGGGCCTGAAGGA

AGATTCTCGTGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTTGGGGAAATTCC

CAAACACTTTCAGGTCACGCCTCGCACTGGCCGTCGTTTTAA

>P00L15_C

AGGAAACAGCTWTGACCTGGCCAGCTTTCCTCCATTCTTCTGGACGTACTGGATGGAGTC

GTGGATGGTTGAAAAGAGCCCAAGCAGTACATTCTCCATGTCATAGATTCTGTACATGCC

ATTCACCAGTTCTTCCAGAGACAGAATGTATTCTCTCCAGTACTTGTCAATCTCCACCAC

GCCCGCCATACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGGCTTAACCATCAT

CAGCCCCTGGCAGTACGAGCAATACCACATTCGGGTAΆGCATTCGGCCGCAGTCCTTACT

GAACTTCAGGTGATCTGTTGTGTTGATCACTTC*R*ATTCCGAGATTCAGGGCCTGAAGGAA

GATTCTCGTGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTTGGGGAAATTCCC

AAACACTTTCAGGTCACGCCTCGCACTGGCCGTCGTTTTA

>POOL27 - reverse complementar

TGACCTGGCCAGCTTTCCTCCATTCTTCTGGACGTACTGGATGGAGT

CGTGGATGGTTGAAAAGAGCCCAAGCAGTACATTCTCCATGTCATAGATTCTGTACATGC

CATTCACCAGTTCTTCCAGAGACAGAATGTATTCTCTCCAGTACTTGTCAATCTCCACCA

CGCCCGCCATACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGGCTTAACCATCA

TCAGCCCCTGGCAGTACGAGCAATACCACATTCGGGTAAGCATTCGGCCGCAGTCCTTAC

TGAACTTCAGGTGATCTGTTGTGTTGATCACTTC!*R*ATTCCGAGATTCAGGGCCTGAAGGA

AGATTCTCGTGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTTGGGGAAATTCC

CAAACACTTTCAGGTCACGCCTCGCACTGSCCGTC

>POOL62_R_resq

CGRTKCCGRGATTSRASGCCWGMAAGAAGATKSTCRWKGCTYGGAKTGGCYTGTWWWCC

TSTTCCTTCAGTGCSCTGAWKCYCGKAATCSRATTGTTCMACACTTCAGAKYACTTGAAG

TTAAATWAGGGCTGGGGC

>POOL65_F

AGCCAGTGGTCAGTCAAATTATTRACAAACGGTCATAGCTRTTTC

>POOL65_R

ATTCTTCTGGACGTMTGGATGGAGTCGTGGATGGTTGAAAAGAGCCCAAGCAGTACATTC

TCCAYGTCATAGATTCTGTACAYGCCATTCACCAGTTCTTCCAGAGACAGAATGTATTCT

CTCCAGTACTTGTCAATCTCCACCACGCCCGCCATACAGCCTTGCATGACCACGTTGCAG

TAGCCACCACAAGGCTTAACCATCATCAGCCCCTGGCAGTACGAGCAATACCACATTCGG

GTAAGCATTCGGCCGCAGTCCTTACTGAACTTCAGGTGATCTGTTGTGTT,*R*ATCACTTCG

ATTCCGAGATTCAGGGCCTGAAGGAAGATTCTCGTGACTTGGAGTGACTTGGAAACCTGG

GTCATAATAAGCTTGGGGAAATTCCCAAACACTTTCAGGTCACGCCTCGCACTGGCCCGT

CGTTT

- Exon 6

>P00L2_F

GAAGGGCTTTGAGCCAGTGGTCAGTCATTTATTRACAAACGGTCATAGCTATTT

>POOL2_R

TGCATATTTGACTGACCACTGGCTCAGTGAGCTTTCATTTTSAGCWCATGAGGGTTGGTC

TCGTKTTTCATTCCATTCCTTACTGCSTATTGGCTACTCATCTGTGGTKTTGCGCATGTC

GGGACTGGCCRTCGTTTTAAAACTACCAGGAGATAAGAAAGCAAAGAAAAAGGGGGGAAA

GAAGAGAACGCAGCAAGAAGAAAGAGACATCGGCTACAAGGGAATTTTAGAGGAAGACAC

ACAGTTTTAAAAAAGGGGGGGGGGGGAGATAAAGCAAGAAAAAAAAACAGCGCCAAAACC

TCCGCCAGAACCAAAAGTCCAAAGGGCAACGACATAAAGCCCACCACCAACCAAAAGAAA

AAGCAAAAGAAAAAAAAAAAAAGAAGACAACGAAACCAACCCAAACCCTAACTCATCAAA

AAGAAGAAGAAAAACGAAACAAGAAGGGAACAGGCAATAAAAAAAAAGATAAAAAAGAAA

AAACACAAAAAAGTACCAAACACAAAAAAACATAAACAAAAAAACTGGCTCGCAGGAAGA CTAAGCCAGGAACTTCACGGGAACGGGCCAGGGGACAAAAGGGGCGTCAAAAAAACCCCC

CTAGACGCAGACTTGCTCACTAAAAAAAGGAGGGAGATCAAGCGGGGGGGAAACGGACAA

AGAACCGTAATGGCACCTTACAGGATAAAA

>P00L15_R

TTCATTTTCAGCTCATAAAGATTGAACWGGTTTTTCATTCCATTGCTGGCWGCCTTTTGG

CTACWGGCAGTAGTTWTGCAAAGACAGAATGTATTCTCTCCAGGACGAGACAATCTCCAC

CACGCCCGMCAGACAGCCATGCATGACCACGTTGCAGTAGCCACCACAARGCGTAACCAA

CATMAGCCCCTGGCAGGACRAGCAATACCACATACGGGAAARCATTCGGCCGCAGTCCTT

ACTGAACTAMAGGTGATCTGATGTGTTRATCACTTCAATTCCGAGATCAGGGCCTRAAGR

AAGATTCTCGAGACCTGGAGTGACTCGGAAACCGGGGGCACAATAAGCTAGGGGAAATTC

CCAAACACTTTCAGGGCACGCCTCRCACTGGCCG

>P00L15_F

CAAGGCGCCAGGAATGGAATGAAAAACAGTTTCTACCTCCATGAGCTGAAAATGAATGGC

TTTGAGCCAGTGGTCAGTCAAATTATTGACAAACGGTCATAGCTRTTTCCTCGACAGTTG

GGGGGTTTTGGGGAAAAATAAACCCTCCCTTCCCCGAGCCCCAAAACTCAATATAGTTAT

CTAGAACCCAAGGGTTGTAGAACGGCCCCGCACGACTTTTTAACACTTTTTTTTAAATTT

TCGCGCCCCCGCTTTTTAAACAACGGGGGGGGGGGGGAGCACAAAAAAAAACAGGCCAAA

CAACGGACCGAACAGGCACCACTAACAACTCCCCCAAAAACCAGCCTAACCACATAGACC

ACCGGACCCGCTCCAACAAACCCCGGCCCCCCCCACAAACCACGGCCGATAACCCCCGGC

AACCCCTGATCCGAAGAGAAACCCTGCCAGCACCGGCCAAGAGAGGAACGTAGCTCAACG

CGCCGCCAGAACACCACCCCCGATCAATGACGAAAAGCACGGCGCACCACCCACGCGCCA

CCGAACGAGAAACCCAGCAAAGTACAACAACAGCAATACAAAAAAGGGCAGGGCCACGGG

CGGAAAACGGGCAACAGAGGAAGGCCCGGAGAGAGGGCGGAGAACATGCCAGAATCAACT

AACCCACGGGTAGCCACACAACACAAGCATCG >POOL27_R_resq

GAACTGGTTTTTCAGTTCCATTCCTGGTCATGCCATTTTGGSTACTGGCYGTCGTT

>POOL27_F

CATTTTCAGCTCATAGAGATTGGACTGGTTTTTCATTCCATTCCTAACTACCTTTTAGCT

ACTAGCCGTCGTTTTGCAATGTATTCTCTCCAGTACTTGTCAATCTCCACCACGCCCGCC

ATACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGGCTTAACCATCATCAGCCCC

TGGCAGTACGAGCAATACCACATTCRGGTAAGCATTCGGCCGCAGTCCTTACTGAACTTC

AAGTGATCTGTTGTGTTGATCACTTCRATTCCGAGATTCAGGGCCTGAAGGAAGATTCTC

GTGACTTGRAGTGACTTGAAAACCTGGGTCAWAATAAGCTTGGGGAAATTCCCAAACACT

TTCAGGTCACGCCTCGCAC

>POOL62

TAWATAGCTATGACCGTTTGTYAATAATTAGACTGACCACTGGCTCAAAGCCCTTCATTT

TCAGCTCATGGAGRTTGRACTGGTTTTTCATTCCATTCCTAACTACCTTTTAGCTACTAG

CCGKCGTTWTGCAAAGAATGTATTCTCTCCAGGACATGGCAAGCTCCACCACGCCCGCCA

TACAGCCGTGSATGACCACGTTGCAGTAGMCACCACAAGGCTTAAAGATCATCAGCCCCT

GGCARTACGARCAATACCACAKTCGGGWAAGCATTCRGCCGMAGTCMTTACTGAACTTCA

GGTGATCTGTTGTGTTGATCACTTCGATTCCGAGATTMARGGCMTGAAGGAAGATTCTCG

TGACTWGGARTGACTTRGAAACATGGGTCATAATAAGCTTGGGGAAATTCCCAAACACTT

TCAGGTCACGCMTCGCAATGGCCGTCGTWWT

>POOL65_C

GAAAYAGCTATGACTGTTTGTYAATAATTTGACTGACCACTGGCTCAGRGCTKTTCATTT TCAGCTCATRRAGATTGGRCTGTTTTTCATTCCATTCCTAGCTGCCTTTTGGCTGCTAGC CGKCGTTWTGCAAACGAATGTATTCTCTCCAGTACTTGTCAATCTCCACCACGCCCGCCA TACAGCCTTGCATGACCACGTTGCAGTAGCCACCACAAGGCTTAACCATCATCAGCCCCT GGCAGTΆCGAGCAATACCACATTCGGGTAAGCATTCGGCCGCAGTCCTTACTGAACTTCA GGTGATCTGTTGTGTTGATCACTTCGATTCCGAGATTCAGGGCCTGAAGGAAGAWTCTCG TGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTTGGGGAAATTCCCAAACACTT TCAGGTCACGCCTCGCACTGGCCGTCGTTTTAA

- Exon 7

>POOL2_F

GACTGCGGCGACGGTGCGGTGCGTGTGTCGGGGGCTCTGGGGATGGACTGATGAGGTCAT

AGCTGTWTAACCTAACCCCGGCCCGCTGGTCCAACTGGCAAGCAAGGAGACAGAGGAGAA

GCTGTAAGTATCCTGAATTCAGTAAAAGCCGCCTTCCCCTGCGCGGTGGGGCTGAGGCAG

CCCCTGGGTTTCCGCAGTCTCTGGACTAAGGAGCAGTGGCCTCAGATGCAGAGGAGGCCC

CCACCTGTCCTGGCTTTTCTCTGACGCTGCGCTCACTCTCTCYTCARGGTGCACAAGGCC

CCATGGGACCCTCAGGACCAGCTGGRGG

>POOL2 R TTATYCATCCAGGCCCCCGAATCACTCATCTTCATCGTCGCCGCAGTCGCCGCTTTCGAG CCCCCCCTCGTCGAAGTTTTTGTCCCGCGCTCTGCCTGCTGGCAGAAGTTWTACGAACAA AGGCGAACTGCTAAACACCCAAGAAAGACAGAGAAGGCGCACACCCGCACACAAATACTA AAAATAGAACAACACCAAACAAACTACAAAΆATCTATAGCTACTTAAGTACACTTTATGT GAAAAACACATTTAAAAAΆAGATTATTTATTATTTATATTAAATAAAGTTATATAAAACA CAATTTGTGTTTATTTAACAΆTTAATGCCAGTAATAAAAATTTTATTCTAATTTATAΆTA AATAACTCAATTTCTAΆAAATCTAAACACATTTTATACTCATTCTTAAAAAAACGCACAA AATACATTTTTCATATTTAATAATTATATCATTATCCAGTGGGCCCCCTTTCCCTAATAA AΆATCGACTTTTTGGCAAATTTTATCCTCGGAGGATGTTTGTGAGAGGAGGAGGGAGTGC TGGGATGGAGACAAGAGGGGGAGGGGTGGGGTAGGGGGCGATACGGTGTTTTTGGTTGGA TGTACTGATGTGGTGGGTTTGGTGAGTGGGAGGGGCTAGTTGGAGTGTGGGGGGTTGGAG GGGGAGGGGGAGTTGTGGTGGGGGGGTGACGGCGGGGATGGGATGGTGTGGTGTGTTTTG TGTTTTTGTGGGATGAGTGGTGATGAAGTGGTGAGTATTTGTGTTGGGGGCAGGTGGCGG GCCAGAGGTGGGGGGAGGAGGGTGGAGAA

>POOL3_F

GCAAGCCGGCAAACCTCGACGAGGAGGTTCTCGCTCGCGGCGATTACGTCGACGGTGCCG ATGCGTGTATCGGGTCCTATAGGGATGGACTGATGAGGTCATAGCTGCTTAACCTAAGGG GAGCAAGGGAAAGCACCAAATTCAGAAAGCCGCCTTCCCTGGGGGTAAGCAGCCTGCTCC GCAGCTCTGGAAAAGGACAGGCTCAATCGGAGCCCCACCTGCCTGGATTTCCAGAGCGCA CCACTCTCACAAGGAAACAGAGCCCAAGGAACCTCAAGACAGGGGAGGGGGGGAATCGGA GGTAACAGGGCAGCAATGAGTCCCACTGGGAAGCTGGGAΆCATCCACACCCCCGGGAACG GGCACCACCAGGGCAACAGGGAAACGGAAAAGGGGGGGACAGGAAGAGACCCCCCAAACA ACAGGAGAATGGCCAAGGTATAAGAACCAAAGAAAAACAAAATGGACACCCCGGACAACT AAAAGGACAGTGGTTAATGGGGAGATAGCAGCCCCGAAGGACAAAGTAGCAACCGGGGCC GGCAAAAACCAΆGAAGGTGCGACAAGTACCCCAAGAGCAGACCCTTAAGGGAAAAAAACA GAAAAACCAAAAAGAAACCACACAACATGCGGAAAGTCGAAAATTACAACACTACCCAGA TTCACAACAACAGCAAACTAGAAAA

