AU2006216122B2 - Detection of DNA sequence motifs in ruminants - Google Patents

Abstract

A method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: (a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and (b) detecting the complex formed between the probe and the target nucleic acid wherein the repeat elements are formed of repeating nucleotide sequences of at least (3) nucleotides.

Description

WO 2006/089366 PCT/AU2006/000240 - 1 "Detection of DNA Sequence Motifs in Ruminants" Field of the Invention The present invention relates to the detection of DNA sequence motifs and their use in genotyping ruminant animals. More particularly, the invention relates to the 5 use of tri-, tetra-, penta- and hexa-nucleotide repeating sequences for genotyping ruminant animals. Background Art Generally, genotyping of ruminants such as sheep and cattle is performed by analysis of variations that occur in regions of repeating dinucleotide sequences 10 within the genomic DNA or by analysing variations that modify the length of a restriction fragment (RFLPs). Commercially available kits for these types of analysis are available and are currently used for establishing parentage of animals within a population. However, methods used to identify and to type RFLPs are relatively wasteful of 15 materials, effort, and time. Moreover, RFLP markers are costly and time consuming to develop and assay in large numbers. Furthermore, dinucleotide repeat sequences are prone to "stuttering" during in vitro amplification processes such as polymerase chain reaction. This stuttering results in a single original fragment being amplified as two or more fragments of 20 different lengths. The amplification products usually appear on an electrophoretic gel, or capillary electrophoretic analysis as additional bands or peaks, referred to as shadow bands or shadow peaks. The presence of shadow peaks makes the automated analysis of dinucleotide microsatellites imprecise. In order to accurately determine the copy number of a dinucleotide repeat motif 25 that has shadow peaks, a skilled operator must manually review the sequence data and make a determination of the true repeat number. This has led to genotyping service providers providing either low-cost services with doubtful WO 2006/089366 PCT/AU2006/000240 -2 precision (as the sequences have not been manually reviewed to correct errors due to shadow peaks), or services with relatively high precision but an associated high cost due to the costs involved in manual checking. Several studies have shown error rates of approximately 10% (Visscher et al (2002) J Dairy Science 85: 5 2368-2375) and even as high as 36% (Baron et al (2002) Genetics and Molecular Biology 25:389-394). Previous studies in ruminants failed to find the tetranucleotide GATA repeat element in the genomes of sheep or cattle. A few repeat regions have been located in sheep and cattle. However, these repeat regions have not been used 10 for genotyping. Thus, there is a need for an alternative method for genotyping in ruminants that can be automated and which permits relatively accurate high throughput analysis. Summary of the Invention The present invention provides a method for detecting a repeat element in a 15 target ruminant nucleic acid sequence, the method comprising the steps of: (a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and (b) detecting the complex formed between the probe and the target nucleic acid. 20 wherein the repeat elements are formed of repeating nucleotide sequences of at least 3 nucleotides. The present invention also provides a method for detecting a plurality of repeat elements in a target ruminant nucleic acid sequence, the method comprising the steps of: 25 (a) contacting a plurality of nucleic acid probes capable of hybridizing with nucleotide sequences flanking said elements; and WO 2006/089366 PCT/AU2006/000240 -3 (b) detecting the complexes formed between the probes and the target nucleic acid. The present invention further provides a method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: 5 (a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and (b) detecting the complex formed between the probe and the target nucleic acid using DNA amplification. The methods of the present invention can be applied to genotyping. Thus, the 10 present invention also provides a method for characterising a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: (a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; (b) extending the complexes formed between the probe and the target nucleic 15 acid and amplifying the sequence containing the repeat element; and (c) characterising the repeat element using the amplification products. The methods herein can be applied to analyse genetic information. Thus, the present invention also provides a method of detecting an association between a genotype and a phenotype in a ruminant using a repeat element in a target 20 ruminant nucleic acid, the method comprising the steps of: (a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; (b) extending the complexes formed between the probe and the target nucleic acid and amplifying the sequence containing the repeat element; WO 2006/089366 PCT/AU2006/000240 -4 (c) characterising the repeat element using the amplification products; (d) determining the frequency of the repeat element in a trait positive population of ruminants; (e) determining the frequency of the repeat element in a control population of 5 ruminants; and (f) determining whether a statistically significant association exists between said genotype and said phenotype. The methods of the present invention may be carried out using kits. Thus, the present invention also provides a kit for detecting a repeat element in a target 10 ruminant nucleic acid sequence, the kit comprising: (a) a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and (b) means for detecting the complex formed between the probe and the target nucleic acid. 15 The present invention still further provides a method for identifying a repeat element in a ruminant nucleic acid sample, the method comprising the steps of: (a) contacting a nucleic acid probe or a plurality of nucleic acid probes, designed to hybridise to repeat elements with at least 3 repeats, with the sample; and 20 (b) detecting the hybrid complex formed between the probe and nucleic acid sample. Brief Description of the Drawings Figure 1 shows a gel of 16 sheep samples, amplified using primers BOS3.4RF:5'AAgCAAAATgCCTTACACAT3' and WO 2006/089366 PCT/AU2006/000240 BOS3.4RR:5'AGCATCAGCTCAAGAACATT3' and analysed on a LiCor DNA Fragment analyzer. Figure 2 shows a gel of DNA samples from 9 cattle amplified using primers 5 BOS3ARF: 5'AAGCAAAATGCCTTACACAT3' and BOS3.4RR: 5'AGCATCAGCTCAAGAACATT3' and analysed on a LiCor DNA Fragment analyzer. Detailed Description of the Invention Methods for detecting a repeat element 10 The present invention provides a method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and b) detecting the complex formed between the probe and the target nucleic 15 acid. The present invention is based on the surprising discovery that ruminants possess repeat elements of at least 3 nucleotides that may be used for genotyping. The repeat elements of the present invention are formed of repeating nucleotide sequences of at least 3 nucleotides and more preferably at least 4, 5 or 6 20 nucleotides. The repeat elements include microsatellites, repeat motifs, simple sequence repeats (SSR), short tandem repeats (STR) and variable number tandem repeat (VNTR). Preferably, the repeat elements comprise a sequence selected from the group of sequences in Tables 1 to 3 hereunder. 