>POOL3_R

TCTTCATCGTCGCCGCAGTCGCCGGTTTCGAGCCCCTTCTTGTCGATTTTTTTCTCCTCC

GCTCTGCCTGCTAGCCRWCGT

>POOL15_R

TCTTCATCGTCGCCGCAGTCGCCGCTTTCGAGCCCTYCCTTGTMGAWTTTKTTCTCMGCC

GCTATGCGTGCTGGCCRWCGTTWT

>POOL15_F

ATCCGGCAAACCTCGACGAGGAGGTTCTCGTAGCGGCGACTGCGGCGACGATGAAGATGC GTGTATCGGGGCCTCTAGGGATGGAATGATGAGGTCATAGCTGTTTAACCAAGGAGAGGG TGGGGGGGCACCCCCCCACCCCCTCCCCACCCCTTCCCCCCACCGCGCGCCCAACCTGAG TTCACAGCTCTATACGCAACAGGAACCCCAAGCAGCAAAACCCCCGGGGCCGGCTACGCT TTTAAGAAAAATTTTTTTTTTTCAGTTTACAAGGCCGCCTTTTCTTTTTACGCTTGTGGG GGGGGGGGTCCTAAAAAAAAAACTATCTGTTTGCTTTGTTCCCCTCTTTTTTGTATTGTA AGGGAGTACGGGAATATGCGTTATTCTGACACTCTCCCCGGCGTCGAGCCGAACACTATC CTCCCCGATGCTTACCTTAGCTTTACCTGGGGGGGACTATTGATTATTCGTGAGGGCCTT GATATAACCTCCTTCGGCTCGCTAGTCCCGGACTATGCTTCGGGCGAGACTGGGTATCTT CTTTCTGATTATTTCCTATACGATTTCTCGCCCATCAGGGTCCTCAGTAGACGAAATATC GCTGAAATTTTCCTGGCTTTTGGCCACAACCCCACTGACAACTCGTGTCACCAGATCCCT GGTACCCCCAATCTTTTTTATTACTCTTTGTTGTCTGTGGAGGTTTCGCACAGGCCCCGC CCCGTCCTGCCGCTCTGCCGTTCTATAΆTAGTCTGAAATCTTCTATTACACAATACACAT CTTCGTCTTATATTGTCTGGTGTTTCGCTTTCTCTCATGTACCTTTTGCT

>POOL27_F

GCCGGCAAACCTCGACGGGCAGGTTCTCGCTCGCTGCGATTACGGCGACGGTGCCGGTGC

GTGTGTCGGGTCCTCTTGGGAYGGACTGATGAGGTCATAGCTCATAACCAGCCCCGCCCC

CCCCATCCAACCACCACAGCACACCGCCCGGGCCAGAGCGCACTTTCGTTAAGAACACCC

GCGCCAGACGCCGACCCCCCCCACCCCCTAGAACGGCCTACGACCAAACCAATTCTTTTT

AGCTGGTGCAGCCGCAGCTTCTCTTTAGAGCAGGTGGGGGGGGGTGCGCATTCAGGATCA

AATCCTACCTTCCCTGTTGATTCCCATCAACGAATTATATTATAGTGTCTCACAGCCTCC

CCTGGGCCCTCGACTCGCCTGTTACCCTCTATTCTTGCGATATCTCCGCGCCATTGACCC

TTTCATCTCCTCTCTTGGGGGCTGAATTACTATTAAATTATTTCTCTCCCCAAATTCTTA

TAGTACTAATCTGTCACACCTGTTTCCTCCGCTTTTCCCCACCTCGTGCCTTCACCATCT

CCCTGCCCGTTCCATGAGTTGGGTAGTGGCACCGCGACTACTGTCCTTCTTCTGCTTTCC

CTTTTCAAATACACTATCCCTCCCTCCCTCACCAATATCTCCCCCCTCTCTTCTGACTCT

CCGTTCACTTTCTTCTCATCTCTCGATCTCTCTTATATTCCTTCACCTACTCCGGATAAT CATCTCACCCTGCGGACTTTCTTGATTATCATATCTTGCTCCCGCGCCCTCCTTATATCA

ATTCTCTCACCCCTATCTCCGTCGTATTCCTGTTCATTATACGCCCTCGACTGCATAGTA

TTCTAAAATCTCTTACCTCTATTTTTCGTTATTTGCGGATCTTGTTGGCAAGGTTGCCGA

GCGGCCTCCTCCT

>POOL27_R

TTTTCATCCCAGAGCCCCGAATACCTCTCTTCATCGTCGCCGCAGTCGCGTTCGGCCCTC TCGTCGAATTTTTCGCCTCTGCTCTACGTCGTTCTGAACGGCCACCACCAAACAAAACCT CTAACACCCCACAACCAGTAAAAACGAAAGACCAGAAAACAAGCAGGAAGCTACTAAACA AAAAAAATAGACAAAΆAAAAAATAAAAACAATAΆAGAGAAAGACACACAAAAAAACAAAG AAAAAACCAACAGACACGAAACAACAAAAAAAGAAAACACACACAAAATTAAACAATACA AAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAGAAAACAAAAAAAACACCAAAAGAGAAA AACAACCCTGAAACAAAACAAAAAAAAAAAAAAAAAATTAAAAAAAATTAAAAAAAAAAA GAAAATTAAAATATTAAATAATAACAAAATATTAAAATTAAAAATATATAATAAAAAAAT

AATAAAAAAAAAAAAAAAATAAAAACTACAAAATTATTAATAATAGTAATATAACATAAA

TATACATCAAAAAAAAAATTAACAAAACTCAAAATAACTAAATAACTAACAATAACAATA

CTAATACAAAATACAAAAAAAACATAGAAAGTATACTGTATACTAACAAAAAACAGTAAA

TACTATAAAAGTATCATAAGCACTTCTATAACTAACAAAATACTCAACACACGCAAACAA

GGGCAACAAAAGAAAAGGCGCACCCTTTGTCCTCTAGTCACAGATACTTGAAATCAAGAA

TGAACGTTTAATAATAAGATAGCAAAA

>POOL62_F

GGTGCCGACGAATGCTAGGCGAGCCAGTTCTAGCTCGCTGCGATTGCGGCGACGGTGCCG GTGCGAGTATCGGRGCCTCTGAGGATGGACTGATGTGGTCATAGCTGTTTAACCAAGTTT GGTGGAAGCACATCCCTTCCCCAACTCTCGACGTGCGGGAACCCTCAAAAACCAACGCCC TTTTGGAGGAAAGAAGCATTCTGGGAAAACGAGAGGCCAACACCTTCCCCGAGCGAATTT TTTGGGCGAGGTTTTTTTTAAGTCTTTTTCAGGACTAGCTTTTTTTTGAATGTGGGGGGG GGGGGGAGTCATCACTTTTTTTTTCTCCCCTTGATCGTCCACTTACTACATCAACTCGCA CCTCACCTGAGTTTTACCTCCCTCTATATATACCGAGATGTCCCTTTTCGTGGTCACCAA AAATGTGATTGATAAΆCGTGCCCCCTAGGACCCCCTCCTCCTAACGAATATAAACTTACT CCATTCACATTATTCTCTCGCCTGGTAGAAACCTTGATAGTGAACTGGGCCGTATTTTAG TTCTGCTAGGGAGGCCTGCATTGTTACGGAATCGCGGGTTCTTTACAATTACTCCTACTG ATGTTTTGTATATCGTTGTATATAGTΆTTTTCTTCTTCTTTCTCTAATTTTTTCCTTTAC TATAATGTCCTCGTAATATTGCACTTTCCTTGTGCTTCCCCTTCGGAGTGGCGTTGGCGG GAGTCCCGTCGCTTTGTATATTGTCTATTATAGTAAGTTTCTATTATCCATTCAACTATT TATTGAGAAGCATCCTCGTTTTTATTTTCTATTTTTTCATCCCTTTTCAAC

>POOL62_R

CGCGCGCTGAAGCGCGGAGTCGCGCRTGTGTAGAKATAGATATCGATCAGGTCGACGCAC TCACGCGCGCCTCGTGTAATAGTGCWCCAGAGAGGCGGTCGCTCTTGATCGTCAGCGCGC TGACCCATGTGACGGTAGGAATATCGCGATAAGACGGCATTCTGTCACACTTAAGACGAT CTCTAACAMCTCAAGTTATACCTAGAGGAGATAGCACACGGAGGTCATAGCTCGACGTAG TSWCAGCGCTGCAGACTAGRCWCTCGGCGCGCTGGGCGTCGGAGAATATGATGTCTTCTA ACTTATATTCATTCCACGGCGCGACTGTACGCTAGTCTCTGTGTAGAGCGACACAGACGT TACTGCAAGATAGGCGGCCGCGCGCGGCCCCACACTGCTCCCTGAGGCCCTTTGTAAAAG ACCACAGTCCCTCTACAGCTCTCACTCATCCACTCTCTTCGCAATCACGAATTGCTCCAA ATACCGCATCAAGCACGCGTGCGCGTATCGGAGACGTCGAGCATGATCCTGGAGATTATG MGATGCGGTACACGGGACATGATAGTGTATTGWSTCTTCGCAGCACGCATTCTCAATACA TGTGGGTCGCAGATCGTACAATCACTATCTATACGCATAGCAGATATGACTAACCTCCAT ATCGGTCTCATCACCTCGAGTTGTGATATATCTCCATGCCAGTGCATACGTATAGTCACC GTTACTACAATCCTAAGTACGAGCTCTGCCGCAGTTGGCGMGAGTGCACTCCATCGATGT GGTACTAGCGTCCGCGCTGTCTACGACAGAGAGTCTTATAACGAKAGCACAGGATCCGTG ACGATGCGATCTCWATCTCTCTYATCAATGTTCTCTTCGCAACTTCACACGATCGTTCGC AGGGCGCACGCGCAATGAGTAGTCGCGCCTCCATGTGCGATGTATCTGCACTACGATACA GCAYACTGTACTCGCGCGCGCAGGAGGASTACCATCGYRCTAGYGCGCGTGCACTCTACM KMRTMTGACTGCAAGACATCAACGCCGCATTCASAAYWCACACACGAGCGTGACGAGATA GCCGACCTCAGTACTGTCCCACGCTCTGATCACTACAGAGTCACGCTCTCTCTCAGCTAC AGCAGTATCTTMCWGTGATCGCTCCTCTATAGTAGTAAGCTTGTGTACGTCTATCTGAΆT GACTACTGAGAAGGACGGTACGCGATACTGACTATGAATGCGACACGACTCGCGCCTCAT ATAACGTGAGTTGTCGATCTATCCCGCAGTCTCCAGMTGTATATCATCTCTCCCCCCTCC GAGAGAGCCTGCCACGCTYTCCGCTACACACGCTCGACGCCTGACGCACGTATCGCTCTC TCTAATCGCAGGTCAGAGGCGATGTCTCGGCTTCGATTGCGTCTSACATCACTACCGCGA CTGATAGTCTACACGTCTGCTCATACAACAGCCGCAYGTTATTCTACACCGATGTCTACW CTAGTGTCGCACAGCGCTTSGAGTGAGATGCGTGACTGCAGCGACGTCTACTGTCCGATG CGCTCACGTATCGCAGATCTACTCTCACAGCGTTCGTTACTATACGTCGTAGCGTAGTGA

CGACGTGAGACGAGCAGGTCACGATCAGTGGGAATTGTGACGTACACTCTCTAGCACGCC

GCKCGACTGCACGACTCACYACATGACGCACGATGAGCGGCATCATTAGCGACGCGAACT

AAGACGTCAGCGACGCCAGCCTGACGTATGTGACCACACACTCGTCTGCGCAGCACCGAT

AGACTCTATCTATTTGCGCCAGACTGCTATCGCTCACATCGCAGAGCCGGGCAGGCATAG

TGCCGTCTTACTGCCAGCATTCAGCCACAGACCGATATCTGCTCGCTATCATGATCTCAG

ATATATATCTRCTTACTCTGATACCCACTCCTCCCACACGCCGCGAGATACTACAGRTCA

CACGACACTGTACCAGACACGTCGWCCGCGCACCACCTACATCACTGGGTCGCCGCCCGC

TCGCGGACGAGCGGACGGCCACGACGTCCGGTACACGCGTCGTGATATTATGTGCACTTA

GGCGCTACAAGACTGCGTATCCCCGGATGACGCAGTAGATCAGGCGCTACCAGGTA

>POOL65_F

CAAGKCCGGCGATGGTCGGCGAGSCGGTTCTCGCTCSATGAGMTTAAAAYGACGGTGCCG RTGCGAGTGTCGGGTCCTCTGGTGGATAAACTGATCATGGTCATWGCTCTATTACCATTT TTAACTTTAAAAAAACGGCCACAAAACTCCCCCCCCCCCACTTACCCAACAACCACCAAC CCCGAAACCCCCCCCACCCACACCACACACACAAAACAACCAΆAACCACACCACCGCACA CCAAACACACACCCAACAAACACCCCACCACAAACTAΆAACCCATACTTCTATAACATTT AAAACCCACACTTTTATAAAAAAAAACTCTTGGGAGAAACAACAAAAACCCCTCCCCCCT GATCTGACCCGCTAGCGCATCCTACACACCCCTAACTCGCCCCCTGCCAATCCAAAACAC AAATAAAAACCAAGCGCAAAGACAAACCCCAAAACACGAAATAAACAAGACGTCAACCCC CTCCCCCCAGACACCCACAAAATAAACAGCCCTAACCCATAAAAACGCACGACAATCTCA ATACTTTAGTAATAGTAACACGATGATACCGATACGCACACCTAGCACACAGCACTCAGC AGGACCGATCCTGATCTGCACGCGCACACTGACCGACAAGACTCTATAGATGTGCCCACA TGCATAAAGCTCAACACGCCGACACACGAGAACAACAGCCTCAGCAACAAAACCTAACAT AGCAACGCTGAGCAGACACCTCACCATACTACTGACGTCGATGATATGTCTCACACGGCA GAAACAGAGAGCAACGCACCACACAAAACCCAACAACTCACACTGATGTACTCCGATCAA GACGCACCGCACGCATGATAGCCGAGCAAGCAGACACCGTAAAGAACTATCATACTATCA CTCATGCACATGCGTGAATGACAGGCAGAATACTACATATACATCAGAATAGCACATAAA AAGAAACTAGACTCGCACACACGATGCCTCTCACACGATATCAATCGCTCACGGCTGATG TCTAGGCGGTGCTGCACTGAGAGGGACACTCGGCGTAGCATAATACCGCGACAGGACGAA GACTAAGCTCTATACTCACATCCGCTACATAGCACACATCCACAGTCACCACTCGTGACT ACGACATCGACCTCAAGCGTAAGACGATCGTGAGACTCACACAGCAGACACCTCAGCGCC ACACGCTACGCTGACCTAAGCATCCACTTTAACAGACATAGCAT

>POOL65_R

GCCACCACAGGCGCACCCAGCGATCCATAACGCGTAGCACCGAACGCGCGCGGACAAGCA GACTACAGTCATAAGCCGCGYCTAGAGAGCACGTCACTRGGCAGCGCTCTCAAACCTTTG CACTATTCCTACTCGCAAATGGCCAGGGCTTGCCGCTYAGGTCGCACAGGGACTGTTATT YTATATAAAATTACCGCAΆGTGTGAGGGGACAAAGCCGCGGTCCCCCGAACAGTCTAGCC CCCACCGACAAACATMTGGATRTCAACATGCTACGAACGGCGCGTGCGTGCCGATTGGCG AGTACTGTAGCGCTGACACCAGTGCGACATAACTAYRCGTACGAACATACGCGGAGCGCC TAGGACACGCGCGTCCAATCCACACTCTAGYAGTCGWCATGCTAGTGTCCASCACACGAG CGCACTCAAGCTAGAAGCGGCAGAACTACTMGAAGGCGTCACGCTCAGCATCGATCGATC GTATGCGACGAAAGCACACGACGCGATGCAGTCCGTGGTACATCACCGAGACCCACGTAC GCACAGCGCAGGAATTGCACTATCCGCGCTAGCGAGACGAGCATGCACAGAKCTCGGTGT ATCCTCTCTATGCTCTCTAGTAGGCTATATAGCACTGCTGAGACCGCCACAGCCGTGGAC TCGAGACGTGACTGTACTTGTASTSACACATCCGCACTRCATGAGTATGAGAGATACGAT ACACGAGCACACGCGTTAGCCAGACTCACGCGCGTCACTCTCAGCACGAACCGAACTCGC GCRSATCGTGACGTCGGTTCGAGCTCGCAGCACAGTSGCGCTCGAGCGACGTCTAGAKGC TGTATAGCTCTATATATGAATATACGCTACTGCTRAKCGAGATCTATACATCGTCGGTGA TGGAGATGCGCAGACGTGCTGAGCGCCAGATATCAACTATGTRCAGCACACTGAAGCTAC GCTGCACTTTATATAYAYAGASACTAGTACTACTCTCGTGATGACTGTGTGTCACTATAC TCCACTCGACTGCGGTCGCAGTCACGTACACTGATGTCTCCGTCGCTCGTACGACTCWCC GCGAGTAGTCGTGGCGTACGACTGMTCGCAGCGCTCTCTGTATCTCTCYCCCCAGCTATG GTACATGACTCCTCATGACTCCGTCTCACTTGTGMMACTATTCATCTCTACTCGCTAGGG TGCCGCCTGTGTAGTCCTCGWATCTCTGCACTCTAGCATCTCACACGACGATATGAGCCR TGCGTCGACGTGTCTCACACTCAGACGACAGCGATCGATGCGTGGATCTATGATACGCGA CGTGAATCTCAGTGCTACGTACTTCAGTCTACACGCGGAGACCGCGCTACGTTCTCCGCA GCGTCKGAGCACGCGTCTCGCTGATGGCGCCGCGCCTGCGACGTCTCCGCGTGCGCACCA TCGASACGATTGGCGTCACTGTGTGGTGTACGMTGSAGACKATGTGTGTCGTCCACGACT GCGATCGMGMGGACGTCTCAGCTCGCCTGAGACGCACGGCTMCGCTATGWCAGATCGACT ATCYGWSWCGTCGCTAGACTCCCATACCGCGACATCCTGATCATCACTSATCGTACACGC TCACTCTACCGCGCCCTCTGCACGCACAYATRCGCCTCACGTACGMTGTACAAGCAGACG TCCACACGCATACATCACCGACTACAGTGCCTACCGGATKACRTCGCKCTAGCCATCCAG CATCGGACGGCAGCGAGGGATRTGGGAGATGAGCWGCACGCGTCCGTMGATTAGATCGTA ACGCTATGCCTGTCTCTGATACCGCATCTCGTGCTGCGCCACACTGCAGTAC

- Exon 8-3' UTR

>POOL2_R

AAAACATGGTAACCATGTGCTAGCAGCCAAACATAGGAAAATAATTTGGCACAATTTGAT

TTTTTTTTTTYYTTTGCAAARGGACAATCTTKSCYCCCMCWAAAWTTTMCCTCCCYCSMA

AAAAYCCWTTTKWTTTYCAAAMMCGYCMCSCRGGGTTAGYYYYCWAYTWAAGGGCGGGGG

RAAKTAAATARGGTAAAAAAAAAWWAAAAAAAAAAAAAAARGGGGGGCCCYTYCAAARRG

GRAAAMCCCSGGGSCYCTRAGRAMMATYCMGKSCCCCMCMAAAAAAWACCMCACCMMAAA

AMCCCCAAACTKGGGGGGGCCCTKGGGCCCCAAGGGSCCYYYCCCCRGGTKKGGGGGGGG

GSCCACCCCTTTKYCYTYYYCTTYCCCCGGCTGYTKTYYYCSGG

>POOL3__F

CGGAAGCAAGCAGCAGGCGAATGAGAAGGACAATGAGCTGGCCGCCTCCCACAACCTGGG

GAACGGCCACTCGGCGCCCAΆGGTCCTCCCCAGCTTTGCGGTCTCTGTGCTGTGCT^'*W*CTT

CTTGCTGGTGCACTGACTGCTCCTCAGTGCCCCGTGGTTCTCCCTCTCTGAAGGGCACCA

CCTTTCTTCTTCTTTTCTATTTTTTTTTTACCTTATTTACTYCCYCCMSCCATTAAGTAR

AARACTAACCCTGCGTGACGTTTTCRAAAATCAAATGGATYTTTGCGAGGAAGGTAAATT

TTAGKGGTAGCAWAAATTGTCCTTTTGSAAARAAAAAΆAAAAAAAAAAWKTTKCCCAAAT

WWTTTYYTTGWKKTKGSGTGCTAGCACAKGGTTMCMAKKTTTTTTYTGTYTKTYTTTCCT

TTT

>P00L3_R

TGGTAACCATGTGCTAGCAGCCAAACATAGGAAAATAATTTGGCACAATTTGATTTTTTT

TTTTTCTTTGCAAARGGACAATCTTGSCTMCCMCTAAAWTTTMCCTYCCYCCCAAAAATC

CATTTGATTTYCAAAAMCGCCMCCCAGGGTTAKTCYYCTAYTTAAGGGCGGGGGRAAKTA

AATARGGTAAAAAAAAAWWAAAAAAAAAAAAAAAGGGGGGGGCCCTYCAAARRGGGAAAM

CCCCGGGSCCCTGGGGAAMATYCCGGGCCCCMCCAAAAATTACCCCCCCMMAAAAMCCSC

AAAACGGGGGARRACCTKGGGCCCCAAGGGSCCTTYCCCCAGGTKKKGGGAGGSGSCCGC

CYMATTGCCCTTCCCATTCCCCGGCTGCTKGCTYCCGG

>POOL15_F

CGGAAGCAGCAGCAGGCGAATGAGAAGGAMATGAGCTGGCCGCCTCCCACAACCTGGGGA

ACGGCCACTCGGCGCCCAAGGTCCTCCCCAGCTTKGCGGTCTCTGTGCTGTGCT*W*CTTCT

TGCTGGTGCACTGACTGCTCCTCAGTGCCCCGTGGTTCTCCCTCTCTGAAGGGCACCACC

TTTCTTCTTCTTTTCTATTTTTTTTTTACCTTATTTACTYCCYCCASCCATTAAGTARAA

RACTAACCCTGCGTGACGTTTYCRAAAATCAAATGGATCTTTGCGAGGAAGGTAAWTTTT

AGKGGTAGCATARΆTTGTCCTTTTGCAAARAAAAAAAAAΆAAMAAATTKGGSCMAΆWWWW

TTTYCCAAGGTKGGSSGSATAAMAMWKGKTAMCMAKTTTTTYTTKYCCCSGTTTYCCYTT

GGSGGGGGGG

>POOL15_R

GGTAACCATGTGCTAGCASCCAAACATAGGAAAATAATTTGGCMCAATTTGATTTTTTTT

TTYYYTTKSMAAARGGCAAAYYAWGSCYCCCMCAAAAWTTTCCYTYCCCCCMAAAAAYCC

WTTTAWTTTYCAAAAMCYCCCCCCGGGKTWACCCYYCCMCTAAGGGSGGGGRGRRRGWAA

WWRGGGWAAAAAAAAAWTAAAAAAAAAAAAAAAGGGGGGSCCCCTYMAAARRGGRAAAMC

CCSGGGGCCYGGGGGGMMWYCCTKSSCCCCCCMAAAAARWACCCCCCCCAAAAMMCGSMA

AACGGGGGGGRGMCYTGGGSCCCAARGGGSCCKTYCCCCGGGGGGGGGGGGCSGCCMSRC

YMTTKGCCYTYYCCTTYCCCSGSSGGTKKSYYYCG

>POOL27_R

CATGGTAACCATGTGCTAGCAGCCAAACATAGGAAAATAATTTGGCACAATTTGATTTTT

TTTTTTTCTTKGCAAARGGACAATCTWKSCTMCCMCTAAAWTTWMCYTYCCCCSCAAAAA

YCCWTTTRATTTYCRAAAMCGYCCCSCAGGGTTAGYYYYCWACTWAAGGGCKGGGGRAAK

TAAWWARGGTAAAAAAAAAWTARAAAAAAAAAAAAAGGGGGGGSCCYTYCAAARRGGRAA

AMCCCCGGGSCCYKRARRACCATYCTKKSCCCCCCCAAAAATWACCMCMSCCCAAAAMCC

CCAAASYGGGGGRGGMCYYGGGGCSCCAAGGGSCCTTYCCCCSGGTKKKGGRRGGSSSCM

SSCCYWTKKCCCTYCYCTTYSCCCGSCKGCTKGCTYCSGG

>POOL27_R_resq

CATGGTAACCATGTGCTAGCAGCCAAACATAGGAAAATAATTTGGCACAATTTGATTTTT

TTTTTTTCTTKGCAAARGGACAATCTWKSCTMCCMCTAAAWTTWMCYTYCCCCSCAAAAA

YCCWTTTRATTTYCRAAAMCGYCCCSCAGGGTTAGYYYYCWACTWAAGGGCKGGGGRAAK

TAAWWARGGTAAAAAAAAAWTARAAAAAAAAAAAAAGGGGGGGSCCYTYCAAARRGGRAA

AMCCCCGGGSCCYKRARRACCATYCTKKSCCCCCCCAAAAATWACCMCMSCCCAAAAMCC CCAAASYGGGGGRGGMCYYGGGGCSCCAAGGGSCCTTYCCCCSGGTKKKGGRRGGSSSCM