25 WO 2006/089366 PCT/AU2006/000240 Table 1 Motif phase I Phase 2 Phase 3 Phase 4 Complement phases 5' - 3' 1. AGC GCA GAG - GCT, TGC, CTG 2. AGG GGA GAG - CCT, TCC, CTC 3. AGT GTA TAG - ACT, TAC, CTA 4. AGA GAA AAG - TCT, TTC, CTT 5. ACC CCA CAC - GGT, TGG, GTG 6. ACG CGA GAC - CGT, TCG, GTC 7. ACA CAA AAC - TGT, TTG, GTT 8. ATC TCA CAT - GAT, TGA, ATG 9. ATA TAA AAT - TAT, TTA, ATT 10. GGC GCG CGG - CCG,CGC,CCG 11. TAGA AGAT GATA ATAG TCTA, ATCT, TATC, CTAT 12. CTGT TGTC GTCT TCTG ACAG, GACA, AGAC, CAGA 13. TTTC TTCT TCTT CTTT GAAA, AGAA, AAGA, AAAG 14. TAGC AGCT GCTA CTAG S GCTA, AGCT, TAGC, CTAG 15. TTGC TGCT GCTT CTTG GCAA, AGCA, AAGC, CAAG 16. GGCA GCAG CAGG AGGC TGCC, CTGC, CCTG, GCCT 17. GGGC GGCG GCGG CGGG GCCC, CGCC, CCGC, CCCG 18. GGCC GCCG CCGG CGGC GGCC, CGGC, CCGG, GCCG 19. GGGA GGAG GAGG AGGG TCCC, CTCC, CCTC, CCCT 20. GGGT GGTG GTGG TGGG ACCC, CACC, CCAC, CCCA 21. ACGT CGTA GTAC TACG ACGT, TACG, GTAC, CGTA 22. TCGA CGAT GATC ATCG TCGA, ATCG, GATC, CGAT 23. TGCA GOAT TGCA GCAT TGCA, ATGC, TGCA, ATGC 24. TACA ACAT CATA ATAC TGTA, ATGT,TATG, GTAT 25. GGAA GAAG AAGG AGGA TTCC, CTTC, CCT, TCCT 26. GGAC GACG ACGG CGGA GTCC, CGTC, CCGT, TCCG 27. TCAT CATT ATTC TTCA ATGA, AATG, GAAT, TGAA 28. TTTG TTGT TGTT GTTT CAAA,ACAA, AACA, AAAC 29. TTTA TTAT TATT ATTT TAAA, ATAA, AATA, AAAT 30. AACG ACGA CGAA GAAC CGTT, TCGT, TTCG, GTTC 31. AACC ACCA CCAA CAAC GGTT, TGGT, TTGG, GTTG 32. ACTG CTGA TGAC GACT CAGT, TCAG, GTCA, AGTC 33. AACT ACTA CTAA TAAC AGTT, TAGT, TTAG, GTTA 34. AGCT GCTA CTAG TAGC AGCT, TAGC, CTAG, GCTA 35. TTGA TGAT GATT ATTG TCAA, ATCA, AATC, CAAT 36. GGAT GATG ATGG TGGA ATCC, CATCH, CCAT, TCCA 37. GCGT CGTG GTGC TGCG ACGC, CACG, GCAC, CGCA 38. CACT ACTC CTCA TCAC . AGTG, GAGT, TGAG, GTGA 39. CAGC AGCC GCCA CCAG GCTG, GGCT, TGGC, CTGG 40. AAGT AGTA GTAA TAAG ACTT, TACT, TTAC, CTTA 41. ACAT CATA ATAC TACA ATGT, TATG, GTAT, TGTA 42. TTAA TAAT AATT ATTA TTAA, ATTA, AATT, TAAT WO 2006/089366 PCT/AU2006/000240 -7 Table 2 Motif phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Complement phases (5'-.3') 43. AAAAC AAACA AACAA ACAAA CAAAA 44.GTTTT TGTTT TTGTT TTTGT TTTTG 45. AAAAG AAAGA AAGAA AGAAA GAAAA 46.CTTTT TCTTT TTCTT TTTCT TTTTC 47. AAAAT AAATA AATAA ATAAA TAAAA 48.TTTTA TTTAT TTATT TTTAT TTTTA 49. AAACC AACCA ACCAA CCAAA CAAAC 50. GGTTT TGGTT TTGGT TTTGG GTTTG 51. AAACG AACGA ACGAA CGAAA GAAAC 52.CGTTT TCGTT TTCGT TTTCG GTTTC 53. AAAGC AAGCA AGCAA GCAAA CAAAG 54. GCTTT TGCTT TTGCT TTTGC CTTTG 55. AAATC AATCA ATCAA TCAAA CAAAT 56. GATTT TGATT TTGAT TTTGA ATTTG 57. AAACT AACTA ACTAA CTAAA TAAAC 58. AGTTT TAGTT TTAGT TTTAG GTTTA 59. AAAGG AAGGA AGGAA GGAAA GAAAG 60. CCTTT TCCTT TTCCT TTTCC CTTTC 61. AAAGT AAGTA AGTAA GTAAA TAAAG 62. ACTTT TACTT TTACT TTTAC CTTTA 63. AAATG AATGA ATGAA TGAAA GAAAT 64. CATTT TCATT TTCAT TTTCA ATTTC 65. AAATT AATTA ATTAA TTAAA TAAAT 66. AATTT TAATT TTAAT TTTAA ATTTA 67. AACAC ACACA CACAA ACAAC CAACA 68. GTGTT TGTGT TTGTG GTTGT TGTTG 69. AACAG ACAGA CAGAA AGAAC GAACA 70. CTGTT TCTGT TTCTG GTTCT TGTTC 71. AACAT ACATA CATAA ATAAC TAACA 72. ATGTT TATGT TTATG GTTAT TGTTA 73. AACCC ACCCA CCCAA CCAAC CAACC 74. GGGTT TGGGT TTGGG GTTGG GGTTG 75. AACCG ACCGA CCGAA CGAAC GAACC 76. CGGTT TCGGT TTCGG GTTCG GGTTC 77. AACCT ACCTA CCTAA CTAAC TAACC 78. AGGTT TAGGT TTAGG GTTAG GGTTA 79. AACGC ACGCA CGCAA GCAAC CAACG 80. GCGTT TGCGT TTGCG GTTGC CGTTG 81. AACGG ACGGA CGGAA GGAAC GAACG 82. CCGTT TCCGT TTCCG GTTCC CGTTC 83. AACGT ACGTA CGTAA GTAAC TAACG 84. ACGTT TACGT TTACG GTTAC CGTTA 85. AACTC ACTCA CTCAA TCAAC CAACT 86. GAGTT TGAGT TTGAG GTTGA AGTTG 87. AACTG ACTGA CTGAA TGAAC GAACT 88. CAGTT TCAGT TTCAG GTTCA AGTTC 89. AAGCC AGCCA GCCAA CCAAG CAAGC 90. GGCTT TGGCT TTGGC CTTGG GCTTG 91. AAGCG AGCGA GCGAA CGAAG GAAGC 92. CGCTT TCGCT TTCGC CTTCG GCTTC 93. AAGCT AGCTA GCTAA CTAAG TAAGC 94. AGCTT TAGCT TTAGC CTTAG GCTTA 95. AAGGC AGGCA GGCAA GCAAG CAAGG 96. CCGTT TGCCT TTGCC CTTGC CCTTG WO 2006/089366 PCT/AU2006/000240 -8 97. AAGGG AGGGA GGGAA GGAAG GAAGG 98. CCCTT TCCCT TTCCC CTTCC CCTTC 99. AAGGT AGGTA GGTAA GTAAG TAAGG 100. ACCTT TACCT TTACC CTTAC CCTTA 101. AAGTC AGTCA GTCAA TCAAG CAAGT 102. GACTT TGACT TTGAC CTTGA ACTTG 103. AAGTG AGTGA GTGAA TGAAG GAAGT 104. CACTT TCACT TTCAC CTTCA ACTTC 105. AAGTT AGTTA GTTAA TTAAG TAAGT 106. AACTT TAACT TTAAC CTTAA ACTTA 107. AATAC ATACA TACAA ACAAT CAATA 108. GTATT TGTAT TTGTA ATTGT TATTG 109. AATAG ATAGA TAGAA AGAAT GAATA 110. CTATT TCTAT TTCTA ATTCT TATTC 111. AATAT ATATA TATAA ATAAT TAATA 112. ATATT TATAT TTATA ATTAT TATTA 113. AATCC ATCCA TCCAA CCAAT CAATC 114. GGATT TGGAT TTGGA ATTGG GATTG 115. AATCG ATCGA TCGAA CGAAT GAATC 116. CGATT TCGAT TTCGA ATTCG GATTC 117. AATCT ATCTA TCTAA CTAAT TAATC 118. AGATT TAGAT TTAGA ATTAG GATTA 119. AATGC ATGCA TGCAA GCAAT CAATG 120. GCATT TGCAT TTGCA ATTGC CATTG 121. AATGG ATGGA TGGAA GGAAT GAATG 122. CCATT TCCAT TTCCA ATTCC CATTC 123. AATGT ATGTA TGTAA GTAAT TAATG 124. ACATT TACAT TTACA ATTAC CATTA 125. AATTG ATTGA TTGAA TGAAT GAATT 126. CAATT TCAAT TTCAA ATTCA AATTC 127. ACACC CACCA ACCAC CCACA CACAC 128. GGTGT GGGTT GTGGT TGTGG GTGTG 129. ACACG CACGA ACGAC CGACA GACAC 130. CGTGT TCGTG GTCGT TGTCG GTGTC 131. ACACT CACTA ACTAC CTACA TACAC 132. AGTGT TAGTG GTAGT TGTAG GTGTA 133. ACAGC CAGCA AGCAC GCACA CACAG 134. GCTGT TGCTG GTGCT TGTGC CTGTG 135. ACAGG CAGGA AGGAC GGACA GACAG 136. CCTGT TCCTG GTCCT TGTCC CTGTC 137. ACAGT CAGTA AGTAC GTACA TACAG 138. ACTGT TACTG GTACT TGTAC CTGTA 139. ACATC CATCA ATCAC TCACA CACAT 140. GATGT TGATG GTGAT TGTGA ATGTG 141. ACATG CATGA ATGAC TGACA GACAT 142. CATGT TCATG GTCAT TGTCA ATGTC 143. ACCAG CCAGA CAGAC AGACC GACCA 144. CTGGT TCTGG GTCTG GGTCT TGGTC 145. ACCAT CCATA CATAC ATACC TACCA 146. ATGGT TATGG GTATG GGTAT TGGTA 147. ACCCC CCCCA CCCAC CCACC CACCC 148. GGGGT TGGGG GTGGG GGTGG GGGTG 149. ACCCG CCCGA CCGAC CGACC GACCC 150. TGGGC TCGGG GTCGG GGTCG GGGTC 151. ACCCT CCCTA CCTAC CTACC TACCC 152. AGGGT TAGGG GTAGG GGTAG GGGTA 153. ACCGC CCGCA CGCAC GCACC CACCG 154. GCGGT TGCGG GTGCG GGTGC CGGTG 155. ACCGG CCGGA CGGAC GGACC GACCG WO 2006/089366 PCT/AU2006/000240 -9 156. CCGGT TCCGG GTCCG GGTCC CGGTC 157. ACCTC CCTCA CTCAC TCACC CACCT 158. GAGGT TGAGG GTGAG GGTGA AGGTG 159. ACCTG CCTGA CTGAC TGACC GACCT 160. CAGGT TCAGG GTCAG GGTCA AGGTC 161. ACGCC CGCCA GCCAC CCACG CACGC 162. GGCGT TGGCG GTGGC CGTGG GCGTG 163. ACGCG CGCGA GCGAC CGACG GACGC 164. CGCGT TCGCG GTCGC CGTCG GCGTC 165. ACGCT CGCTA GCTAC CTACG TACGC 166. AGCGT TAGCG GTAGC CGTAG GCGTA 167. ACGGC CGGCA GGCAC GCACG CACGG 168. GCCGT TGCCG GTGCC CGTGC CCGTG 169. ACGGG CGGGA GGGAC GGACG GACGG 170. CCCGT TCCCG GTCCC CGTCC CCGTC 171. ACGGT CGGTA GGTAC GTACG TACGG 172. ACCGT TACCG GTACC CGTAC CCGTA 173. ACGTG CGTGA GTGAC TGACG GACGT 174. CACGT TCACG GTCAC CGTCA ACGTC 175. ACTCC CTCCA TCCAC CCACT CACTC 176. GGAGT TGGAG GTGGA AGTGG GAGTG 177. ACTCG CTCGA TCGAC CGACT GACTC 178. CGAGT TCGAG GTCGA AGTCG GAGTC 179. ACTCT CTCTA TCTAC CTACT TACTC 180. AGAGT TAGAG GTAGA AGTAG GAGTA 181. ACTGC CTGCA TGCAC GCACT CACTG 182. GCAGT TGCAG GTGCA AGTGC CAGTG 183. ACTGG CTGGA TGGAC GGACT GACTG 184. CCAGT TCCAG GTCCA AGTCC CAGTC 185. AGACG GACGA ACGAG CGAGA GAGAC 186. CGTCT TCGTC CTCGT TCTCG GTCTC 187. AGACT GACTA ACTAG CTAGA TAGAC 188. AGTCT TAGTC CTAGT TCTAG GTCTA 189. AGCCC GCCCA CCCAG CCAGC CAGCC 190. GGGCT TGGGC CTGGG GCTGG GGCTG 191. AGCCG GCCGA CCGAG CGAGC GAGCC 192. CGGCT TCGGC CTCGG GCTCG GGCTC 193. AGCGC GCGCA CGCAG GCAGC CAGCG 194. GCGCT TGCGC CTGCG GCTGC CGCTG 195. AGCGG GCGGA CGGAG GGAGC GAGCG 196. CCGCT TCCGC CTCCG GCTCC CGCTC 197. AGCCT GCCTA CCTAG CTAGC TAGCC 198. AGGCT TAGGC CTAGG GCTAG GGCTA 199. AGGCC GGCCA GCCAG CCAGG CAGGC 200. GGCCT TGGCC CTGGC CCTGG GCCTG 201. AGGCG GGCGA GCGAG CGAGG GAGGC 202. CGCCT TCGCC CTCGC CCTCG GCCTC 203. AGGGC GGGCA GGCAG GCAGG CAGGG 204. GCCCT TGCCC CTGCC CCTGC CCCTG 205. AGGGG GGGGA GGGAG GGAGG GAGGG 206. CCCCT TCCCC CTCCC CCTCC CCCTC 207. AGTAT GTATA TATAG ATAGT TAGTA 208. ATACT TATAC CTATA ACTAT TACTA 209. ATCCC TCCCA CCCAT CCATC CATCC 210. GGGAT TGGGA ATGGG GATGG GGATG 211. ATCCG TCCGA CCGAT CGATC GATCC 212. CGGAT TCGGA ATCGG GATCG GGATC 213. ATCCT TCCTA CCTAT CTATC TATCC 214. AGGAT TAGGA ATAGG GATAG GGATA WO 2006/089366 PCT/AU2006/000240 -10 215. ATCGC TCGCA CGCAT GCATC CATCG 216. GCGAT TGCGA ATGCG GATGC CGATG 217. ATCGT TCGTA CGTAT GTATC TATCG 218. ACGAT TACGA ATACG GATAC CGATA 219. ATCTC TCTCA CTCAT TCATC CATCT 220. GAGAT TGAGA ATGAG GATGA AGATG 221. ATCTG TCTGA CTGAT TGATC GATCT 222. CAGAT TCAGA ATCAG GATCA AGATC 223, ATCTT TCTTA CTTAT TTATC TATCT 224. AAGAT TAAGA ATAAG GATAA AGATA 225. ATGCC TGCCA GCCAT CCATG CATGC 226. GGCAT TGGCA ATGGC CATGG GCATG 227. ATGCT TGCTA GCTAT CTATG TATGC 228. AGCAT TAGCA ATAGC CATAG GCATA 229. CCCCG CCCGC CCGCC CGCCC GCCCC 230. CGGGG GCGGG GGCGG GGGCG GGGGC 231. CCCGG CCGGC CGGCC GGCCC GCCCG 232. CCGGG GCCGG GGCCG GGGCC CGGGC 233. CGCGG GCGGC CGGCG GGCGC GCGCG 234. CCGCG GCCGC CGCCG GCGCC CGCGC 235. CTCCT TCCTC CCTCT CTCTC TCTCC 236. AGGAG GAGGA AGAGG GAGAG GGAGA 237. CTGCT TGCTC GCTCT CTCTG TCTGC 238. AGCAG GAGCA AGAGC CAGAG GCAGA 239. CTTCT TTCTC TCTCT CTCTT TCTTC 240. AGAAG GAGAA AGAGA AAGAG GAAGA 241. CTTGT TTGTC TGTCT GTCTT TCTTG 242. ACAAG GACAA AGACA AAGAC CAAGA Table 3 3-base motifs 4-base motifs 5-base motifs 6-base motifs ATT CCCT ACCCC ACTTTC AGG TGGC CAGTT GGC CCTT ACTGA AGT GACA TGAAA ACG GAAT GTT AGAA GAA TAAA CAG GTGG TGG GGGC ATTA GATA TGAA ATGG TCTA