SSCCYWTKKCCCTYCYCTTYSCCCGSCKGCTKGCTYCSGG

>POOL62_R

TGGTTACCATGTGCTAGCAGCCAAACATAGGAAAATAATTTGGCACAATTTGATTTTTTT

TTTTYYYTKSCAAARGGACAWYCTWKSCTCCMMCWAAAWTTWMCYTYCCCCCCAAAAAYC

MWTTGRWTTTYCAAAAMCTYMMCCCAGGGTWAKYYTYCWMYTWAAGGGCGGGGGRAAKTA

AWWARGGTAAAAAAAAATWAAAAAAAAAAAAAARGGGGGGGCCCYYYMAAARRGGRAAAM

MCSSGGGGMACGRRGRRCMATYCTKGCCCCCCCMAAAAAAARCCCMCCCCAAAAMCCCCC

AAMSGGGGGGGGGCCTTKGGSSSCMRKKGGSCCTYYCCCMRGGGGGGGGGGGGGSSCCSC

YYTTTKKCCYTYYCAWTYSCCGG

>POOL62_R_resq

ATGCATRASGACMAWRCAGTMTGGACARYCTTYYASRMMCTGKKKRACKKYYAYTYKGCC

CYCAAAGTCCTCCCCAGCTTTGCGGTCTCTGTGYTGYGYTMCYTCTTGCTGGCGCWYTGA

YTKYTCMWMASTKCCCCGTGGKTCTCCYTYYCTGWAGGKGCCGCSTTTMATCTTCTTTGC

TATTTTTTTTTTCCMWWATTWACTMCGGGGTYKCYCMWYAAAAAAAWAAAMCCAGGKKGC

MTTTTWCAAAAAYCAATKG

>POOL62_F_resq

CTCCCGGAAGCAGCAGCAGGCGAATGAGAAGGACAATGAGCTGGCCGCCTCCCACAACCT

GGGGAACGGCCACTCGGCGCCCAAGGTCCTCCCCAGCTTKGCGGTCTCTGTGCTGTGCT*W*

CTTYTTGCTGGTGCACTGACTGCTCCTCAGTGCCCCGTGGTTCTCCCTCTCTGAAGGGCA

CCACCTTTCTTCTTCTTTTCTATTTTTTTTTTMCCTAWTTWMYTYCYYCCASCCWTWAAK

WARAAAACTAMCCCKGSKTGASKTTTYCAAAAWYMAAAGGRAYYTTTSCAAGRAAGGAAA

ATTTWAKKGKWASMAWAAWTKGYCYTTTTGMAARRAAAAAAAAAAAWCAAATTKKGCCMA

AWTWWTTYYCCAWTKTKGGSCGSCAAMCMMTGGKTAMCCAGGTTTTTYCGGCCKTYYTTY

CCYTT

>POOL65_C

AAAGGGRAAAGCCGAACARAAAAACATGGTAMCATGTGCTAGCAGCCAAACATAGGAAAA

TAATTTGGCACAATTTGATTTTTTTTTTTTYTTTGCAAARGGACMATYTWTGCTACCMCT

AAAATTTMCCTTCCTCSCAAARATCCATTTGATTTTCGAAAACGTCMCSCAGGGTTAGTY

TTYTACTTAATGGCKGGRGGAAGTAAATAAGGTAAAAAAAAAATAGAAAAGAAGAAGAAA

GGTGGTGCCCTTCAGAGAGGGAGAACCACGGGGCACTGAGGAGCAGTCAGTGCACCAGCA

AGAAG*WΛAGCACAGCACAGAGACCGCAAAGCTGGGGAGGACCTTGGGCGCCGAGTGGCCGT

TCCCCAGGTTGTGGGAGGCGGCCAGCTCATTGTCCTTCTCATTCCCCGGCGGCTKGCTYC

SGG

0 Validated SNPs

>Exon 3

TATGACCTGGCCAGCTTTCCTCCATTCTTCTGGACGTACTGGATGGAGTCGTGGATGGTTGAAAAGAGC

CCAAGCAGTACATTCTCCATGTCATAGATTCTGTACATGCCATTCACCAGTTCTTCCAGAGACAGAATG

TATTCTCTCCAGTACTTGTCAATCTCCACCACGCCCGCCATACAGCCTTGCATGACCACGTTGCAGTAG

CCACCACAAGGCTTAACCATCATCAGCCCCTGGCAGTACGAGCAATACCACATTCGGGTAAGCATTCGG

CCGCAGTCCTTACTGAACTTCAGGTGATCTGTTGTGTTGATCACTTC*^*ATTCCGAGATTCAGGGCCT

GAAGGAAGATTCTCGTGACTTGGAGTGACTTGGAAACCTGGGTCATAATAAGCTTGGGGAAATTCCCAA

ACACTTTCAGGTCACGCCTCGCACTGGCCGTC

>Exon 8-3'UTR

GGAAGTAAATAAGGTAAAAAAAAAATAGAAAAGAAGAAGAAAGGTGGTGCCCTTCAGAGAGGGAGAACC

ACGGGGCACTGAGGAGCAGTCAGTGCACCAGCAAGAAG*W*AGCACAGCACAGAGACCGCAAAGCTGGG

GAGGACCTTGGGCGCCGAGTGGCCGTTCCCCAGGTTGTGGGAGGCGGCCAGCTCATTGTCCTTCTCATT

CCCCGGCGGCT

• GPC4

0 ESTs [public]

The following ESTs were extracted from the public databases: 40477470, 40802561, 40803618, 46179606, 46179967, 40798192, 41139802 and 15036802.

0 Contigs

>Contigl

GAGGCTGTCTGGCCAACCAAGGGGATCTGGATTTCGAGTGGAACAATTTCATAGATGCTA

TGCTGATGGTGGCCGAGAGGCTGGAGGGTCCTTTCAACATTGAGTCGGTCATGGATCCCA TCGACGTGAAGATTTCTGATGCCATCATGAACATGCAGGAGAACAGCGTACAGGTGTCTC

AAAAGGTGTTCCAGGGCTGTGGCCCCCCCAAACCCCTGCCGGCCGGCCGCCTCTCCCGCT

CCATCTCCGAAGGTGCCTTCAGCGCCCGCTTCAGGCCATTTCACCCCGAGGAGCGCCCGA

CCACGGCAGCCGGCACCAGTTTGGACCGACTGGTTACTGATGTCAAGGAGAAGCTGAAAC

AGGCCAAGAAGTTCTGGTCCTCTCTTCCCAGCAACGTCTGCAATGACGAGAGGATGGCTG

CAGGCAATGGCAATGAGGACGACTGCTGGAATGGAAAAGGCAAAAGCAGGTACCTGTTTG

CAGTGACAGGAAATGGATTAGCCAACCAGGGCAACAATCCAGAGGTCCAGGTCGACACCA

GCAAACCAGACATACTGATCCTTCGTCAAATCATGGCTCTTCGAGTTATGACCAGCAAAA

TGAAGAATGCTTACAATGGGAACGACGTGGACTTCTTTGATATCAGTGATGAAAGCAGTG

GAGAAGGAAGTGGGAGTGGCTGTGAGTATCAGCAGTGCCCTTCGGAGTTTGAGTATAATG

CAACTGAGCACTCTGGGAAGAATGCCCATGATAAAGCCAGCAGCGCTGGGGCCCCTGCCA

GGGCCCAGCCCTACCTCCTCACTGCCTTTGGCATCTTGTCCCTGGTCATGCCGAGAGAGT

GGAGATAATTCGCAACCTTAGAGAAAAAGTGTTCATCATCAAAAAGCTAACAAGGCGCCG

GTTATCACTTTTATACCATCCTAGTGACTTTGCTTTTTAAATGAATGGACAACGATGTAC

AGTTTTTACTACATGGCCACTGGTTTAAAAGATGCTGACTTTGTTGTTGTTGTTGTTGTT

TTTTTTCCATTCAGTTTTGCGGGCAAAGGGGACTTGGAACTTAAGGTCAGTCCTGCCCCC

CTCCGAATCATGTTAAAAGTGGCTAACGGTGTAGGTACAGAACTCTAGTTAGTTGTGCAT

TTGTGATTTTTATCACTCTGTTTGTTTGTTTGTTTTTTCTCTTTTTGTTTGTGGGTTTTT

TCCCTTCGATACGATCTCACCTTGTTTCTTACAAGAAAACCAGGGTCCTTTCTTGGCATG

TAACATGTACGTATTTCTGGAATATTAAATAGCTGTACAGAAGCAGGCTTTATTTATCAC

GTTATCTCATTAAAAGAAAAAGCCCAACAAGCAGTAACATTGCCATCTATCCCTGTTGCT

TTAGCTGCCTTCTCTGGAGAGAAGAGGAGGCGGTTTTGGGTTTTTTTGTTATTTTTATGA

AGCTAGGACAGAATGTGAAGGCACAAGGAGGCTGTGGACCCACTTGTCGGGTTGCACTCG

AGCATCCATACAAAACAAAGGCTTTGCTTCATTGAACGCTGGTGACCGGA

>Contig2

TTTTTAGGACATGCCTATTTTATTAACACCATGCTTTAATAAATAACTGGCTACTTCTAA TAAAATTAAGCCTCTGTTTACAACAGCCCCCGATATTCCATTTTGTCCATTCTGCAGAAT TTGGTGTCAAAAGTTGAGTGAΆGTGTAGACCCTGAGCTATCAAGTAATCATGTTTCAΆTA TAAAAATAGAGGATTACTCTTACACCCGAAGATTGAACACTAACACAAACAACCGCTCTG TGGCTTTCAGCCTCCTGTGGAACTGGCTGGGACCGTGATGGCCATCTCAAGCTCGAAGAG ACTTGCAGCATTTGATGCTTTCTGCAGACTGCTGGGAACTCCTTTTACAGGGATGGGCCA CACTCTCCTAGTTTTATGAGCTGGCATTTGGTAGGAAGGATACAGGGAATGGGCTAGTCC TAAGGGGGGGACCGAGGCAGTGGCTGCTCTTTGGCCTGGGACAAGAGACAATGCCTTTGT CAGTTCACCCACGTGGCTAAGGCTCCCCACTAAGATGCTGTCTTCTCAGCGCTACCTACT CCCCCGTCTCGCCCATCCCCCACCCCCCAAAATAAAAGCAACAACACATGAAAAAACCTG GACACTCA

0 Primers

- Exon 3

EX3_F: TGCAGAGAAATCCCTGAATGA EX3_R: AGGCGAGCCCAGAAGTCA

- Exons 5-6

EX5-6_F: TGCTGATGGTGGCCGAGA EX5-6_R: CGGAGATGGAGCGGGAGA

- Exon 7

EX7_F: GGAGAAGCTGAAACAGGCCAAG EX7_R: CCAGCAGTCGTCCTCATTGC

- Exons 8 & 9-3'UTR

EX8_EX9_utr_F: GGTACCTGTTTGCAGTGACAGGA EX8_EX9_utr_R: CCTTAAGTTCCAAGTCCCCTTTGC

- Exons 8 & 9-3'UTR_redesigned EX8_EX9_utr_F: GGTACCTGTTTGCAGTGACAGGA EX8_EX9_utr_R: CCGTTAGCCACTTTTAACATGATTCG

-Exon 9-3'UTR: EX9_utr_F: TGACTTTGCTTTTTAAATGAATGGA EX9_utr_R: GTCCACAGCCTCCTTGTGC

- 3'UTR: utr_F: GGCACAAGGAGGCTGTGG utr_R: TGTCTTCTCAGCGCTACCTACTCC

-3'UTR2: utr2_F: GGAGTAGGTAGCGCTGAGAAGACA Utr2_R: TGCCTATTTTATTAACACCATGCTT

0 Sequences

- Exons 5-6

>P00L2_R

GCCGGCRGGGTTTGGGGGGGCCACAGCCCTGGAACMCTGCGTCGGAAWAGCGCRGTCACC

CGYGGGAAGGGACAGGGCAGGCTGGAGGGCCCCCCCGCYCCGCSACACCTCTCCGGTACC

SGGCCTCACGTACCTTTTGAGACACCTGTACSCTGTTCTCCTGCATGTTCATGATGGCAT

CARAAAYCTTCACGTCGATGGGATCCATGACAGACTCAATGTTRAAAGGACCCTCCAGCC

TCTSGGCCACCATCAGCACTGGCCGTCGT

>POOL3_R

GGAGAGGCGGCCAGCCRGCATCAGTTTGGGGGGGCCACAGCCCTGGTGCAYCTGCGKCSG AMTMWSCGCGGTCACCCGTGGGAAGGGACAGGGCARGCTGGAGGGCCCCCCCGCCCCGCC ACACCTCTCCGGTACCCGGCCTCACGTACYTTTTGARACACCTGTACSCTGTTCYCCTGC ATGTTCATGATGGCATCARAAATCTTCACGTCGATGGGATCCATGACAGΆCTCAATGTTG AAAGGACCCTCCAGCCTCTCGGCCACCATCAGCACTGGCCGTCGT