ATCC

WO 2006/089366 PCT/AU2006/000240 More preferably, the repeat elements comprise a sequence selected from the group of sequences in Tables 4 hereunder. Table 4 3-base motifs 4-base motifs 5-base motifs 6-base motifs ATT CCCT ACCCC ACTTTC AGG TGGC CAGTT GGC CCTT ACTGA AGT GACA TGAAA ACG GAAT GTT AGAA GAA TAAA GTGG GGGC ATTA GATA TGAA 5 Preferably, the method for detecting a repeat element in a target ruminant described above is carried out using probes selected from group described in the results section of any one of Examples 1, 2 or 3. Alternatively, the method may be carried out using probes selected from the group consisting of the nucleotide sequences that are identified by bold, italics and underlining in the clones 10 described in the results section of any one of Examples 1 or 2. The target ruminant nucleic acid sequence may be varied as there are different locations in the genome that contain repeat elements amenable to detection using the method of the present. Preferably, the target ruminant nucleic acid sequence is selected from the group of DNA sequences in the clones described in the 15 results section of any one of Examples 1, 2, 3 or 4 herein that also represent a separate aspect of the present invention. The target nucleic acid sequence may comprise a single repeat element or a plurality of repeat elements. When there is a plurality of repeat elements they may comprise the same nucleic acid sequence or they may comprise different 20 nucleic acid sequences. For example, the target ruminant nucleic acid sequence may contain a trinucleotide repeat element and a tetranucleotide repeat element.

WO 2006/089366 PCT/AU2006/000240 -12 When there are a plurality of repeat elements it may be desirous to detect more than one repeat element to provide more detailed information on the genome. Thus, the present invention also provides a method for detecting a plurality of repeat elements in a target ruminant nucleic acid sequence, the method 5 comprising the steps of: a) contacting a plurality of nucleic acid probes capable of hybridizing with nucleotide sequences flanking said elements; and b) detecting the complexes formed between the probes and the target nucleic acid. 10 Whilst the detection of multiple repeat elements could be done separately it is preferable for the detection of different repeat elements to be carried out simultaneously. The "ruminant' of the present invention is any ruminant or ruminant-like animal. Ruminants include bovines, ovines, caprines, or cervines, while the ruminant-like 15 animal include llamas, camels, alpacas and vicunas. Preferably, the ruminant of the present application is an ovine or a bovine. Most preferably, the ruminant is sheep or cattle. The nucleic acid probes referred to herein can be used in the method of the present represent but also represent a separate aspect of the invention. The 20 probes are capable of hybridising to regions of the nucleotide sequence flanking the repeat element. The term probe used herein is used in the traditional technical sense of the term and/or refers to primers for nucleic acid amplification. Thus, it will be appreciated that when used herein the term "probe" also refers to "primer" insofar as the 25 context permits. Furthermore, probes used in the method described herein include variants that hybridize under stringent hybridization conditions to the particular probes described herein.

WO 2006/089366 PCT/AU2006/000240 - 13 Preferably, the probes are isolated, purified, and/or recombinant or synthesised as oligonucleotides. Even more preferably, the probes are complimentary to a sequence flanking a repeat element in any one of the clones described in the results section of any one of Examples 1, 2, 3 or 4 herein. 5 In one form of the invention, the probe is selected from the group consisting of the probes as described in the results section of any one of Examples 1, 2 or 3. In another form of the present invention the probe is selected from the group consisting of the nucleotide sequences that are identified by bold, italics and underlining in the clones described in the results section of any one of Examples 1 10 or 2 herein. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the probe, the ionic strength of the solution and the G+C content. The higher the G+C content of the probe, the higher is the melting temperature because G:C pairs are held by three H bonds 15 whereas A:T pairs have only two. The G+C content in the probes of the invention usually ranges between 10% and 75%, preferably between 35% and 60%, and more preferably between 40% and 55%. A probe according to the invention is between 8 and 1000 nucleotides in length, or is specified to be at least 8, 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 20 500 or 1000 nucleotides in length. More particularly, the length of these probes can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30 nucleotides. Shorter probes tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are 25 expensive to produce and can sometimes self-hybridize to form hairpin structures. The appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art. Preferred probes of the present invention have a 3' end that is complimentary to a fragment of the sequence flanking the repeat element. Such a configuration 30 allows the 3' end of the probe to hybridize to a selected nucleic acid sequence WO 2006/089366 PCT/AU2006/000240 - 14 and dramatically increases the efficiency of the probe for amplification or sequencing reactions. The 3' end of the probe of the invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500 or 1000 nucleotides upstream of the 5 repeat element. The probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al. (1979), the phosphodiester method of Brown et al. (1979), the diethylphosphoramidite 10 method of Beaucage et al.(1981) and the solid support method described in EP 0 707592. Probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids (disclosed in W092/20702) and morpholino analogs (described in U. S. Patents 5,185,444; 5,034,506 and 5,142,047). 15 The probes may be "non-extendable" in that additional dNTPs cannot be added to the probe. Nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise 20 block the hydroxyl group. Alternatively, the 3' hydroxyl group can be cleaved, replaced or modified. U. S. Patent Application Serial No. 07/049,061 filed April 19, 1993 describes modifications, which can be used to render a probe non extendable. The probes of the present invention may be labelled and thus further comprise a 25 label detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Useful labels include radioactive substances ( 32 P, 35S, 3 H, 121), fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) or biotin. The probes may be labelled at their 3' and 5' ends. Examples of non-radioactive labelling of nucleic acid fragments are described in 30 the French patent No. F7810975 or by Urdea et al (1988) or Sanchez-Pescador et WO 2006/089366 PCT/AU2006/000240 - 15 al (1988). In addition, the probes may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. (1991) or in the European patent EP 0 225 807 (Chiron). 5 A label can also be used to capture the probe, so as to facilitate the immobilization of either the probe or its extension product. A capture label is attached to the probe and can be a specific binding member that forms a binding pair with the solid phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a probe, it 10 may be employed to capture or to detect the target DNA. Further, it will be understood that the probes provided herein may themselves serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a probe to thereby immobilize the probe 15 to the solid phase. in cases where a polynucleotide probe itself serves as the binding member those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to the target. In the case where a polynucleotide probe itself serves as the capture label at least a portion of the probe will be free to hybridize with a nucleic acid on a solid phase. DNA labelling 20 techniques are well known to the skilled technician. The probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or 25 other animal) red blood cells, duracytes and others. The solid support is not critical and can be selected by one skilled in the art. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material that is insoluble, or can be made insoluble by a subsequent WO 2006/089366 PCT/AU2006/000240 -16 reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor that has the ability to attract and immobilize the capture reagent. The additional receptor can include 5 a charged substance that is opposite charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. 10 The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatised plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle. chip, sheep (or other animal) red blood 15 cells, duracytes and other configurations known to those of ordinary skill in the art. The probes of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20 or 25 distinct probes of the invention to a single solid support. In addition probes other than those of the invention may be attached to the same solid support as one or more 20 polynucleotides of the invention. The hybrid complex may be detected in a variety of ways. Ultrasensitive detection methods that do not require amplification are encompassed by the present invention as are methods in which the sequences of interest are directly cloned and then sequenced. However, preferably, the complex is detected using DNA 25 amplification. Thus, the present invention also provides a method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and WO 2006/089366 PCT/AU2006/000240 -17 b) detecting the complex formed between the probe and the target nucleic acid using DNA amplification. Preferably, the repeat elements are formed of repeating nucleotide sequences of at least 3, at least 4, at least 5 or at least 6 nucleotides. In another form, the 5 repeat elements are formed of repeating nucleotide sequences selected from any one of Tables 1, 2, 3 or 4. The probe used to form the complex may be selected from group described in the results section of any one of Examples 1, 2 or 3. Alternatively, the probe may be selected from the group consisting of the nucleotide sequences that are identified 10 by bold, italics and underlining in the clones described in the results section of any one of Examples 1 or 2. DNA amplification techniques utilise the hybrid complex as a source of double stranded DNA for extension. It will be appreciated that a single strand is able to function as "template" for PCR, since the first amplification cycle converts it to a 15 double strand. DNA amplification techniques are known to those skilled in the art and may be selected from the group consisting of: ligase chain reaction (LCR) e.g. EP-A-320 308, WO 93/20227 and EP-A-439 182, the polymerase chain reaction (including PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) described in Guatelli J. C., et al. (1990), 20 Q-beta amplification e.g. European Patent Application No 4544610, strand displacement amplification as described e.g. EP A 684315 and target mediated amplification as described in WO 93/22461. PCR is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. 25 Following DNA amplification the amplification products can be visualised by any convenient means apparent to those skilled in the art. For example, the nucleic acids can be applied to PAGE or some other similar technique that separates the nucleic acids, at least on the basis of size. The detection of complexes can also be carried out using detectable labels bound to either the target or the probe. 30 Typically, complexes are separated from unhybridized nucleic acids and the WO 2006/089366 PCT/AU2006/000240 - 18 labels bound to the complexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the complexes using the labels present on the 5 probes. Genotypinq Variations in the number of repeats within repeat elements can be used to type individuals and thus establish pedigree and/or parentage. Thus, the present invention also provides a method for characterising a repeat element in a target 10 ruminant nucleic acid sequence, the method comprising the steps of: a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; b) extending the complexes formed between the probe and the target nucleic acid and amplifying the sequence containing the repeat element; and 15 c) characterising the repeat element using the amplification products. Preferably the repeat element is characterised according to the number of repeating nucleotide sequences (repeats) of at least 3, at least 4, at least 5 or at least 6 nucleotides, therein. There are various methods that can be used to determine the number of repeats including: sequencing, hybridisation, 20 electrophoretic separation on the basis of length and single strand conformational polymorphism analysis (SSCP). Preferably, sequencing is automated. For example, dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol can be applied. The results from such reactions can be electronically analysed and thus 25 are particularly amendable to high throughput screening protocols.

WO 2006/089366 PCT/AU2006/000240 -19 Hybridization assays including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization can be used. When using hybridisation, allele-specific probes can be used in combinations, with each member of the combination showing a perfect match to a target sequence 5 containing one allele. it will be appreciated that hybridization conditions should be sufficiently stringent so that there is a significant difference in hybridization intensity between alleles. These conditions can be determined by one skilled in the art. Hybridization assays may also be based on multiple probes (arrays) that rely on 10 the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., a micro-chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic 15 DNA probes arranged in a grid-like pattern and miniaturized. Chip technology has already been applied with success in numerous cases. Chips of various formats can be produced on a customized basis by Affymetrix (GeneChip

TM

), Hyseq (HyChip and HyGnostics), and Protogene Laboratories. In general, these methods employ arrays of oligonucleotide probes that are 20 complementary to target nucleic acid sequence segments from an individual wherein the target sequences include a polymorphic marker. The hybridization data from the scanned array may be analysed to identify which alleles of the DNA repeat region are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 25 and US patent No. 5,424,186. Thus, the present invention also provides a method for characterising a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting a nucleic acid probe capable of hybridizing with a nucleotide 30 sequence flanking said element; WO 2006/089366 PCT/AU2006/000240 - 20 b) extending the complexes formed between the probe and the target nucleic acid and amplifying the sequence containing the repeat element; and c) characterising the repeat element using the amplification products by contacting said amplification products with a chip comprising at least one 5 probe selected from the group consisting of the probes described in the results section of any one of Examples 1, 2 or 3. The present invention further provides a method for characterising a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: 10 a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; b) extending the complexes formed between the probe and the target nucleic acid and amplifying the sequence containing the repeat element; and c) characterising the repeat element using the amplification products by 15 contacting said amplification products with a chip comprising at least one probe selected from the group consisting of the nucleotide sequences that are identified by bold, italics and underlining in the clones described in the results section of any one of Examples 1 or 2 herein. The chips that can be used in the present invention also represent an aspect of 20 the invention. Thus, the present invention also provides a chip comprising at least one probe selected from the group consisting of probes described in the results section of any one of Examples 1, 2 or 3 and the complements thereof. The present invention further provides a chip comprising at (east one probe selected from the group consisting of the nucleotide sequences that are identified by bold, 25 italics and underlining in the clones described in the results section of any one of Examples I or 2 herein and complements thereof.

WO 2006/089366 PCT/AU2006/000240 -21 Multicomponent integrated systems may also be used to characterise the repeat element. These systems miniaturise and compartmentalise processes such as amplification (e.g. PCR) and capillary electrophoresis reactions in a single functional device. An example of such a technique is disclosed in US patent 5 5,589,136 which describe the integration of PCR amplification and capillary electrophoresis in chips. Integrated systems can be envisaged where microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz or plastic wafer included on a microchip. The movements of the 10 samples are controlled by electric, electro-osmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts. For the present invention the microfluidic system may integrate nucleic acid amplification, sequencing, capillary electrophoresis and a detection method such 15 as laser induced fluorescence detection. The methods for characterising DNA repeat regions described herein can be applied to pedigree analysis, genotyping case-control populations, in association studies, as well as individuals in the context of tracing products from that animal or detection of alleles of DNA repeat regions which are known to be associated 20 with a given trait, in which case both copies of the DNA repeat region present in individual's genome are investigated to determine the number of repeats within a given repeat element so that an individual may be classified as homozygous or heterozygous for a particular allele. Genetic Analysis 25 Various methods are available for the genetic analysis of complex traits. The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for co-segregation between a locus and a putative trait locus using family studies and the association approach in WO 2006/089366 PCT/AU2006/000240 -22 which evidence is sought for a statistically significant association between an allele and a trait or a trait causing allele. In general, the methods described herein may be used to demonstrate a statistically significant correlation between a genotype and a phenotype in 5 ruminants. More specifically, the repeat elements may be used in parametric and non-parametric linkage analysis methods or identical by descent (IBD) and identical by state (IBS) methods to map genes affecting a complex trait. Preferably, the methods of the present invention are applied to identify genes associated with detectable traits in ruminants using association studies, an 10 approach which does not require the use of affected pedigrees and which permits the identification of genes associated with complex and sporadic traits. One embodiment of the present invention comprises methods to detect an association between a haplotype and a trait. Thus, the present invention also provides a method of detecting an association 15 between a genotype and a phenotype in a ruminant using a repeat element in a target ruminant nucleic acid, the method comprising the steps of: a) contacting a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; b) extending the complexes formed between the probe and the target nucleic 20 acid and amplifying the sequence containing the repeat element; c) characterising the repeat element using the amplification products; d) determining the frequency of the repeat element in a trait positive population of ruminants; e) determining the frequency of the repeat element in a control population of 25 ruminants; and WO 2006/089366 PCT/AU2006/000240 -23 f) determining whether a statistically significant association exists between said genotype and said phenotype. Optionally, said ruminant control population may be a trait negative population, or a random population. The method may be applied to a pooled biological sample 5 derived from each of said populations or performed separately on biological samples derived from each individual in said population or a sub sample thereof. The repeat elements of the present invention can also be used to identify individuals whose genotype increases their likelihood of developing a detectable trait at a subsequent time. These methods are extremely valuable as they can, in 10 certain circumstances, be used to initiate preventive treatments or to allow detection of warning signs such as minor symptoms in an individual carrying a significant haplotype. The methods can also be used to determine which individuals from a population will possess advantageous characteristics such as increased wool production, finer wool, increased milk production etc 15 Kits The methods of the present invention can be conveniently carried out using a kit. Thus, the present invention also provides a kit for detecting a repeat element in a target ruminant nucleic acid sequence, the kit comprising: a) a nucleic acid probe capable of hybridizing with a nucleotide sequence 20 flanking said element; and b) means for detecting the complex formed between the probe and the target nucleic acid. The kit may contain a plurality of probes selected from the group consisting of the probes described in the results section of any one of Examples 1, 2 or 3. 25 Alternatively, the kit may contain a plurality of probes selected from the group consisting of the nucleotide sequences that are identified by bold, italics and underlining in the clones described in the results section of any one of Examples I WO 2006/089366 PCT/AU2006/000240 -24 or 2 herein. Preferably, the probe is labelled with a detectable molecule. Even more preferably the probe is immobilized on a substrate. As indicated above a plurality of probes may be used in the methods of the present invention. Thus, the present invention also provides an array comprising 5 a plurality of probes described herein attached in overlapping areas or at random locations on a solid support. Alternatively the probes of the invention may be attached in an ordered array wherein each probe is attached to a distinct region of the solid support that does not overlap with the attachment site of any other polynucleotide. Preferably, such 10 an ordered array of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each 15 polynucleotides location makes these "addressable" arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the probes of the invention. One particular embodiment is known as the Genechips T M , and has been generally described in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. 20 These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., 1991). The immobilization of arrays of probes on solid supports has been rendered possible by the development of a technology generally identified as 25 "Very Large Scale Immobilized Polymer Synthesis" (VLSIPSTM) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPSTM technologies are provided in US Patents 5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95111995, which describe methods for forming oligonucleotide arrays through 30 techniques such as light-directed synthesis techniques.

WO 2006/089366 PCT/AU2006/000240 - 25 In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies have been developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation 5 strategies are disclosed in PCT Publications WO 94/12305. WO 94/11530, WO 97/29212 and WO 97/31256. The means for detecting the complex in the kit can be varied and includes the detecting means described herein. Preferably, the kit comprises one or more of the reagents necessary to carry out DNA amplification such as a polymerase 10 enzyme. Methods For De Novo Identification Of DNA Repea Rgegions As indicated above, the present invention is based on the identification of a number of repeat elements in the genome of ruminants. Thus, the present invention also provides a method for identifying a repeat element in a ruminant 15 nucleic acid sample , the method comprising the steps of: a) contacting a nucleic acid probe or a plurality of nucleic acid probes, designed to hybridise to repeat elements with at least 3 repeats, with the sample; and b) detecting the hybrid complex formed between the probe and nucleic acid 20 sample. The probes used in this method are designed to hybridise to repeat elements with at least 3 repeats and can be designed according to the repeat element of interest. Preferably, the probe is capable of hybridising to 3 to 10 repeats of a repeat element selected from the repeat elements listed in Tables 1 or 2. More 25 preferably, the probe is capable of hybridizing to 3 to 10 repeats of a repeat element selected from the repeat elements listed in Table 3. Most preferably, the probe is capable of hybridizing to 3 to 10 repeats of a repeat element selected from the repeat elements listed in Table 4.

WO 2006/089366 PCT/AU2006/000240 -26 The nucleic acid sample may be obtained from any ruminant source and include biological samples such as body fluids e.g. blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas 5 and the like; biological fluids such as ruminant cell culture supernatants, fixed tissue specimens including tumour and non-tumour tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of ruminant genomic DNA used in the present invention is peripheral venous blood. Techniques to prepare genomic DNA from biological 10 samples are well known to the skilled technician. General Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and 15 modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. The present invention is not to be limited in scope by the specific embodiments 20 described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein. The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are 25 hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates. As used herein the term "derived" and "derived from" shall be taken to indicate that a specific integer may be obtained from a particular source albeit not 30 necessarily directly from that source.

WO 2006/089366 PCT/AU2006/000240 -27 Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 5 Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. 10 Where this invention describes particular nucleotide sequences such as probes it will be appreciated that the invention extends to variants of the particular sequences described. A variant of a nucleotide may be a naturally occurring variant such as a naturally occurring allelic variant or it may be a variant that is not known to occur naturally. 15 Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. 20 Variants of nucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical to a nuc)eotide described herein and preferably at least 99% identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a nucleotide described herein. 25 A hybridizing nucleic acid according to the invention is one that hybridizes to the polynucleotides of the present invention under highly stringent conditions. The following is an example of stringent hybridization conditions: WO 2006/089366 PCT/AU2006/000240 - 28 - hybridization is carried out at 650 C in the presence of 6 x SSC buffer, 5x Denhardt's solution, 0,5% SDS and 100 pg/ml of salmon sperm DNA; - followed by four washing steps: -two 5 min washes, preferably at 650 C in a 2 x SSC and 0.1% SDS buffer; 5 -one 30 min wash, preferably at 650 C in a 2 x SSC and 0.1% SDS buffer, -one 10 min wash, preferably at 650 C in a 0.1 x SSC and 0.1% SDS buffer. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. The hybridization conditions described above are to be 10 adapted according to the length of the desired nucleic acid following techniques well known to the one skilled in the art. For example, if an oligonucleotide is made of e.g. CCGG, then the washing temperature may be higher for a 20-base molecule. If it is e.g. AATT, then a lower wash temperature may be required to avoid removing fully hybridised molecules, 15 The present invention will now be described with reference to the following examples. The description of the examples in no way limits the generality of the preceding description. Examples Example 1 - Locating Microsatellites in Sheep DNA 20 Materials/Methods A modified version of the method of Hamilton, M.B.; Pincus, E.L.; Di Fiore, A. and Fleischer R.C. 1999, Universal Linker and Ligation Procedures for Construction of Genomic DNA Libraries Enriched for Microsatellites. BioTechniques 27:500-507 was used as summarised hereunder.