>POOL15_C

AAMAGCTATGACCGGAGATGGAGCGGGAGAGGCGGCCAGCCGGCAGGGGTTTGGGKGGGC

CACAGCCCTGGAACACCTGCGTCGGAATAAGCGCGGTCACCCGTGGGAAGGGACAGGGCA

GGCTGGAGGGCCCCCCCGCCCCGCCACACCTCTCCGGTACCCGGCCTCACGTACCTTTTG

AGACACCTGTACGCTGTTCTCCTGCATGTTCATGATGGCATCAGAAATCTTCACGTCGAT

GGGATCCATGACAGACTCAATGTTGAAAGGACCCTCCAGCCTCTCGGCCACCATCAGCAC

KGGCCGTC

>POOL27_R_rsq

KGCCRGCCGGYTGGGGGCTTGRGKSTGTCAYGGCATCCGATCGACSTGAAKATTTCTGAT

GCCRTCATGAACTTGCAAGAGACCCSCYTMCAGGKGTCTCACAAGGTACSTGMGGCCSGG

TMCCGGAAAGGTTTGSCGGGGCKTTTTAAACCCCTGTACSCYGTTCTCCTGCATGTTCAT

GATGGCATC

>POOL27_C

ACAGMTATGACCGGAGATGGAGCGGGAGAGGCGGCCAGCCGGCAGGGGTTTGGGGGGGCC

ACAGCCCTGGAACACCTGCGTCGGAATAAGCGCGGTCACCCGTGGGAAGGGACAGGGCAG

GCTGGAGGGCCCCCCCGCCCCGCCACACCTCTCCGGTACCCGGCCTCACGTACCTTTTGA

GACACCTGTACGCTGTTCTCCTGCATGTTCATGATGGCATCAGAAATCTTCACGTCGATG

GGATCCATGACAGACTCAATGTTGAAAGGACCCTCCAGCCTCTCGGCCACCATCAGCACT

GGCCGTCGT

>POOL62_C

AACAGCTATGACCGGAGATGGAGCGGGAGAGGCGGCCAGCCGGCAGGGGTTTGGGGGGGC

CACAGCCCTGGAACACCTGCGTCGGAAAAGCGCGGTCACCCGTGGGAAGGGACAGGGCAG

GCTGGAGGGCCCCCCCGCCCCGCCACACCTCTCCGGTACCCGGCCTCACGTACCTTTTGA

GACACCTGTACGCTGTTCTCCTGCATGTTCATGATGGCATCAGAAATCTTCACGTCGATG

GGATCCATGACAGACTCAATGTTGAAAGGACCCTCCAGCCTCTCGGCCACCATCAGCACT

GGCCGTCGT

>POOL65_C

AMAGATATGACCGGAGATGGAGCGGGAGAGGCGGCCAGCCGGCAGGGGTTTGGGGGGGCC

ACAGCCCTGGAACACCTGCGTCGGAATAAGCGCGGTCACCCGTGGGAAGGGACAGGGCAG

GCTGGAGGGCCCCCCCGCCCCGCCACACCTCTCCGGTACCCGGCCTCACGTACCTTTTGA

GACACCTGTACGCTGTTCTCCTGCATGTTCATGATGGCATCAGAAATCTTCACGTCGATG

GGATCCATGACAGACTCAATGTTGAAAGGACCCTCCAGCCTCTCGGCCACCATCAGCACT

GGCCGTC

- Exon 7 >P00L2_C

ATGACCAGCAGTCGTCCTCATTGCCATTGCCTGCRGCCWTCCTCTCGTCATTGCAGACGT

TGCTGGGAAGAGAGGACCAGAACTTCKTGGCCTGTTTCAGCTTCTCCGCTAGCCGTCGT

>P00L3_F

CAGCAACGTCTGCAATGACGAGAGGATGGCTGCAGGCAATGGCAATGAGGACGACTGCTG

GTCATAG

>P00L15_C

TCATTGCCATTGCCTGCAGCSATCCTCTCGTCATTGCAGACGTTGCTCGGAAGAGAGGGC CΆGAACTTCKTGGCCTGTTTCAGCTTCTGGAGTGGCCGTCGTTTT

>POOL27_C

YAGCTATGACCAGCAGTCGTCCTCATTGCCATTGCCTGCAGCCATCCTCTCGTCATTGCA

GACGTTGCTGGAAGAGAGGACCAGAACTTCGTGGSCTGTTTCAGCTTCTCCGCTGGCCGT

CGTTTT

>POOL62_C

TTWAAYAGCTATGACCAGCAGTCGTCCTCATTGCCATTGCCTGCAGCCATCCTCTCGTCA

TTGCAGACGTTGCTGGAAGAGAGGACCAGAACTTCTTGGCCTGTTTCAGCTCCTAAACTG

GCCGTMGT

>POOL65_R_rsq

GCCATTGCCTGCGGCCMTCYYCTCCTCATTGCAKMCGTTGCTGGGAAGAGAGGACCRSAA

CTTCKTGGCCTGTTTCAGCTTCTCCRCTGGCCGTCGTTTT

>POOL65_F_rsq

TWGCTATGACCAGCAGTCGTCCTCATTGCCATTGCCTGCAGCCATCCTCTCGTCATTGCA GACGTTGCTCGGAΆGAGAGGACCAGAACTCCTTGGCCTGTTTCAGCTTCTAAACTAGCMG

TCGTT

- 3'UTR

>POOL2_C

CCCCAAATTAAAGGCAACAACAMATGAAAAAACCTGGACACTCRGATCTCCCCCAGTGGG

CTGCCTGACTGCTGGAGTGGTACCCAGCTGGGAACCACCACAAGTCCTTTGCTGTTCTGA

TGAGAGGAGGSCGGGCAAGAAAAAGACGCAAGTAΆGACΆCCTTAGAATGGCCGTTTTAAA

AAAACCAACCCAGCTGGGATCCAAACCAAATGCAAATCCGTATGCACGGACAAAGAGTTT

GCTTGAGTGTTGACAGCGACTCCCTGAGCCCAGGCAGTACCCCAATTCTAGCAGGGCCCC

CAGACAGCTCCCTCTCTAGGACCCGACTTGAAAACTGGAACATGTGGTACCCAATGAGAT

CGTTGGTCACTTAACTTCTGCCATCGGGATATCAGTTCCAAGAGGGCCAAGTGTGGAAAC

AAAGACCAGCACACAGCGGCTTGCTAATTTGCCTGGATTTAAAACCAAAACCAAAAGCAC

AACAACAACCCCCACCCCCAAACTACGTGTGAACATGTGGCGACTGGGAGATTCTGAATG

CCTTCTTTCTTTCTAGGAAGAATGCATCAAGAATCTCCTGGATGGGTTACACTTTGAGTA

CAAGTGGCTTTCAGAA^Y*-TGGACCGCTCTGTGATCTTAGTTCATGCAATTCGACTCAACCA

AAACGGGTAGTCTGCRGTCTCTTCCGGTCACCAGCGTTCAATGAWGCAAAGCCTTTGTTT

YGTATGGATGCTCGAGTGCAACCCGAAAAGTGGGTCACAGCTTCCTTGTGCAATTGGCC

>POOL3_C

GTCTTTCAGGCTACCTACTCCCCGTCTCGCCCATCCCCCACCCCCCAAAATAAAAGCAAC

AACACATGAAAAAACCTGGACACTCAGATCTCCCCCAGTGGGCTGCCTGACTGCTGGAGT

GGTACCCAGCTGGGAACCACCACAAGTCCTTTGCTGTTCTGATGAGAGGAGGCCGGGCAA

GAAAAAGACGCAAGTAAGACACCTTAGAATGGCCGTTTTAAAAAAACCAACCCAGCTGGG

ATCCAAACCAAATGCAAATCCGTATGCACGGACAAAGAGTTTGCTTGAGTGTTGACAGCG

ACTCCCTGAGCCCAGGCAGTACCCCAATTCTAGCAGGGCCCCCAGACAGCTCCCTCTCTA

GGACCCGACTTGAAAACTGGAACATGTGGTACCCAATGAGATCGTTGGTCACTTAACTTC

TGCCATCGGGATATCAGTTCCAAGAGGGCCAAGTGTGGAAACAAAGACCAGCACACAGCG

GCTTGCTAATTTGCCTGGATTTAAAACCAAAACCAAAAGCACAACAACAACCCCCACCCC

CAAACTACGTGTGAACATGTGGCGACTGGGAGATTCTGAATGCCTTCTTTCTTTCTAGGA

AGAATGCATCAAGAATCTCCTGGATGGGTTACACTTTGAGTACAAGTGGCTTTCAGAA*Y*T

GGACCGCTCTGTGATCTTAGTTCATGCAATTCGACTCAACCAAAACGGGTAGTCTGSAGT

CTCTTCCGGTCACCAGCGTTCAATGAAGCAAAGCCTTTGTTTTGTATGGATGCTCGAGTG

CAACCCGACAAGTGGGTCACAGCYTCCTGTKCACTTGGCCGTC

>POOL15_C

CCCCGTCTCGCCCATCCCCCACCCCCCAAAATAAAAGCAACAACACATGAAAAAACCTGG ACACTCAGATCTCCCCCAGTGGGCTGCCTGACTGCTGGAGTGGTACCCAGCTGGGAACCA CCACAΆGTCCTTTGCTGTTCTGATGAGAGGAGGCCGGGCAAGAAAAAGACGCAAGTAAGA CACCTTAGAATGGCCGTTTTAAAAAAACCAACCCAGCTGGGATCCAAACCAAATGCAAAT CCGTATGCACGGACAAAGAGTTTGCTTGAGTGTTGACAGCGACTCCCTGAGCCCAGGCAG

TACCCCAATTYTAGCAGGGSCCCCAGACAGCTCCCTCTCTAGGACCCGACTTGAAAACTG

GAACATGTGGTACCCAATGAGATCGTTGGTCACTTAACTTCTGCCATCGGGATATCAGTT

CCAAGAGGGCCAAGTGTGGAΆACAAAGACCAGCACACAGCGGCTTGCTAATTTGCCTGGA

TTTAAAACCAAAACCAAAAGCACAACAACAACCCCCACCCCCAAACTACGTGTGAACATG

TGGCGACTGGGAGATTCTGAATGCCTTCTTTCTTTCTAGGAAGAATGCATCAAGAATCTC

CTGGATGGGTTACACTTTGAGTACAAGTGGCTTTCAGAA*Ϊ*ΙTGGACCGCTCTGTGATCTTA

GTTCATGCAATTCGACTCAACCAAAACGGGTAGTCTGCAGTCTCTTCCGGTCACCAGCGT

TCAATGAAGCAAAGCCTTTGTTTTGTATGGATGCTCGAGTGCAΆCCCGACAAGTGGGTCA

AAGCCTCCTTGTGCACTGGGCSGTC

>POOL27_F

CGACTGAGTACAACACACAGTAGCAGACATCTRTAAATTCGATATGCTAATTACATAACA

TCTTAATCAAACTCTGCTGTCGTCTGGCTGAGTATKATGTATATCTAGAGATAGGTGGCG

TTTTCCGTCTAATCAATATATATATCATCTTCTCACCTCACGACACATCTAGGCGCGGGG

CGATATCAATCAGYACTTTGCTCCWCATCCACAACACAATAGYGTTATACTTAACAGCTT

GTCACATACAGCAAGAACAGAAAAATAAGAGATGCTGTAGAGAATACACAGCGACCGCCC

CCACTCACCCCCCGCGCCTCTAGTTAATAAAGACAGGTGWATACATCACCGAAACCAAAA

TGTCCTCCCCCCAAGCACACCAATCATTAAATTTCCTCTCACTTAGTATCATGCACTTCG

ATACAAAAAAAAAACCCTATATAAGGCCGCATAMGCTATCCTCTACCTTCTTTATACGCG

AGGAAACTTACCGTGACAAGAAATAATACAATAAACCCAACTGACATTATACTATAACCT

TCTTGCGACATGCGAGTCTACACCAGACACGGTTCACATCCAGCTTGCCGTCCTCTAATA

CAAAGAGATACTGACCTTCTATATTTGTCTTGATTGTGTTACTATACTCATATATATACA

AGATGATACTCTACTGAGCTACCCATATCCGTCTCCAAATATACCTATACTCATACMSTT

AACCGCTATCATMTCTCTCGTCTTCTTAGTAGTACTATCTTCTACCGCTAACATGCTCGT

GCWTATCASACGAGCGCACGACATGTTCRTAATATGATACGGMGACTTGACTCTCAAYTG

GATCCATGTCGCTATGTCTCTTTCTCTCTCTTTTCCTTCTCTCTCTCCTCTGACACACAT

CGCTAGCAACTCGCTATCTCTATGWCTCATGGACTTAGAACAGATRCTCACCGCACCATA

TTCTCAACGCGTCACACAATAGGATCATATCTTAAGAGACACTATAACACCWATGTGACA CGTCTATGCATATCTCACGACTGCATATATCACTATATACATACACACWCTACTCTAGAG AACACATCGCTAGAGCTGCCATCCTCAAACGAGATAGGAGAGRCGTTCTCCGAGTCCAAC CGTCAGAATCGTCTTATAATGATATCTCAATCAGCTCAACAGCAGCTATACACCATCGTC GTCATAAGCCTACCTTCCATCTACGACATCCCACCACGATACTCTACTCAAAAGTCATTG GTTTACWAYATAATTCTGTGTCTCTTTCCTAΆGAGATATCTATAGACRCATCTRTACTGA GAATCTTACAGTGCTATCTTCTACACAAWGTCTGCCTACGCACGAGAACACAGCTSAGGT ATCACAATACGGTWTATGCTACATAAGAAKCACTGGACGTAYACCCGCCACGACATTGRC TACACAATCAACATTTACGCCATGTTACAYACCGC

>POOL27__R

CAAATAACTGGAGATATCAGCTTGGCGCTCATGACGCTCAGTTGACCACACTGTCGCGGA

GTCATCTGTGTCCTATTTATGACCTATTMCATMTCTCTGATRTTGACCTTGTCGTGAGTA

CTTGAGACGATGTACATAGAACATCCAAACACCTATGTACCWCCACCATATGCATCTTTT

CGCATACCGCGTAGGTTCATTGAGACGCGCCTATACGCGCACGATGTGCATACTCGCGTA

AGCATCGTCGTGCGCCGCCGGGCGCTGATGACGCAAGCTSGCACGARTGAMCACCGACGA

ATTATATGAAAATTAGACGCTCCGATCTAGCTTATCGTGCTATGTATGTCGCGAGCGATG

AGCAGACTCTACACATAMTCTTACTCTTCTGTACGACGTTCCGTCTACTACTCCATTCAT

ATAACAATTAGAGATATATATGTMKGTAATAGAGCAGATAAAGTCACGTGACAGACTTTT

GTCGCTCTGCGTTGTTCAATCCGACCACCAGATATCGATACGATTCTAGGCTGAGTATGT CTTGATATAAATGAATATAGATTATGTTCGATGCTACCAGCTATCTTCTCTGTGTATCWA TTACACATGTGACGTGCAAATGTCTCACGCTCTCCTACACATCKATATGACTCGCAYAYA CACACATGCACAGTATATAGATATGTGAATGATCGTAGTGTATAGTACATAGAGAGCAAT AGACAAGAAGTCGCGCACAGCACTACGCCTCGCGGCACCGTATTGWSTCGTATGAGCAAC GTACGAYATCTTCATGATAGCATAAGAGGTGTGCGTWGTACTATAGACGAGCACGAGCGC ACCACACGAGTACACAGAGAGTCCCAGTCAAGATGACTTGCCTGAGCGCTGCTGAGTGCA GCGCGAGCGGGACGCGCGGCGAGCAGTGCGCGGCGCTGTGTGAGGCGTACGCGGAATCGG CGTTAGTCGTCTAGCGTATATAGATTGTACTTACTAATACGTCATGTCCGTCTTCTCTTC ACTCGAATGACGCGMGTCTGTCGTACCAAYACAATAGTCAYACTAAGATGCCKCTCTGTT AACTACCGCGTGCTCTACGAGCTACGACGATGAATAACAAGAGGCGGTCCAGCGGCGCAC TACTGATCACACTGTCGGGTGACATGTATCATAGATTTCCTATACCTCTCAGAAAGGAGA TGATKGGTATTAGGTGTCAGAGASACGAGAGCTGACATAGCATCACGCTACACCAGCACA GCATGCGTCCATACACACTCGCTMTATCTATCAGAGCCTCATAGTCAACCGCACGACCTC GGTGCATTKCCGCAGACGTGACCTGCCGTCGTCGACGATMGTSAAAAGTAAGTATCGTCG CRTGTATAACGAGATGCACTATGTGTACGCGCCTGTGCTACTTAATCACAΆTTTATCGTA CGACTGTAGTGAGTTGTGATTGTGTCTACATGACACTACAGAASTTATATAGACTACAGA