WO 2006/089366 PCT/AU2006/000240 -29 1. Sheep chromosomal DNA was digested with two restriction endonucleases adapted to form sticky ends compatible with the 3' overhang of linkers Eco-top and Eco-bottom. Eco-top: 5' CTCGTAGACTGCGTACC 3' 5 Eco-bottom: 5' CATCTGACGCATGGTTAA 3' 2. The linkers were annealed to form short double-stranded "linkers" and the linkers were ligated to the digested fragments of chromosomal DNA by ligation reactions. 3. Chromosomal fragments were amplified by polymerase chain reaction, using 10 linker oligonucleotides as primers to make amplification independent of chromosomal sequences. 4. The amplified preparation of the chromosomal DNA fragments was heated to separate the strands and a biotinylated selection probe was added to the mixture and allowed to anneal to the chromosomal fragments. 15 5. The selection probe (annealed to the chromosomal fragments) was removed from the mixture using magnetic metal nanobeads coated with the complementary affinity binding agent, streptavidin. 6. After washing to remove non-specifically bound DNA, the "captured" chromosomal fragments were eluted by heat denaturation and separated from 20 the capture beads. 7. Eluted fragments were re-amplified using priming sites in the linker molecules and the products ligated to a plasmid cloning vector for cloning in E coli. 8. Clones were screened by hybridisation to identify those containing the appropriate DNA fragments and then sequenced to establish the identity of the 25 repeating sequence motif and to characterise the flanking DNA for potential priming sites for amplification from the genome.

WO 2006/089366 PCT/AU2006/000240 - 30 Results The following repeats were identified in the clones: ATGG, CCTT, ATCC, AGAA, TGGC, ACCOC, CCCT, GATA, GACA, GTGG, ATTA, TCTA, AGAG and AGG The entire sequences of the clones are set out hereunder. The primer sequences 5 are underlined, bold and in italics. KM- (complete, see KM25 for forward primer for CSO6) CSO6 (tggc)/ CS01 (acccc) GATCCCACGTGCTACAGAGCCACGAAGCCCATAGGCCTCGCCGATGGAATCCGTGCTCTG 10 GATCAAAACCAACCCGGTCAGCCTCCTCCCGGCCCCGGCCGGGGGGCGGGCGCCGGCGGC TTTGGTGACTCTAGATAACCTCGGGCCGATCCCTTCAAGGAAACTCCTGGGGTGACTCCT GTCCAGGGAATCATCCAAATGGGCCTGTTTCTGAAAAAGGCCCGAGTCACAGCTGTGACA GATTCTGTGGATCGTGGCTGGCTGGCTGCGGCGCTGGCTGGCTCGGCTGGCTGGCTG GCTGGATTCCCATGAGAGTCTGAGGATGGAACACATGGACAGAAAAGCATCCGATTCCCT 15 TTGGTCAAGAATCGGTCTCGCCTTCTGCGCCTGGTGTCTTTCCTACGTCTGGATGATTCC CTCCCCCACCCCACCCCACCCCACCCCACCCCGCCCCCGCTCCGCTCCCAGCTTGAAGGT GCTCTCAAGGTCCCGCCGGAACGCTCTCTTCCTCTCTTCGGAGCGCCCTTCTGAAGGGGA ACGTTTTCTTCCACGTCATCGCCCCGAGACAGCTTCAGCCTGGCCCTCCCCTCCACCCCC GCCTCCCTCTCTCCCTCCTGCTCCTCTTCCTCCTCCTCTGAACTCTTGAGCTCTCCTCGC 20 ACCGGCCTCTCACCCCACACGGTGGCAGTGTTGGCCTAGGTATGCTCAGGCGTCTCCTCC CCGCATCCCAGTGGACTGCCACTGGCTCTCTCTCGACTGCGTCGTCCTGGGACCATGTGT TTCCTGGCCCTTTCTGCGGGTGGGGGGAGACCCGGACGGGCCNGGCGGGGGTGTGGGGGA GCCTGCATGCGGGGGGAAGGGTGGGGGCAGAGAGGAGGAGGAGGAGGTGGNCGAGGAGGA GGAGGAGCAGGAGGACGAGGAGGAGGAACGACACAACTCCCGAGGTGCCAGTGTGTGCCT 25 GTGGCCCGGGAAACAGACGACGCACCGGGCTGGCTCCGAAAAGGGGATCCCCGTCCTTTG CGACCCATACCCTGTGTCCTTGCTATGTCAACATGTCACTCGATC KM2 (complete) C TCTTTCCCGCTNNANGGGGNAGCTTNAGGCCAACGTGTTCACTCTCCTCTTTGGGTTT 30 CCTCAAGAGGCTTTTTAGCCCCTCTTCCCTTGCTGCCATAAGGGTGGTGTCATCTGCATA TCTGAGGGGATCCGTTTCCGGAAAGACGGATACCCCCACGTCGCTTCTTTCTTTCTTGCT CCCCGTTTCTCTGGCCGAATTCCAAGTGATTCAGCCTCTTTTCCTCCACTCGTTTTCCTA CGACACGATCCCCCATGTTGTGCAAAAAAGCGGTTAGATCATCGACACTTCGAACGCACT TGCGGCCCCGGGTTCCTCCCGGGGCTACGCCTGTCTGAGCGTCGCTTGGCGATCGCCGAC 35 TCACTGAACGGAG KM6 (complete) CS02 GA TCGTGTCGCTCCTTTTCTGTTGTCTACGTGTTTCACGGCGAGTGAGTGAGAGAGTCTT TCGATGGTTTGCTAGGATGTGTGAATGTCGTGAGACCATGGTACTTGTCAGCCGTGGATG 40 AACAGAACGGCTTCAGCTTTCAGGGTGATCTCAAGTGCACTTTCCCCACCCAGCGGCGCC TGCTTGGGTTTGTTGTCTTCGGACTTTGTCACGGTCTCTACCCAGGTTGAGTTGTGTCTT CTCTCGGTGGGGGTTCCGAGTGTGTCTCCTCCTTTTCCTTTCTTGCTCCTGGGCTTGCTT GTCTGCGTCTGCTTTCCAAAGTCCTGCTTTGTTCTCCGAGCAGCGCTCGCCTTGGTTTCG

CTTTGCCGGCCCCTCCCTCCCTCCCTCCCTCTCTTTCGGGGGAGGGGGGGCCGGGGGAGT

WO 2006/089366 PCT/AU2006/000240 - 31 CTGCGATGCCGCTCGCTGGTGCCCCTCTCTCCGCCGACCCCGCCCCGAGCCCCCACCGCC CGCCGGCGTCTCCGTGGAATGTCCCCCCAGCACCCCGGAATCGCGTGOGGGAGTGAGTCT CCTTCGTGGCAGCCTCCTGAGGA 5 KM8 (complete) GATCTCGGcGAAGCACAGAAAGCCAGAC-AGTTGCATGAACCTGACCGTCACGCTTTCAGAA GCCAAGGGAACCAGAAATGAGGTTCACTCGCGTGTGGGTCTGTCTTTCCACGGGACGAAT CCTCTCTTTGAGCAGATGAGGGTTCCGGGGGCCCCGTGGAGCAGAGAGGATAGAGAGTTC CCTCAGGTCCCCTGCTCCTCCCATGCACGCGCACGCTCCCCAACGGTCCTAGGAACAGCC 10 TGCCCCAGAGGAIGCGTGCTGGCCACAACCCACCTCCACGGAGACGGABGACG.GCAGTGTCC GTCCGCGTCAGTCATCCTCGTCCAG:-GTCCCCGGGCCGTGGGCCCTCGCCTTCACGCCTG GCA.CCGTCCGTTCTGTAGGTGTGTGTCGAACCTGCCCGGAGCCCTGTGGCATCGTCCCG KM9 (incomplete, centre missing) '15 GATCATCNTCNCGCTCCWTNGAANGCNGTCCTCNNC2\AAAATGACCCANAGCGCTGCCGG CNCCTGTCCTACTAGTNGCATGATAAATAANACAGTCATAAGTGCGGCGACGATAGTCAT GCCCCGCGCCCACCGGAAGGANCTGACTGGGTTGAAGGCTCTCAAGGGCNTCNGTCGANG CTCTCNCTTATGCGACTCCTGCATTNNGAAGCANCCNNTTAGTAGGTTGANGCNGTTGAG CACCNNCGCNNCANGG2L/TGGTGCATGCAAGGAGATGGNGCCCANNAGTCNCNCGGNCAC 20 GGGGCCTGCCACCATACCCNCGNCGAAACAAGCGCTCATGAGCCCGAAGTGGNGAGCCCG ATCCAAAGAGTGGACAGGACGGTCZGGTGAGTGCCATATGCz-.GGAA-AGGA- GNCAACC CACNAACACCCTCCCN.ACGGTGGTTGNGTTCANTCCAA&GATCAGNTCCTTTGACTAGCGT TGGTACGACGGCNACCACNNGGGGGATGGAGAAACACAACNGTTGGTTTCTTTTGGACGA NGAGCCCCCCTCTGTGTGTGTGTGTgTGTGTGTGTGTGTgTgTGtGt gTGTgtgTgAGAg 25 A........ACGCCAGAGTTTTCCCGANAGAGAGAGAGAGAGAGAGAGA( ACAGAGAGAGAGA GATGCGGATGGGGATGGGAGGAGGGGTGCGTGGGTGGGGCGGATC KMI1 (complete) GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC 30 GAGCGTGACACNACGATGCCTGCAGCAATGGCACAACGTTGCGCAAACTATTAACTGGC OAACTACI'TACTCTGCTTCCCGGCACAATTAATAGACTGGATGGAGGCGGATAAAGTT GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGA GCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCC CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATG.AGCGAAGTGGGCAG 35 GCAGGGGGCCCCCCGAGCAGACACCTTCCTTCCAAAG~AAAGGGAGAACAGACAGACACCC AGAGCACAAGGAGACAACAAATCANCGGCAGGGCTGGGCCGGGCTGGGCTGGG4CTGC TGGGGGTGGGGGCGGGCTCACGGAAGCACCCCGGGGCGTTCATCTGGACATTGATCGTGT CGCTCCTTTTCTGTTGTCTACGTGTTTCACGGCGAGTGAGTGAGAGAGTCTTTCGATGGT TTGCTAGGATGTGTGAATGTCGTGAGACCATGGTACTTGTCAGCCGTGGATGAACAGAAC 40 GGCTTCAGCTTTCAGGGTGATCTTGGACTGAACACAACCACCGTGGGGAGGGTGTTCGTG GGTTGGCTTCCTTTCCTTTCCCTATGGCACTCACCTGACCGTACCTGTCCACTCTTTGGA TCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGAGTATTCTATAGTGTCANCTAAGNAT CAANCTT 45 KMI2 (complete) GATCTGTCTATTTCGTTCATCCATAGTTGCCTQACTCCCCGTCGTGTAGATAACTACGAT ACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACC GGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC

TGCAACTTTATCCGCCTCCATCCAGTCTATTA-ATTGTTCCGGGAAGCTAGAGTAAGTAG

WO 2006/089366 PCT/AU2006/000240 - 32 TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCA TCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC KMI 5 (complete) CS03 5 GATCAATGTGTCCTGCAATTCACATTAATTCTCGCAGCTAGCTGCGTTCTTCATCGACGC ACGAGCCGAGTGATCWITCACGATAAGGGCAGGGAATATGGAGATGGAGCAGCGACCATCA GCCCAACACATGAA2AATCCTTTCCCCAATGTGGCCCTGAAGGTCTATTGAGTCTTCAGAG AGTGATCCGTTTCTGGAAAGACGGATACCCCCACGTCGCTTCTTTCTTTCTTGCTCCCCG TTTCTCTGGCCGAATTCCAAGTGATTCAGCCTCTTTTCCTCCACTCGTTTTCCTACGACA 10 CGATCGATTCCCAAAGAGAACGTTCCTTCACTCAAAAAGTCTAGGATTGCTCCCCTTCA GACGACTCTCTTTCCTTTTCTACATTCCAACGACATGGATTCATCTATTCCCAGGTGCCT AAGGATATGGAGGCCTGGCGGCCATCACGGACTCGACCGTGAGAAAAGCCCTGTGCTCGC GAAGACTCTCCAGAGACTCCCAGACTCTCTGTGCTGTTTACGGTGGAGAGGGAGCCGACG OTCGTGTGCGTCGTGGCGGGAGGGTGGGTGACCCTGTCACGCGAGCTAGTCTGTCAGCAG 15 AGAGGTTTGACCCGAGACGCCCTTGTCACACCCAGGGCCGGGCGTGAGCCGTCATGACTG GNCCGACACGTGAAACACCCTTCACCCACGTCATTCCTGACCAACCCACTAGACTCATCA TTTCTAGGTAGACGCTGGCTTTGGGGGGAGAGCTTGGGGAACGGGGGGNTTCCTGAGGCT T 20 KM25 CS06 TCGGNACTCTCATGG1NTAATCCAGCCAGCCAGCCAGNCAGCCAGCCAGCCAGCCAGCCAG CCAGCNAGCCNCGATCGTGTCGCTCCTTTTCTGTTGTCTACGTGTTNNACGGNGAGTGAG TGAGAGAGTCTTTCGATGGTTTGCTAGGATGTGTGAATGTCGTGAGACCATGGTACTTGT CACGGAGAAACGTCGTTCGGGTCGATCAGCA 25 GGAGGCCCTGTGCGTCCCTGTGTGCTGCAGGACACCGTGCTACCCACATCTTGATCTTGG ACTGAACACAACCACCGTGGGGAGGGTGTTCGTGGGTTdGCTTCCTTTCCTTTCCCTATG GNACTCACCTGACCGTCCTGTCCACTCTTTGGATCCTT( ATCTCCCCCTCGCCCTCGAGG CCATCGGTCGGTCCTTTTCTTTCTCCTCCTCCTGCTCCCCGTCCTCCTACTCACCCTAGT TTCTCTCCCCGCCTCCCCACTCCCCGCCCCTCCACACACACACACACACACACACACACA 30 CACACACACACACACACACACACACACACGCAAGTCCCG;CTCTCTCA ATGGATCTCTCG CTGACGGCCGACGTTTTCCTTTCGCCTTCTTTCCTTCCTCCCGTCCTGCTTCCTTTCCCT TTGAGTGNGTGTGTGNGTGTGTGNGTGTGTGTNTGTGAGTGTGTGTGTGTT KM27 (complete) 35 ATCCCCTGGAGAAGGA21ATGGCAACCCACTCCAGTACTATTGCCTGGAAAATC!CCATGGA CAGAGGAGCCTGGTAGGCTACAGTCTATGGGGTCGCTAAGAGTTGGACATGACTGAGCGA CTTCACTTCACTTCACTTCACTTCATAAGGTATTGAAAATGCTGAGTGCTCCATTCCTTT TAAAGGAZATTTAAATGTTTTGTTGTCTTTATTCCTAATGACAAGGACCATGATGGAATT TAGACCCACTGTCCGCCCACCTATCCATCCATCCAGGCAGCCACCATCCACCTGTCCATG 40 ATC KM30 (complete) GATCCCATTGCAGCCCCAGCTCTCATCTCCTAAGTGGCTGGGGCGTTTTGTTTACTGTTA CTCAGCCTCTATTTCCTCACACGTACGTGCAGATATAAGAACACATTCCAGTTGTCTGG 45 CTGTAGTGTTCAGTTCAGTTCAGTCCAGTCGCTCAGTCATGTCCGACTCTTTGCGACCCT ATGAATCGCAGCATTCCAGGCCTCCCTGCCCATCCATCTCATGTCCATCCAGTCAGTGAT GCCATCCAGCCATCTCATCCTCTGTCATCTCTTTCTCCTCCTGCCCCCAATCCTTCCCAG CATCAGGGTCTTTTCCAATGAGTCAACTCTTCACATGAGGTAGCC.AAAGTATTGGAGTTT CAGCTTTAGCATCAGTCCTTCCAATGAACACCCAGGACT GATC 50 WO 2006/089366 PCT/AU2006/000240 - 33 KM31 (complete) GATCTCTGATAGATAAGCAAAGGTTAGACCTGTCCTCAGAACTTTTCTGTATGCTGTGAA TGGTTCAGTTCAGTTCAGTCGCTCAGTCGTGTCCGACTCTTTGCGACCTCATGAATTGCA GCATGCCAGGCCTCCCTGTCCATCACCAGCTCCCGGAGTTCACTCAGACTCATGTTCATT 5 GAGTTGTAGTTGTACCTTTTACTAAAAGTTAATTACTGTCACACACAAGCGTAGTACCA CTTAGTAATCATTTATTAAGTGTTGTTGTTCAGTCGCTAAGTTGTGTCCGATTCTTTGTG ACCCTAAGGACTGCAGCACGCCAAACTTCTTTGTCCTTCACTATCTCTCAGAGTTTGCTC AAACTCATGTCCATTGAGTTAGTGATGCCATCCATCCATCCCATCCTCTGTCATCCCCTT TCTCCTCCCGCCTTCAATCTTTCCCAGCATTAGGGTCTCTTCCAATGAATCGGCTAAATC 10 TATTCAAATATATCTTTCATTTACATGGTACGCTTCATCCGACTTGGAATGATTCAGAAC CTTTCTAAAAAtAAACACTAGGTAAAGAGTAATTTCCTCCCAGATACACAtATGGGGAAA CAGtAAGAATTCACAGGCAACCCTGGGAGTAAACAGAATGGA TC KM32 (complete) 15 GATCCCATGGAATCGCAGCACGCCTGGCCTCCCTGTTCATCACCATCTCCCAGAGTTCAC TCAGACTCACGTCCATTGAGNCAGTGATGCCATCCAGCCATCTCATCCTCTGTCATCCCC TTCTCCTCCTGCCCCCAATCCCTCCCAGCATCAGAGTCTTTTCCAATGAGTCGACTCTTC GCATGAGGTGGCCAAAGNACTGGAGTTTCAGCTTCAGCATCATTCCTTCCAAAGAAATCC CAGGGCT GATC 20 KM33 (complete) GATCCCTACATTGTATTTCCTAGAATTTTATAAAAGTAGAATCATATAGTCTGAAAAAAA TCTTTGTATGGATATATACTTTTATTTCTCTTACGAAGGCAACTTTTTTATGTCTTTGTC CTCTCTCCCTTCCTTCCTTCCTTCCTAACTTCTCTCTCCCTCTCTCTTTACCATGTCGTT 25 CTACAATTGTTCTGGTACTATTTGTTGAAAAAGCAAATCACACTTTCAATTTTGTCAAAA ATGTTTGACACTCTT KM35 (complete) GATCCCGTGAACTGCAGCAGTCCTAGCTTCCCTGTCCTTCCCTAGCTCCTAGAGTTTGCT 30 ACAACTCATGTCAGTTGAGTCAGTGATGCCATCCATCCATCTCATCCTCTGTCTCTCCTG TCTCCTCTTG KM37 (complete) GATCCCATTGCAGCCCCAGCTCTCATCTCCTAAGTGGCTGGGGCGTTTTGTTTACTGTTA 35 CTCAGCCTCTATTTCCTCACACGTACGTGCAGATATAATGAACACATTCCAGTTGTCTGG CTGTAGTGTTCAGTTCAGTTCAGTCCAGTCGCTCAGTCATGTCCGACTCTTTGCGACCCT ATGAATCGCAGCATTCCAGGCCTCCCTGCCCATCCATCTCATGTCCATCCAGTCAGTGAT GCCATCCAGCCATCTCATCCTCTGTCATCTCTTTCTCCTCCTGCCCCCAATCCTTCCCAG CATCAGGGTCTTTTCCAATGAGTCAACTCTTCACATGAGGTAGCCAAAGTATTGGAGTTT 40 CAGCTTTAGCATCAGTCCTTCCAATGAACACCCAGGACTGATC KM49 (incomplete) ATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGG ATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGG 45 ATGGATGGATGGATGNNNTNCAGCTAGGNANGCCTTCCTTCCTTCCTTCCTTCCTTCCTT

CCTTCCTTCCTTCNTACTTNNNTTNNTT

WO 2006/089366 PCT/AU2006/000240 - 34 KM61/62/63164/65 (complete) GATCCCAGGGACAGACCTAAAACACTGCTTTACACACAGCCTTGGCTTTCACTGTTCAGC CATCTCTCTCTACCAATGGACAGTGAGTTGTGGGGGTGAGGACCATGCCCATATCATTTC TACATTTCCACCTCCCAGCAAGGCACCCAGGAGGACCCTGGAATAATCTGTCAGATGGAT 5 GGAAGGATAGATGGATGGATGGATGGACGGATGGATGGACGGATGGACGGACAGATGAAT GGATGGATGGACAGATGGATGGGTGGACGGACGGATGGATGATGGATGGACAGATGGATG GATGGATGGATGGATGGATGGACAGATAGGTGGACAGATGAATGGATGGACAGACAGATG GATGGATGGACAGACAATGGATAGATGGATGGATGGATGGATGGATGGATGGACAGATGG GTAGATC 10 KM75 (complete) GATCAATTATTAGAACTCTATTGCATATGTCCAAAAAATTTAAGTAGAGCCATCAGTCCA GTTCAGTTTAGTTCAGTTCAGTCGCTCAGTCGTGTCTGACTCTTTGCGACCCCATGAATC GCAGCACGCCAGGCCTCCCTGTCCATCACCAACTCCCGGAGTTCACTCAGACTCACGTTC 15 ATCAAGTCAGTGATGCCATCCAGCCATCTCATCCTCTGTCGTCCCCTTCTCCTCCTGCCC TCAATCCCTCCCAGCATCAGGGTCTTTTCCAATGAGTCAACCCTTCTTATGAGGTGCCCA AAGTACTGGAGTTTCAGCTTTAACATCATTCCTTCCAAAGAAATCCCAGGGCTGA TCCAA CCAGTCCATTCTAAAGGAGATCTGTTAGTGCAGGGAGCCCACTGTGTTGCCTGTATGTTC TGTGTCTTGGTTCAGCCGCTGTGGACCCTGAGTGAGCTCTTCTTTTGGGACGCAGCTACA 20 GTTGGATTATCTGGGCCACATGCGCTCATCAAGCTTCCCAGTTGGCTCAGTGGTAAAGAA TCCCCTGCAATGCAGGAGACACAGAAGCCTCGGGTTCAATTCCTGGGTCAGAAAGATC MNS242 (incomplete) GATCATATTCAGAAGAAATTATTAAAACCATAAATTTCTATAAGGGAAGCATGGGTTTCC 25 CTTGTGGCTCAGCTGGTGAAAGAATCCGCCTGCAATGCAGGAGACCTGGGTTCGATCCCT GGGTTGGGAAGATCCCCTGAAGAAGGAAACGACAGCCCACTCCATTACTAGTGCCTGGAA AATCCCATGGACGGAAGAGCCTGGTTAGGCTGCAGTCCATGGGATCGTAAAGAGCCAGAC ACGACTGCGTGACTTCACTTTCACTTTCATAAGGGGAGCATATTAGTTCTAAAGCATTAG TTAACAACACCTTGCTGATCTTTTTGCAAAATTTCAGAAAATAATTGTATGTGCGCTCTC 30 TCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACACACACACAGTTTC TTTTCTGAGGGACCTTGAGAGTAAGTGATCTTAATGCTTCCCTTTGCAGACAGCACAATT CGGGGTGAGGGGGTGTTGTCCATGGTGCTGAAGTTGTCAGGGGCAGAACTAGAAATAATT TCTTGACTGCAGTCCATTTCTTTTCCGTGTGATTATGTTGCCTCATCCAGTATATTGTGG GTCAGGGTCAATCTGTTGTCTCCTTTGCTCTGAAATCTCTGAAATGCTCCTAGGGTGCAT 35 CCTCACGCCAACCAGCAGCTGCTTTCTAAAAGGAGCATTTGAATGCAACTCTGAATCCTG AGGAGGAAATGGTTTTCACTGTGGTTTGAAATCTTTTCTATACTCTCTCCACCCACGTAT A KM85 (incomplete) 40 ATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGG ATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGG ATGGATGGATGGATGGNNNNCTGCTANNNNNNNCTTCCTTCCTTCCTTCCTTCCTNNNTN NNTNANTTANTNNNTNNNTNNNTNCNTNNNT 45 KM86 (complete)

AGGCCTTCCTTCCTTCCTTCCTTCCTTA

WO 2006/089366 PCT/AU2006/000240 - 35 KM87 (complete) AGG4-AGCi-- AAggagg-AGGGGGAGGTGGAGGGAGGGGTCTCTCTGGCTGTCTCTC TAGG.AGTCTATTCAAGTCAALAGTATGATAGAGCTGGAG-GAACTTGATTCCAATGTGGT CTAAGCCTGTGCTTTCATGTAg/ cATATGA2TGGATCTTCTATAGTTGAGGTA -GGCTCA 5 a /gAGATGCTTCTCAAAAGTCACACAGCAAGAGTGTTGATATGTCTTCTTGATTCTG~g/ tGGAGTGTTCCCTTCCCTACGTTAGGTTTCATTTGAGACATTTCACATTTCCTTCCATAT GTCCATCCATCCACCCATCCACCCATcATTGCATCTATGGTTCTATCCATCCATCCGcC aTcCATCGCCATCCACCCATACACCCATCCATCCATCATCCALTCTATCCATCATCCATCC ATCCATCATCCACCCATCCACCCATCATTGCATCTATGGTTCTATCCATCCATCCATCCA 10 TCCATCCATTGCCATCCACCCATACACCCATCATCCATCCATCCaCCCATTCATCCATCC aTcCATCCATTcaTTCATTCaTCTATCCATCCaTCCATCCATCCATTCATCACCATCCAc Cc aTCCATCCaTCCaTCCATcCaTA KM89 (complete) 15 z-k~-AOAGLG-AG kGGGGTGGGGGTTTTGTTTT TAGGAGTCTATTCAAGTCA2XAGTATGATAGAGCTGGAAGGGAACTTGATTCCAATGTGGT CTAGCCTGTGCTTTCATGTAGATATGAATGGATCrTCTATAGTTGAGGTAAGGCTCAGA GATGCTTCTCAAAAGTCACACAGCAAGAGTGTTGATATGTCTTCTTGATTCTGGTGGAGT GTTCCCTTCCCTACGTTAGGTTTCATTTGAGACATTTCACATTPCCTTCCATATGTCCAT 20 CCATCCACCCATCCACCCATCATTGCATCTATGGTTCTATCCATCCATCCACCCATCCAT CGCCATCCACCCATACAOCCATCCATCCATCATCCATCTATCCATCATCCATA KM92 (complete) Z4CC-ATGGATGAGTGGATGGAG-CA.2GG-AAAGATGGATGGGTGGGTAAAA- GGATGGATGGA 25 TGGGAAAOAGAAAGAGGTATCTCTCT~kCTT AGACCGTCATGGGCGCTGGTCZ--C-GAGCCTTCAC-GGACTGGACTTGGACTGAACTTGGT TGAGAGAGAGCCCAGAGTGGTGGGAGTCTC%C-flGTGCTGCGG IGGATCCATGACTTTGT CCACAALGACCATGCTCCCCCCA.TCCAGCATGTGGTCTTCCAGAGTCACTGACTCAGCTTC TCTCCTGCTCTGGACGGAACCC P-GTGCCC-GAGCTGACC4G-GG 30 KM93 (complete) ATCGATAGATAGATAGATAGACAGATAGAAAATAGACGTATAGATAGATAGATAGATAGA TAGATAGATaGATAAATAGATAGATAGATAGATAGATAGATAGATAGATAGACAGAGAGA CAAAAAAAAAAAAAAAGTGTGCGCGCGTGCG 35 TAGATAGATAGATAGACAGATAGGCAGATAGATAGATAGATAGACAGATAGATAGAGAGA GAGAGAGACAGACAGACAGAGAGACTGACACTAGCTGATGGCGCAATGAA-AAGTGATCc KM94 (complete) GATAGTTAGATAGACTGCGTGGATGGATGGATGTATGGACAGACAGATAGACTGGATGGA 40 TGGATGGATGGATGGATGG;ATGGATAAATAGATAGACTGGGTGGATGGATGGATGGATAG ATAGACTTGATGGATGGATGGATGGACAGACAGATAAACTAGATGGATGGATGGATGAAT GGATAGATGGGTAGATAGACTGGGTGGATGGATGGATAGACAGATAGATAGACTGGGTGG ATGGATGGATGGATGGACAGACAGACTGGATGGATGGATGGATGGATAGATGGGTAGATA GACTGOTGATGGATGGAGGATGGATAGTTAGATAGACTGGGTGGATGGATGTATGGA 45 TGGACAGACAGATAGACTGGATGGATGGATGGATGGATGGACAGACAGACTGGATGGATG GATGGATGGATGGATGGATGGGTGGATGGGTAGATAGACTGGGTGGATGGATGGATGGAT

GGATGGATGGATGGATGGATG

WO 2006/089366 PCT/AU2006/000240 - 36 KM95 (incomplete) AGATAGCCACCAGCTAGCCAGACAGACAGAAAGACAGCCAGGCACCCAGACAGACAGAC AGACAGACAGACAGCCAGGCAGCCTGACAGACAGACAGACAGACAGCCAACCAGCCACAC AGCCAGCCAACCAGCCAGCTAGACAGCCAACCAGCTAGCCAGACAGACAGAAAGACAgCC 5 agAcagACAGAcagacaGacaGAcagACagacagaCagCCAACcagaCagaCaGCCagcc age cagac KM96 (complete) atGGATGGATGGATGGACGGGCGGATGGATGGGTGGACGGATGGGCAGATGGATGGATGA 10 CAGATGGATGGATGGATGGATGGATGGATGGATGGTTGGACAGACAGATGGATAGGCAGA TAGATGGTTGAATGGACAGATGGATGGATGCATGGATAGATGAATGGATGGATGGACGGA TGGACAGATGGATGGACGGATAGACGGATGGATGGACAGATGGATGGACAGGTGGACAGA TGGATGGATGGTGGGTGGATGGATGGATGGATGGATGGACAGATGGATGGACAGAtggat GGATGGACAGACGGATGGATGGGTGGATGGGCAGATGGATGGATGGATGGATGGGCAGGC 15 AGGCACTTGGGAACCCACAGGTTTCCCCGGAAGCTACAGGCAGGAGGTGGCATGTATGTG AATGGTAGATGGGATCTGGGTGAGAGAAAGGACAGAAGGTCACACCTCTGGAGACCCAGT GAACCGAGGTGCCTGATGGGTTTCTAAG KM98 (complete) 20 GATTCAGACAGGCAGAGAGATTATATGTACCAgAAGAAATAgACaGACAGAGAACATATG TATATaCAGAGACAAACAGGCAGAGATTGTTGTAGAAGAACAGACAGGCAGACAGACAGA CGGCAAACGAGATTGTGAGGGAGGGACAAAGAACCACAGAGGGATTATAGGCCTGAGGCG ATGAAGAGTGTGTGTTTGGTGTGAGGTCCTCGAGCGTTGAGTTCCCCAGCAGCACTCGAC CACTGACCATCTGCCACGCCCCAACCTACTACCCTCCTCCTCCCTCTT 25 KMIOI (complete) AAGGGGTCGCTCCTCTTTGCAGCTGCCGTTCATATGTTTGGGGGAGTTTGGCTCTAGAGA AGCCAGGGTCACGAGTTTAGGCTCCATGATGTGGGGGAGCAGACCAAGAAAGTAATTTGG TGCTGGTCTACAGCGCCTGGGCAGAGCTCTGTCCATGCCTGCCTTGGTCCTCAGGTGGGA 30 ATCAGGATGGTTCACTGTAGCTCCCCATGGGTGCAGATAAAACTGCTTAGAGCACCAGCG TAGAGAGATAGGCAGAAATGATAGAATAGATTAGATATAGAGGATGGGTGGATGGGTTAG GTGGGTAGTTGCATGCATGGGTTGaGGGGTGGCTTGGTGGATGGATATGAATGGATGGAT GGTAGCTACGTGGATGGATGTATAGATGGGTGGATAGGTGAATGTAGATGGGTAGATAAT AGATGGATGGATGGATGATGGATGGATGAATGGG 35 KM102 (complete) GATTCAGACAGACAGAGAGATTATATGTACCAgAAGAAATAGACAGACAGAGAACATATG TATATACAGAGACAAACAGACAGAGATTGTTGTAGAAGAACAGACAGACAGACAGACAGA CGGCAAACGAGATTGTGAGGGAGGGACAAAGAACCGCAGAGGGATTATAGGCCTGAGGCG 40 ATGAAGAGTGTGTGTTTGGTGTGAGGTCCTCGAGCGTTGAGTTCCCCAGCAGCACTCGAC CACTGACCATCTGCCACGCCCCAACCTACTACCCTCCTCCTCCCTCTT KMI04 (complete) ACACACAGGATAATCTTCGTAATGTCTTCGTAGTATGAGTTGCTTTGTGCGAGCGGTGGT 45 TACAGAACTGTTTGCCTGTGCAAGACTGGTAGTGGAAGGCTGGAGTGAAAATTCCGAAGT GGTGCGTCTAATTCTATATTAGCTTCTGTTTTTTCATTATGGGGTCTCTCGTGATGTGGA AGATAGTGAAACTAAACTACGTTTCAGGATTGTATGGAAGACACGTCTCTCTCTCTCTCT