TTGTTAGTAAGCTCGTGTGCGTACTCATGGTGCTTATACTCAATCATAGTAGTATGGATG

TAGTGCTACTTATCGAGCTGTCTAGCAGYCTCTGACTCGCTCTCCAAGATCGCAGTTCCG

CTCTCWCTGAGACACTCMTCTMTCTCTATAGTGTCTACGACWGAGTATCTATGATATTCT

ACCTAGGCGGTACTTCACACATAAGTGACGCTTGGCATCCTGACTCTTTGCATTACGATC

TAATGCGATGACTACCTCGCAAWTCGCGAAGAGGATTGGCAGTGATGCGCAGGTGTGAAT

AGAATGATCTTTGTAGTCTCTACTGTACTGTGATCACTCGCGTCGCTCTCGCTTGCTGGT

GGCGSTGGTGAGAATTGGAGTGGCGCGTCAGCGCTGTGGATTAACATMTCATGAAGACTC

TCCAWTGATTGATAAGACTATATACGTACAGGATGGCTAGTCTGCTTATACCCACTACGT

AGATATAGAGACTGACTCCTATCCATAGAGTTCTC

>POOL27_R_rsq

CATMCCTGTTTCCWAAGGCTTTGCTTCATTGAACSCTGGTGACCGGAAGAGACTGCAGAC

TACCCGTTTTGGKTGRGTCGRRTTSCMTGAACTAARATCACASARCGGYCYWGTTCTGAA

AGMCRCTTGTACTCAARGTGTAACCCATMCASGAGATTCTTGATKCATTMTTCCTAKWWA

GAAAGAASGMAYYCAGAATCTCCCARWCSCMACATGTTCACACGTAGTTTGGGGGWGGGG

GTGGTTGAGTGTTGACAGCGACYCCCTGAGCCCAGGSAGTACCCCAWTTYTAGCAGGGCC

CCCAGACAGCTCCCTCTCTAGGACCCGACTTGAAAACTGGAACATGTGGTACCCAATGAG

ATCGTTGGTCACTTAACTTCTGCCATCGGGATATCAGTTCCWAGAGGGCCAAGTGTGKTA

ACAAAGACCAGCACACAGCGGCTTGCTAATTTGCCTGAATTTAAACCCAAAACCAAAAGC

A

>POOL62_F

GCTGTGGMCCACTTGTCGGGTTGCACTCGAGCATCCATACAAAACAAAGGCTTTGCTTCA

TTGAACGCTGGTGACCGGAAGAGACTGCAGACTACCCGTTTTGGTTGAGTCGAATTGCAT

GAACTAAGATCACAGAGCGGTCCA*Y*TTCTGAAAGCCACTTGTACTCAAAGTGTAACCCAT

CCAGGAGATTCTTGATGCATTCTYCCTAGAAAGAAAGAAGGCATTCAGAATCTCCCAGTC

GCCACATGTTCACACGTAGTTTGGGGGTGGGGGTTGTTGTTGTGCTTTTGGTTTTGGTTT

TAAATCCAGGCAAATTAGCAAGCCGCTGTGTGCTGGTCTTTGTTTCCACACTTGGCCCTC

TTGGAACTGATATCCCGATGGCAGAAGTTAAGTGACCAACGATCTCATTGGGTACCACAT GTTCCAGTTTTCAAGTCGGGTCCTAGAGAGGGAGCTGTCTGGGGGCCCTGCTAGAATTGG GGTACTGCCTGGGCTCAGGGAGTCGCTGTCAACACTCAAGCAAACTCTTTGTCCGTGYRT ACGGATTTGCATTTGGTTTGGATCCCAGCTGGGTTGGTTTTTTTAAAACGGCCATTCTAΆ

GGTGTCTTACTTGCGTCTTTTTCTTGCCCGGCCTCCTCTCATCAGAACAGCAAAGGACTT

GTGGTGGTTCCCAGCTGGGTACCACTCCAGCAGTCAGGCAGCCCACTGGGGGAGATCTGA

GTGTCCAGGTTTTTCCAAGGGTKKTGCCTTTAATTTGGGGGGTGGGGAATGGCCAAACGG

GAATWGTAACCTTAAAACCGGCCACCGGT

>POOL62_R

CCGTCTCGCCCATCCCCCACCCCCCAAAATAAAAGCAACAACACATGAAAAAAMCYKGAM

AAYYCGATYYYCCCCMGTKGGGTGGCYKRMTGGTKGAGTGGKWMCCCGSYKGGRAMCMMC

MCCARKYCYTTKSYKTTTTGRWKAAARGRRGSCSGGSMARRAAAAAAMSCCAGTWARRMM

MCYTWAAAWKGSCCTTTTWAAAAAAMCMAMCCCGCYKGGRWYCMAAMCMAAWKSMAAWYC

CTWTGCCCGGRMMAAAAATTTKGTTGAATKKTTAMMGCCACTYCCYKRACCCMRGSMAKW

MCCCMAWTYYWGCMRGGSCCCCMAAMMGCYYCCTYYYYTRGRMCCCACTTKRAAACTKGR

AMMWKKKGKWMCCMAWGAAAWYCTTKGKYCAYTWACTTTTKSCMWYSGGRWTAYCGTTYC

MAAARGGSCMAGTKKTGAAACCAARRMCCASMCCCARSSGSYTKCYTAWTTKSCYGGRTT

TWAAAMCMAAAMCMAAAACMCCACCACCACCCCCMMCCCCMAA

>POOL65_C

AYMSGTCCTAGCTGTTKCYTTGCTTCATTGAACGCTGGTGACCGGAAGAGACTGCAGACT ACCCGTTTTGGTTGAGTCGAATTGCATGAACTAAGATCACAGAGCGGTCCΆ*X*|TTCTGAAA GCCACTTGTACTCMAAGTGTAACCCATCCAGGAGATTCTTGATGCATTCTTCCTAGAAAG AAAGAAGGCATTCAGAATCTCCCAGTCGCCACATGTTCACACGTAGTTTGGGGGTGGGGG

TTGTTGTTGTGCTTTTGGTTTTGGTTTTAAATCCAGGCAAATTAGCAAGCCGCTGTGTGC TGGTCTTTGTTTCCACACTTGGCCCTCTTGGAACTGATATCCCGATGGCAGAAGTTAAGT GACCAACGATCTCATTGGGTACCACATGTTCCAGTTTTCAAGTCGGGTCCTAGAGAGGGA GCTGTCTGGGGGCCCTGCTAGAATTGGGGTACTGCCTGGGCTCAGGGAGTCGCTGTCAAC ACTCAAGCAAACTCTTTGTCCGTGCATACGGATTTGCATTTGGTTTGGATCCCAGCTGGG TTGGTTTTTTTAAAACGGCCATTCTAAGGTGTCTTACTTGCGTCTTTTTCTTGCCCGGCC TCCTCTCATCAGAACAGCAAAGGACTTGTGGTGGTTCCCAGCTGGGTACCACTCCAGCAG TCAGGCAGCCCACTGGGGGAGATCTGAGTGTCCAGGTTTTTTCATGTGGAAAACGACGGC CAGTGGCACAAGGAKG

- 3'UTR2 >POOL2_C

AGGAAACAGCTATGACCTGCCTATTTTATTAACACCATGCTTTAATAAATAACTGGCTAC

TTCTAATAAAATTAAGCCTCTGTTTACAACAGCCCCCGATATTCCATTTTGTCCATTCTG

CAGAATTTGGTGTCAAAAGTTGAGTGAAGTGTAGACCCTGAGCTATCAAGTAATCATGTT

TCAATATAAAAATAGAGGATTACTCTTACACCCGAAGATTGAACACTAACACAAACAACC

GCTCTGTGGCTTTCAGCCTCCTGTGGAACTGGCTGGGACCGTGATGGCCATCTCAAGCTC

GAAGAGACTTGCAGCATTTGATGCTTTCTGCAGACTGCTGGGAACTCCTTTTACAGGGAT

GGGCCACACTCTCCTAGTTTTATGAGCTGGCATTTGGTAGGAAGGATACAGGGAATGGGC

TAGTCCTAAGGGGGGGACCGAGGCAGTGGCTGCTCTTTGGCCTGKGACAAGAGAYAATGC

MTTTGTCCCTTCACCCAC

>POOL3_C

TGGGTGAACTGACAAAGGCATTGTCTCTTGTCCCAGGCCAAAGAGCARCCACTGCCTCGG

TCCCCCCCTTAGGACTAGCCCATTCCCTGTATCCTTCCTACCAAATGCCAGCTCATAAAA

CTAGGAGAGTGTGGCCCATCCCTGTAAAAGGAGTTCCCAGCAGTCTGCAGAAAGCATCAA

ATGCTGCAAGTCTCTTCGAGCTTGAGATGGCCATCACGGTCCCAGCCAGTTCCACAGGAG

GCTGAAAGCCACAGAGCGGTTGTTTGTGTTAGTGTTCAATCTTCGGGTGTAAGAGTAATC

CTCTATTTTTATATTGAAACATGATTACTTGATAGCTCAGGGTCTACACTTCACTCAACT

TTTGACACCAAATTCTGCAGAATGGACAAAATGGAATATCGGGGGCTGTTGTAAACAGAG

GCTTAATTTTATTAGAAGTAGCCAGTTATTTATTAAAGCATGGTGTTAATAAAATAGGCA

GGTCATAGCTGTTTCCT

>POOL15_C

GAAACAGCTATGACCTGCCTATTTTATTAACACCATGCTTTAATAAATAACTGGCTACTT

CTAATAAAATTAAGCCTCTGTTTACAACAGCCCCCGATATTCCATTTTGTCCATTCTGCA

GAATTTGGTGTMAAAAGTTGAGTGAAGTGTAGACCCTGAGCTAYCAAGTAATCATGTTTC

AATATAAAAATAGAGGATTAYTCTTACACCCGARGATTGAACACTAACACAAACAACCGC

TCTGTGGCTTTCAGCCTCCTGTGGAACTGGCTGGGACCGTGATGGCCATCTCAAGCTCGA

AGAGACTTGCAGCATTTGATGCTTTCTGCAGACTGCTGGGAACTCCTTTTACAGGGATGG

GCCACACTCTCCTAGTTTTATGAGCTGGCATTTGGTAGGAAGGATACAGGGAATGGGCTA

GTCCTAAGGGGGGGACCGAGKCAGTGGCTGCTCTTTGGCCTGGGACAAGAGACAATGCCT

TTGTCAGTTCACCCACGTGGCTAAGGCTCCCCACTAAGATGCTGTCTTCTCAGCGCTACC

TACTCCACTRGCCCGTCGTTTTA

>POOL27_C

AGGAAACAGCTATGACCTGCCTATTTTATTAACACCATGCTTTAATAAATAACTGGCTAC

TTCTAATAAAATTAAGCCTCTGTTTACAACAGCCCCCGATATTCCATTTTGTCCATTCTG

CAGAATTTGGTGTMAAAAGTTGAGTGAAGTGTAGACCCTGAGCTATCAAGTAATCATGTT

TCAATATAAAAATAGAGGATTACTCTTACACCCGAAGATTGAACACTAACACAAACAACC

GCTCTGTGGCTTTCAGCCTCCTGTGGAACTGGCTGGGACCGTGATGGCCATCTCAAGCTC

GAAGAGACTTGCAGCATTTGATGCTTTCTGCAGACTGCTGGGAACTCCTTTTACAGGGAT

GGGCCACACTCTCCTAGTTTTATGAGCTGGCATTTGGTAGGAAGGATACAGGGAATGGGC

TAGTCCTAAGGGGGGGACCGAGGCAGTGGCTGCTCTTTGGCCTGGGACAAGAGACAATGC

CTTTGTCAGTTCACCCACGTGGCTAAGGCTCCCCACTAAGATGCTGTCTTCTCAGCGCTA

CCTACTCCACTGGCCGTCGTTTT

>POOL62_F

CTTWKCCACGTGGGTGAACTGACGAAGGGATTGTCTCTTGTCCCAAGCCMAAGAGCAGCC

ACTGCCTCGGTCCCCCCCTTAGGACCAAGCCMWTYCCYKKWWYCYTYCYWMCMAAWGGCC

RGCTMWWAAAATARGGAARGGKKGSCCAAYCCYGTWAAARGGAKTYCCMRSCATTCTGAA

AAARSMWYMAAWKSCGGMARKYYYYTYCAASYTKRAAWKGSCMTTMMSGKCCCCGCCMGT

TYCCAMAGRRGSYGAAARSCMCCAARCCGTTKKTTKGTTTAAKKKTYMATCCTCCGGKKK

TAAAATWAWTCTYTWWTTTTWTTTTKGAACCTGATTWCCTKRTTGCYCAAGGTYCTAMCY

TYAATYAAMYTTTGAMCACCAATTCCGSCGGAKKGRCCAAATKGGATTTCCGGGGCCGGT

GGAAACCGAAGCCTAAATTTAATAAAAGKWRCCCGGTAATTAATAAAGCMAGGTGGTAAT

TAAATTGGCCGG

>POOL62_R

TGGCTACTTCTAATAAAATTAAGCCTCTGTTTACAACAGCCCCCGATATTCCATTTTGTC CATTCTGCAGAATTTGGTGTCAAAAGTTGAGTGAAGTGTAGACCCTGAGCTATCAAGTAA TCATGTTTCAATATAAAAATAGAGGATTACTCTTACACCCGAAGATTGAACACTAACACA AACAACCGCTCTGTGGCTTTCAGCCTCCTGTGGAACTGGCTGGGACCGTGATGGCCATCT CAAGCTCGAAGAGACTTGCAGCATTTGATGCTTTCTGCAGACTGCTGGGAACTCCTTTTA CAGGGATGGGCCACACTCTCCTAGTTTTATGAGCTGGCATTTGGTAGGAΆGGATACAGGG AATGGGCTAGKYCYWARGGGGGGRMCCAAGCARKKGSYKCYYYTTKGSCYGGGRMMAAAA AMMAWKSCYTTKKYCATTYAACCAACTKGGTWAAGGCCCCCMMYWAAAWKGTKKYYTYCT ARSSSTWMCTTMYTCAAYTGCCCTYCTTTT >POOL65_C

TCKTAGTGGGGCAGCCTTAGCCACGTGGGTGAACTGACAAAGGCATTGTCWCTTGTCCCA GGCCAAAGAGCAGCCACTGCCTCGGTCCCCCCCTTAGGACTAGCCCATTCCCTGTATCCT TCCTACCAAATGCCAGCTCATAAAACTAGGAGAGTGTGGCCCATCCCTGTAAAAGGAGTT CCCAGCAGTCTGCAGAAAGCATCAAATGCTGCAAGTCTCTTCGAGSTTGAGATGGCCATC ACGGTCCCAGCCAGTTCCACAGGAGGSTKAAAGCCACAGAGCGGTTGTTTGTGTTAGTGT TCAATCTTCGGGTGTAAGAGTAATCCTCTATTTTTATATTGAAACATGATTACTTGATAG CTCΆGGGTCTACACTTCACTCAACTTTKGACΆCCAAATTCTGCAGAATGGACAAAATGGA ATATCGGGGGCTGTTGTAAACAGAGGCTTAATTTTATTAGAAGTAGCCAGTTATTTATTA AAGCATGGTGTTAATAAAATAGGCAGGTCATAGCTGTTTCC

0 Validated SNPs >3^λUTR - sequence reversed complemented

TTGCTTCATTGAACGCTGGTGACCGGAAGAGACTGCAGACTACCCGTTTTGGTTGAG TCGAATTGCATGAACTAAGATCACAGAGCGGTCCA'*R*|TTCTGAAAGCCACTTGTAC TCMAAGTGTAACCCATCCAGGAGATTCTTGATGCATTCTYCCTAGAAAGAAAGAAGG CATTCAGAATCTCCCAGTCGCCACATGTTCACACGTAGTTTGGGGGTGGGGGTTGTT GTTGTGCTTTTGGTTTTGGTTTTAAATCCAGGCAAATTAGCAAGCCGCTGTGTGCTG GTCTTTGTTTCCACACTTGGCCCTCTTGGAACTGATATCCCGATGGCAGAAGTTAAG TGACCAACGATCTCATTGGGTACCACATGTTCCAGTTTTCAAGTCGGGTCCTAGAGA GGGAGCTGTCTGGGGGCCCTGCTAGAATTGGGGTACTGCCTGGGCTCAGGGAGTCGC TGTCAACACTCAAGCAAACTCTTTGTCCGTGYRTACGGATTTGCATTTGGTTTGGAT CCCAGCTGGGTTGGTTTTTTTAAAACGGCCATTCTAAGGTGTCTTACTTGCGTCTTT TTCTTGCCCGGCCTCCTCTCATCAGAACAGCAAAGGACTTGTGGTGGTTCCCAGCTG GGTACCACTCCAGCAGTCAGGCAGCCCACTGGGGGAGATCTGAGTGTCCAGGTTTTT TCATGTGGAAAACGACGGCCAGTGGCACAAGGA

Bos taurus

• GPC3

0 ESTs[public]

The following sequences were extracted from the public databases: Sl1837014, S1181108 , S11867676, S11898677 , S11900122, S11926992, S11934918, Sl1956084 , S12047690, S12047772 , S12048249, S13574299, S13575420, S13575588 , S17484871, S17485763 , S19024800, S19680007, S19682734, S19682786, S19682917, S19685763, S19685906, S19687763, S19689375 and S19689390.

0 Contig

>Contigl

TGAGTGGGGCGATGGAGAGAAGGACTATTTGAACACCTAAGAGTAAGAATAGGAAGACAG ACATGTGACTGTCTGCTGAGAGGGGAGCCTGTGAGAAGAGGATGGCAGACTGATACATCA GAGAGATTGGCAAGTTATGTGCCCATTCTCAACAACGTCAATATAGATCTGCTTATTATC CTGAAGATCTATTTATTGACAAGAAGGTATTAAAAGTTGCTCGTGTAGAACGTGAAGAAA CCTTATCCAGCCGAAGAAGGGAACTAATTCAGAAGTTAAAATCTTTCATCAGCTTCTATA GTGCTTTGCCTGGCTACATCTGCAGCCATAGCCCTGTGGCTGAAAATGACACTCTTTGCT GGAATGGACAAGAACTCGTGGAGAGATACAGCCAAAAGGCAGCGAGGAATGGAATGAAGA ATCAGTTCAACCTCCATGAGCTGAAAATGAAGGGCCCTGAGCCAGTGGTCAGTCAAATTA TTGACAAACTGAAGCATATTAACCAGCTCCTGAGGACCATGTCTGTACCCAAAGGCAGAG TTCTGGATAAAAACCTTGATGAGGAAGGGCTTGAAAGTGGAGACTGTGGCGATGATGAAG ATGAGTGCATCGGAGGCTCTGGTGATGGAΆTGATGAAAGTGAAGAACCAGCTCCGCTTCC TTGCGGAACTGGCTTATGATCTGGACGTGGACGATGCTCCTGGAAGCAAGCAACATGTGA ATCAGAAGGACAATGAGATAGTTGCCTCTCACAACCTTGGGAATGGCCATCCTCCCCTGA AGCTTCTCACCAGCTTGGCAATCTCTGTGCTGTGCTTTTTCCTCCTGGTGCACTGACTGC CAAGCTCACAGGATATGTGCTGCCCTTCAGCACCCTGTGGTCTCCCTCTATAAAGGGAAC CACCTTTTTCTTTTTCCCTCTTTTTTTTATCTTCTATACCTCCTTCAGCCATTAAGTAGA AAACTAACCCTGCGTTACGTTTTCGAAAATCACGTGGCATCTCCATGAGGAGGGCAAATT TTAGTGGTAGTATAGATTGTCTTTTGCAATACAAAAAATCAAATTGTGCCAAATTATTTT CCTATGTTTGGCTGCTAGAACATGGTTGCCATGTCTTTCTCTCTGTCTTTCCCTTTGCAT GGATTTCTTTGAAAAAAAAAATAATAAACACTCAAATAAAAAAGAAAAAAAAAAAAAAAA AAATCGG

0 Primers

Use of the pig primers for the other exons.

- 5'UTR

5utr_F: GCCGCCAAGCGGTTCC 5utr_R: TGCTTCGCAGGGAGCTAGG

- Exon 4

EX4_F: TGCCCATTCTCAACAACGTCA EX4_R: TTCGGCTGGATAAGGTTTCTTCA

- Exon 5

EX5_F: TTCATCAGCTTCTATAGTGCTTTGC EX5_R: TCTCCACGAGTTCTTGTCCATTCC

- Exon 6

EX6_F: GCCAAAAGGCAGCGAGGA EX6_R: TGGTTAATATGCTTCAGTTTGTCA

- Exon 7

EX7_F: TCCTGAGGACCATGTCTGTACCC EX7_R: CGCAAGGAAGCGGAGCTG

- Exon 8-3'UTR

EX8-utr_F: TCTGGACGTGGACGATGCTC EX8-utr_R: CAAAGAAATCCATGCAAAGGGAAA

0 Sequences

- Exon 1

>Exon l_AngHol

GKAAACAGCTATGACCGGGTTTCTGGCACCCACTTGAGTCCGGGCTGCAGTCTCTGGAAG AAGGAGCGGACCTGGTGACAGGTGGCGTCCGGCGGCGGCGGCGGGGGCTGCGCCTGTCCC GGGCAGTCCAAGCTAAGCAGCATCGCCACTGGCCRTCRTTTT

- Exon 2

>Exon 2_AngHol

TAAAACGACGGCCAGTCCCGACATGCTGCTCAAGAAAGATGGAAGAAAAGTATCAACTAA CAGCACGATTGAACATGGAACAACTGCTTCAGTCTGCAAGTATGGAGCTCAAGTTCTTAA TTATTCAGAATGCTGCGGTTTTCCGGTCATAGCTGTTWCCTAAAA

- Exon 3

>Exon 3_AngHol

TTAAAAYGACGGCCAGTGCGAGGCGTGACCTGAAAGTATTTGGGAATTTCCCCAAGCTTA TTATGACCCAGGTTTCCAAGTCACTGCAAGTCACTAGGATCTTCCTCCAGGCCCTGAATC TTGGAΆTTGAAGTGATCAACACAACCGATCACCTGAAGTTCAGTAAGGACTGTGGCCGAA TGCTCACGAGGATGTGGTACTGCTCTTACTGCCAGGGACTGATGATGGTGAAGCCATGTG GCGGCTACTGCAATGTGGTCATGCAGGGCTGTATGGCAGGTGTGGTGGAGATTGACAAGT ACTGGAGAGAATACATCCTGTCTCTGGAAGAACTGGTGAACGGCATGTACAGAGTCTATG ACATGGAGAATGTACTGCTTGGTCTCTTTTCAACGATCCATGACTCCATCCAGTACGTCC AGAAGAATGGAGGAAAGCTGGCCAGGTCATAGCTGTTTCC - Exon 4

>Exon 4_AngHol

AAAAYGAYGGCCAGTCCTGAGGACCATGTCTGTACCCAAAGGCAGAGTTCTGGATAAAAA CCTTGATGAGGAAGGGCTTGAAAGTGGAGACTGTGGCGATGATGAAGATGAGTGCATCGG AGGCTCTGGTGATGGAATGATGAAAGTGAΆGAACCAGCTCCGCTTCCTTGCGGTCATAGC TGTTTCCTCA

- Exon 5

>Exon 5_AngHol

AGGAAACAGCTATGACCTCTCCACGAGTTCTTGTCCATTCCAGCAAAGAGTGTCATTTTC AGCCACAGGGCTATGGCTGCAGATGTAGCCAGGCAAAGCACTATAGAAGCTGATGAACTG GCCGTCGTTTTAAA

- Exon 6

>Exon 6_AngHol

AGGAAACAGCTATGACCGTTTGTCAATAATTTGACTGACCACTGGCTCAGGGCCCTTCAT TTTCAGCTCATGGAGGTTGAACTGATTCTTCATTCCATTCCTGGCTGCCTTTTGGCTACT GGCCGTCRTTTT

- Exon 7

>Exon 7_AngHol

GAGGAAACAGCTATGACCTTCGGCTGGATAAGGTTTCTTCACGTTCTACACGAGCAACTT TTAATACCTTCTTGTCAATAAATAGATCTTCAGGATAATAAGCAGATCTATATTGACGTT GTTGAGAATGGGCACTGGCCGTCGTT

- Exon 8

>Exon 8_AngHol

AGGAAACAGSTATGACCTCCATGCAAAGGGAAAGACAGAGAGAAAGACATGGCAACCATG TTCTAGCAGCCAAACATAGGAAAATAATTTGGCACAATTTGATTTTTTGTATTGCAAAAG ACAATCTATACTACCACTAAAATTTGCCCTCCTCATGGAGATGCCΆCGTGATTTTCGAΆΆ ACGTAACGCAGGGTTAGTTTTCTACTTAATGGCTGAAGGAGGTATAGAAGATAAAAAAAA GAGGGAAAAAGAAAAAGGTGGTTCCCTTTATAGAGGGAGACCACAGGGTGCTGAAGGGCA GCACATATCCTGTGAGCTTGGCAGTCAGTGCACCAGGAGGAAAAAGCACAGCACAGAGAT TGCCAAGCTGGTGAGAAGCTTCAGGGGAGGATGGCCATTCCCAAGGTTGTGAGAGGCAAC

TATCTCATTGTCCTTCTGATTCACATGTTGCTTGCTTCCAGGAGCATCGTCCATGTGCAG GTCACTGGCCGTCRTTTTA

0 Validated SNPs

>Exon 4

TtcggctggataaggtttcttcacgttctaCACGAGCAACTTTTA*p*TACCTTCTTGTCAATAAATAG ATCTTCAGGATAATAAGCAGATCTATATTGACGTTGTTGAGAATGGGC

• GPC4

0 Contigs

The following sequences were extracted from a public database: BE479857 and btcn28013

0 Primers

- 5 ' UTR-exon 1

5utr-EXl_F: CGCTCTCGAGCCCCTGTC 5utr-EXl_R: ACCGTTGATCTCGTGCATGG

- Exon 2

EX2_F: TCCAGGGTTATACCTGCTGCTCTC EX2_R: TTCTTGTAGCGGGAGGCAAA

- Exon 3

EX3 F: TGCAGAGAAATCCCTGAATGATATG EX3_R: CCGGCAGTGGGAGCAGTA

- Exon 4

EX4_F: GTTTGGCCAACCAAGG EX4_R: AGCATCAGAAATCTTCACATCG

- Exons 5-6

EX5-6_F: CAAGTGTCTCAGAAGGTTTTCCA EX5-6_R: CCAGTCGGTCCAAACTAGTGC

- Exon 7

EX7_F: TGATGTCAAGGAGAAACTGAAGCA EX7_R: GCTTTGGCCTTTCCCATTCC

- Exons 8 & 9-3'UTR EX8-9-3utr_F: GCCAACCAGGGCAATAATCC EX8-9-3utr_R: GAGGAGGCCGGACTGACC

- Exon 9-3'UTR EX9-3utr_F: TGGCCACTGGTTTAAAAGATGC

EX9-3utr_R: TTTGCTTGTTGGGCTTTTTCTTT

0 Sequences

-Exon 2

>GPC4_EX2_AngHol

TTTTAGGAAACAGCTATGACCTTCTTGTAGCGGGAGGCAAAGACAGCTTGTAAATGATTG CACTGTTCACTGACCACACTTTTGAAATCCTCCTTACT*Ϊ*TGGAGGCTGTACTTTTCCTCC ATCTCTTGAGAGCAGCAGGTATAACCCTGGACTGGCCGTCRTTTTAAA

-Exon 7

>GPC4_EX7_AngHol

AGGAAACAGCTATGACCAGCAGTCGTCCTCATTGCCGCTCCCTGCAGCCATCCTCTCATC ATTGCAGACACTGCTAGGAAGAGAGGACCAGAACTTCTTGGCCTGTTTCAGCTTCTCCAC TGGCCGTCGTTTTA

-Exons 8 S 9 - 3'utτ

>GPC4_8-9utr_AngHol_F

YTTTTTTTTWRGKKKTRRRWTTWTTYCCCCYYYYTTKSSYAAMCCCGGGGWWAAARCCRR

ARAMACGGGGGGGGGGGGGSCCCSSCCCCSSGGGGGSCCCCCSSSSSSSGGGKTTTTTYY

TKKSSCYYYTTCCCCRRGGGKSMSSKKSMYTTWWWWYMMMAWYCCRRRGGSMCCGGKKGG

RRRYMCMMYCSSSGCCCCYTYCYTTTTCCCCGGYTTTKKKCCCCWWTTTTTTKKGRRRAA

AAAAAAAAAAGGAATGTTTATTGTGCTCTTTCTCATGTCAAGGTGTACAAACAAACATAT

AACAATTTGTTCTTTAGAAGCACACATGCTTACTGATATCAAAGAAGTCCACATCGTTCC

CATTGTAAGCATTCTTCATCTTACTGGTCATAACTCGAAGAGCCATGACTTGACGAAGGA

TCAGTATGTCCGGCTTGCTGKGTCAACCTGCACCTCTGRTATTG

>GPC4_8-9utr_AngHol_R

RRRAAKYTTTTMAWATGAAAGRRRTGGAATTTTTTTTWAWTWRRRRGGSSGKKKKKTTTT

WAAAAAAAAAAWTTTTWWWKTTTKTTTKTTMMCCCYYSCCCGGRRRAARGSSCCMAAWAM

CCYYYYYYTTTTTTTTTTTTTCCCAAAAAAATAGGTGACGAAAGCAGTGGAGAAGGAAGT

GGTAGCGGATGTGAGTCTCAGCAGTGTCCTTCGGAGTTTGAGTATAATGCAACTGACCAC

TCTGGGAAGAGTGCCAATGATAAAGCCAGCAGCGCTGGGGTCCCCGCTGCGGCCTGGCCC

TACCTCCTCCCTGTCTTCTGCATCTTATCCCTGGTTATGCAGAGAGAGTGGAGATAATTC

TCAACCTTAGAGAACAAGTGTTCATCGTCAGTTACAAAGGCACTGGTTATCACTTTTATA

CCATCCTAGTGACTTTGCTTTTTAAATGAATGGACAACAATGTACAGTTTTTACTATGTG

GCCACTGGTTTAAAAGATGCTGACTTTGTTTTTGCATTCAGTTTTGTGGGAGAAGGGGAT

TKATKAAGG

- Exon 9-3 'UTR

>GPC4_9utr_HolAng GAAACAGCCTATGACCTTTGCTTGTTGGGCTTTTTCTTTTAATAAGATAACGTGATAAAT AAAACCTGCTTCTGTACAGCTATTTAATATTTCAGAAATACGTACATGTTACATGCCAAG AAAGGACCCTGGTTTTCTTGTAAGAAACAAGGTGAGATCATATTGAAAGGAAAAAAACCC CACAAACAGAAAGAAAACAAACAGACAGAGTGATAAAAATCACAAATGCACAACTAACTA CGCTCTGTACCTACACTGTTAGCCACATTTAACATGACTGAGGAGGCCGGACTGACCTTA CATACAATCCCCTTCTCCCACAAAACTGAATGCAAAAACAAAGTCAGCATCTTTTAAΆCC AGTGGCCACTGGCCGTCGTTTTAAAAAARATGCTGACTTTGTTTTTGCATTCATTTTGTG GGAGAAGGGGATTGTATGTAAGGTC

- Exon 9-3'UTR - rsq

TTTTTKGAAAMYCWAAARGMGGGKKGTTTTAAARAAAAAAAWTTTWWWAKKTTTTTTTKK CCCCYYKGCCTGGGRRAAGGSCCCMAAAAMMYTYYYYTTTTTTTTTTTTTCCCAAAAAAA TAGGTGACGAAAGCAGTGGAGAAGGAAGTGGTAGCGGATGTGAGTCTCAGCAGTGTCCTT CGGAGTTTGAGTATAATGCAACTGACCACTCTGGGAAGAGTGCCAATGATAAAGCCAGCA GCGCTGGGGTCCCCGCTGCGGCCTGGCCCTACCTCCTCCCTGTCTTCTGCATCTTATCCC TGGTTATGCAGAGAGAGTGGAGATAATTCTCAACCTTAGAGAACAAGTGTTCATCGTCAG TTACAAAGGCACTGGTTATCACTTTTATACCATCCTAGTGACTTTGCTTTTTAAATGAAT GGACAACAATGTACAGTTTTTACTATGTGGCCACTGGTTTAAAAGATGCTGACTTTGTTT TTGCATTCAGTTTTGTGGGAGAAGGGGATTGTATGTAAGGTCAGTCCGGCCTCCTCAGTC ATGTTAAATGTGGCTAACAGTGTAGGTACAGAGCGTAGTTΆGTTGTGCATTTGTGATTTT TATCACTCTGTCTGTTTGTTTTCTTTCTGTTTGTGGGGTTTTTTTCCTTTCAATATGATC TCACCTTGTTTCTTACAAGAAAACCAGGGTCCTTTCTTGGCATGTAACATGTACGTATTT CTGAAATATTAAATAGCTGTACAGAAGCAGGTTTTATTTATCACKTATCTTATAAAARAA

AAGC

-3'utr2

>GPC4_utr2_Ang_F

TAGCTGTTTCCTTCATGGGTGTTAATAAAATAGGCAGGKCATAGCTGTTT

>GPC4_utr2_AngHol_F

KAGCTGTTTCCTKCATGGGTGKTAATRAAATAGGCAGGWCMTAGCTGTTTCCTTSYYA

>GPC4_utr2_Hol_R

TTSTCAGYGCYACCTAYTCCWMTGGCMGTCGTTTTAA

>GPC4_utr2_Ang_R

CTTGCGCCTGCCACAGCACGCCTTGTGGACAGCCTATKTCCTGGTCTTCTTCAGCGCTAC

CTACATCCACTGGCCGGTCGTTTTAATTTAAAAATATCTT

>GPC4__utr2_Hol_F

RTRAAATAGGCAGGACATAGCTGTTTMCTTSYYA

>GPC4_utr2_Hol_R

TTSTCAGYGCYACCTAYTCCWMTGGCMGTCGTTTTAA

0 Validated SNPs

>Exon 2__F ccagggttatacctgctgctctcaagagaTGGAGGaaAAGTACAGCCTCCA*] r*AGTAAGGAGGATTTCAAAAGTGTGGTCAGTGAACAGTGCAATCATTTACAAGCTG TCTTTGCCTCCCGCTACAAGAA References

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Supporting Online Material: www.sciencemag.org

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Fleischwirtschaft, 67(9), 1098-1102.

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Comparison of different methods for determination of drip loss and their relationships to meat quality and carcass characteristics in pigs. Meat Science, 68(3), 401-409.

Milan, D., Jeon, J.T., Looft, C, Amarger, V., Robic, A., Thelander, M., Rogel- Gaillard, C, Paul, S., Iannuccelli, N., Rask, L., Ronne, H., Lundstrom, K., Reinsch, N., Gellin, J., KaIm, E., Roy, P. L., Chardon, P., and L. Andersson. 2000. A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 288, 1248-1251.

Van Laere, A-S., Nguyen, M., Braunschweig, M., Nezer, C, Collette, C, Moreau, L., Archibald, A.L., Haley, CS. , Buys, N., Tally, M., Andersson, G., Georges, M., and L. Andersson. 2003. A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig. Nature 425: 832-836.

Claims

claims:

1. A method of screening non-human animals for potential inclusion in or exclusion from breeding programs said method comprising the steps of: a) providing a nucleic acid sample from a non-human animal; and either

(i) comparing the level of expression of the quantitative trait locus genes Gpc3 and/or Gpc4 with a reference nucleic acid sample; or

(ii) detecting a polymorphism associated with modulated Gpc3 and/or GpcA expression and/or modulated activity of the products of the Gpc3 and/or GpcA genes; wherein modulation of the level of expression of the quantitative trait locus (QTL) genes, Gpci and GpcA and/or modulation of the activity of the products of said genes, is associated with effects upon the phenotype of non-human animals.

2. The method of claim 1, wherein the non-human animals are agricultural livestock, such as pigs, sheep, goats, poultry and cows.

3. The method of claim 1, wherein the non-human animals are rodents, such as rats and mice.

4. The method of claim 1 wherein the effect on phenotype results in a commercially important trait.

5. The method of claim 4, wherein the commercially important traits are selected from the group consisting of: i) increase in growth generally; ii) increased rate of growth; iii) faster onset of maturity; iv) greater overall size; v) increased lean content of the muscle tissue; vi) improved killing out percentage; vii) improved yield of a particular cut or joint; viii) superior overall meat quality (e.g. drip loss, tenderness, colour); ix) improved early age mortality; x) increased birth weight; xi) increased juvenile weight; xii) reduced incidence of dystocia; xii) increased lean meat content; and/or xiii) increase in the size and/or quality and value of certain meat joints.

6. The method of claim l(i), wherein the comparison between the level of expression of the quantitative trait locus genes Gpc3 and/or Gpc4 with a reference nucleic acid sample uses quantitative and/or qualitative analysis techniques.

7. The method of claim l(i), wherein the level of expression of the quantitative trait locus genes Gpc3 and/or Gpc4 is compared with a reference nucleic acid sample by means of restriction fragment length polymorphism analysis.

8. The method of claim l(i), wherein the level of expression of the quantitative trait locus genes GpcS and/or Gpc4 is compared with a reference nucleic acid sample by real-time PCR.

9. The method of claim l(i), wherein the level of expression of the quantitative trait locus genes Gρc3 and/or Gpc4 is compared with a reference nucleic acid sample by quantitative reverse transcriptase polymerase chain reaction (RT-PCR).

10. The method of claim 9, wherein the products of the RT-PCR are resolved by agarose gel electrophoresis and the intensity of the bands compared with a reference sample.

11. The method of claim 9, wherein the RT-PCR is conducted in real-time.

12. The method of claim 9, wherein the RT-PCR products are quantified by chromatography systems, for example dHPLC

13. The method of claim l(ii), wherein polymorphisms are detected by comparing the sequence of a nucleic acid sample, or amplified target gene with a reference nucleic acid sequence.

14. The method of claim l(ii), wherein polymorphisms are detected by a process selected from the group consisting of i) restriction fragment length polymorphism analysis; ii) heteroduplex analysis; iii) single strand conformation polymorphism analysis; iv) denaturing gradient electrophoresis; v) temperature gradient electrophoresis; vi) single base extension; vii) pyrosequencing; viii) minisequencing; and/or ix) mass spectrometry e.g. MALDI-TOF.

15. Use of non-human animals, selected in accordance with the method of claim 1 , in breeding programs.

16. A kit for detecting animals exhibiting reduced expression of Gpc3 and/or Gpc4 and/or animals possessing polymorphisms associated with reduced expression of Gpc3 and/or GpcA, said kit comprising oligonucleotide primers designed to amplify a region of the QTL comprising the Gpci and/or Gpc4 genes.

17. The kit of claim 16 further comprising reagents required in a polymerase chain reaction.

18. The kit of claims 16 and 17, further comprising tools to facilitate the collection and/or storage of samples from which nucleic acid is to be obtained.

19. The kit of claims 16 to 18 further comprising a reference nucleic acid sample.

20. The kit of claims 16 to 19 further comprising the sequence of a reference nucleic acid sample

21. A method of identifying and storing reproductive material from a non-human animal, wherein said non-human animal exhibits reduced expression of the genes Gpc3 and/or GpcA comprising the steps of: a) providing a nucleic acid sample from a non-human animal; and either (i) comparing the level of expression of the quantitative trait locus genes Gpci and/or GpcA with a reference nucleic acid sample; or

(ii) detecting a polymorphism associated with modulated Gpc3 and/or GpcA expression and/or modulated activity of the products of the Gpc3 and/or GpcA genes; c) identifying suitable animals and obtaining semen or egg samples from said animal for breeding purposes.

22. A method of conducting an animal breeding business comprising the steps of identifying and storing reproductive material from a non-human animal according to the method of claim 21, and providing samples for breeding purposes.

23. A method of obtaining non-human animals for inclusion in breeding programs, said method comprising the steps of; c) screening animals for inclusion in breeding programs by the method of claim 1 d) identifying animals with either i. modulated Gpc3 and/or GpcA expression; and/or ii. modulated activity of the products of the genes Gpc3 and/or

GpcA c) providing said animals for breeding purposes.