CTCTCTCTCTCTCTCTCTCAATCTATCTTATCTATCTATCTATCTCACTCTGTCTGTCTA

WO 2006/089366 PCT/AU2006/000240 - 37 TCT-ATCTATCTATCTATCTGTCTATCTgt cTATCTATCTATCTATCTATCTATCTATCTA 'CTATCTATCTZATCTATCTATCTTTCTACTGACTTTCGGC KMI 05 (incomplete) 5 GATAGTTAGATA~GACTGGGTGGATGGATGGATGTATGGACAGACAGATAGACTGGATGGA TGGATGGATGGATGGATGGATGGATAAATAGATAGACTGGGTGGATGGATGGATGGATAG A.TAGACTTGATGGATGGATGGATGGACAGACAGGTAAACTAGATGGATGGATGGATGAAT GGATAGATGGGTAGATAGACAGGGTGGATGGATGGATAGACAGATAGAAGACTGGGTGG ATGGATGGATGGATGGACAGACAGACTGGATGGATGGATGGATGGATAGATGGGTAgaTA 10 GACTGGGTGGATGGATGGATGGATGGAtaGTTAGATAGACTGGGTGGATGGATGGATGGA TGGACAGACAGATAGACTGGATGGAT4A1TGGATGGATGGACAGACAGACTGGATGGATG GATGGATGGATGGATGGATGGGTAGATAGACTGGGTGGATGGATGGATGGATGGATGGAT GGATGGATGA 15 KMI06 (complete) CCAATGGATGAATGAGTGGATGGGAGGATAGACAGGgagATGATGCaCTGATAgACGCa / gT2AMAGATGGGTGAGTAZAATGGATGGATGGGCAGATGGAAGAaTG~a tGGatGGGTGG ATAGAAZATATGGGCAGGTAAAGGGAGGAAGGGATGGGGAGACGGATGAATGGATAGGTGG ATAGGZAAGATTGCTGAGTGGATGGATGGATGGGTGGATGGATGAATGGATGATGGACGGT 20 CCAGTAGCAAGGTGGATGGGCGGGTGGCTAGATGTATGGATGGAGAGGAGTGAATGTcaa aaGGAAGACC KM 107 (complete) ggggatgGAGGAGTGGAACAGTGAATGGACAGCAGCCGAgAGAGAGGAGCAGCTGGAGAT 25 GGCGGacGatggatgGgCGGGTGGATGGATGGC-TGGATGGATGGatGGGcGGATGGaTGA ATGGGCGGATGGATTA.ATGGAtGGAtGGAtGGATTAATGGGTGGaTGGATGGATTAATGG GTGGaTGGGTGGATGAATGGGTGGATGGATTAATGGATGGATGGGTOGGTTAATGGGTGG ATGGATAAATTAATGGGTGGATGGATGGATTAATGGATGGATGGGTGGATTAa tgggtgg aTGGATGGATGAATGGGCGGATGGatgaatgggCGGATGGATGAATGGGCGATGGATTA 30 GTcGG'GGATGGATAACAGtgaGtGaaTGAgTGAAAGGATGG KM108 (complete) ACCGTTCCCAGTTAAGTAATTCAGCTGTATCGTGACTTGCAGAAGGTAGAGAGAGAGAGA AAGAGAGAGAGAGAGAGAAAGGGAGAAA2&GATAGATAGATAGATaGaTAGATAGAGAT 35 AGAGGAGAGGGAGAAAAGGTAGATAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAA GGGAGAAAAGGTAGATAG-ATAGATAGATAGATAGAGAGAGGGAGAAAAGGTAGATAGAGA GAcAGAGAGAGAGAGAGAGAGAGAGGAGAATACATGGCGGAAGTTGAGGCGAAGAGAGga cagcaGCGAGTGTTTATGTTTGTGCC 40 KMI09 (complete) ACTCTCTTAGTTTCTGCGATGAACTCACTATTCTTATCTTTTCAACCGACATGGCTTAGA CTGGCATACCTTCGCCTGTGCCATGGAGGTTACAGTGAGTzg2-a,- GACAGG-,--2-AACAG ACG-.z 2.CGCGCGCTCGCAAAR21 -AAAAAGCGCG CAGACAGAGCGACAGACG2AC2AG-F2U-ZAGCAGACAGACAGACAG-A2-,jACAAACAGATAGA 45 CAGACTGACAAGCzRAGA2AGC KMII10 (complete) ATCAAQCAGAATATTAATGACGAGTTCTGAATTTTTGGTCTGTCGACCTCTTTTCCTTC

TTTTTTACCTATTTCTTTCCTCAGTGAAGCGAATAT.AATGTCTATCTGTTTATCTGCCTA

WO 2006/089366 PCT/AU2006/000240 - 38 TCTGTCTATCTATCTATCTATCTATCCGTCTGTCTGTCTGTCTACCACGCCTACCATACA TAAGGTCCCGTGTTCGAGCCCTGGCTGTTGGAGGGCTTGTGTTCTAAAAAAGCGTGCTTT TATATGCACTGTATTCGTGTGTGTATC 5 KM111 (incomplete?) atGaAAGCACAGGcTTAGACCACATTGGAATCAAGTTCCCTTCCAGCTCTATCATACTTT GACTtgaatAAACtCCTAGAGAGACAGCCAGAGAGACCCCTCCCTCCACCTCCCCCCTCC TTCCTTCCTTCCTTCCTTCCTTAATCGAATTCCCGCGGCCGCCATGGcGGCCGGGAGCAT GCGACGTCGGGCCCAAt TCGCCCTATAGTGAGTCGTATTACAaTTCACTGGCCGTCGTTT 10 TACAACGTCgTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGcagcacatc cCCCTTTCGCCAGCTGGCGtaaT KM113 (complete) aGAGAGAGAGACAGACAGAGAGAGAGAGAGAGAGACAGACAGACAGACAGACAGACGGAC 15 agacagAcAgACAGACAGACAGACAGACAGACAGACAGAcaGacagAGACAGAGACAGTC AGACAGAGACTGACAGACAGAGACAGAGACAGTCAGACAGAGACAGAGACAGTCAGACAG AGACTGACAGACAGACAGACAGACAGACAGACAGACAGACAGAGAGTGAGTC KMI14 (complete) 20 ACATATGOATAGTAACTTATATGATGACCAAATGAAGAACAAGAAATATTACGAAGTGAA AAGAATAATAAAGCAGGCGAACCAAGAGGCTGAGCAGCGTTcATAAAGTCATGATAATCA TAGACTGACTAATTATGGGATATGAGGGTATTGATGCCTTAAACAGAGAGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGACAGAGAGAGAGAGAGAGAGAGACACAGACAGACAGACAG ACAGACAGACAGACACACAGACAGACAGACAGACAGAGACAGAGACAGAAAGATTTATAA 25 TGAATGCAATGCACAATAGAGAGGGAGATACTAATAAGTCAGAGAAAACACGTAGCATCC TGAGGCAGACCTACAGATGGAGCAaGTCGGTGTTGTGAATATAAGGAGAGCCC KMI 15 (complete) GAGATGAATAGGTGGATGGATGGAGAGATGAATGAATAGATGGATGGATGGATGGATGGA 30 TGGATGACGGATGGTGATGGGTGGATGATGGGTGGATGACGGGTGGGtGATGGGTGGATA GATGAATAGGTGGGTGGATGGAGAGATGAATAGGTGGATGGATGGATAGATGGATGAATG ACTAGATGGGTGATGGATGGATGAATAGATGGATGGATGGAGAGATGAATGAATAGGTGG ATGGATGGATGAGGGATGGATAGGTGAATAGGTCGATGGATGGACAGATAGATGGATGGA TGGATGATGGGTGGATGATGGATGAaTagatGGaTGGATGGATGATGGATGGATGAATAG 35 ATGGATGGATAGAGAGATGAATGAATAGGCAGATGGATGGATGATGGATGTATAGATGGA TGGATGAATGAATAGATGGATGGATGGATAAATGGATGGATGCC KM 116 (complete) ATGATGAAGCCGACGCTGAAGGTGAt /ggATGGAGACGCAGATGAATACa/ga/gGGGGA 40 GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACAGACAGACAGACAGACAGACAGACA GACAGACAGACAGACAGAgACAGAGAGACAGAGACAGAGAGAGACAGACAGAGAGACAGA TGANNGACCCCTTGGAAGNNAGACCTTcTCTCAGTGATACNNTCTCNCTaANNGNGACNA CTCNCTCTCGATGTCACTTCCTACNGACGGAATCTGCTTCTAAACGNANCCNACNTTNAN NTAAAACTCTCTCTCTACAAACNNNNN 45 KMI18 (complete) GGATGATGGGATGGATGAGTGGATGATGGGATGAATGGGTGGGTAGATGATAGAATGAAT GGGTGGGTGGATGATGGGATGGATGGATGGGTGGATGATGGATGGATGGGTGGATGATGG

GATGAATGGATGGGTGGATGATGGGATGGATGGATGGGTGGATGATGAGATGGATGGATG

WO 2006/089366 PCT/AU2006/000240 -39 GGTGGATGATGGGATGGATGGTTGGGTGGGTGATGGGATGGATGGGTGGATGATGGGATG AATGGGTGGATGATGGCTGGATGACAGGTTGACGATGCTGGATGGGTGGGTAGGAAGGCT GCTATGCCCTGAGTGTTTGTGCCCCaccGGGTCTCACGTCTGGACTCTGGGACCACCGTC ACACTCACCTGGGTGTAGGTCTAtc tGGAAATTAGCGTCGTGAGGGTTTCTGGCTTCTGT 5 CCTGCGAGGTGACTGACCCAGTAGTCTAGTTTGTCCCCAGGAGCTTCTGTGCACTGAGGC ATCCTCGCCGCCCCAGTAACTAAGCAGCACCCCACTGTCAGGTAAGGGG KM19 (complete) GATCaTAgCATCAGTGGCAAATGAgATTCTTAAGAAATTGCTGTCTGt /gCTCAGTCTGt 10 CTGTCTGTCTGTCTGTCTCTCTGTCTGTCTGTCTCTGTCTGTCTGTCTGtCTGTCTGTCT GTCTGTCTGTCTGTCTATCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCT CTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTCTCTCTCTCTCTTTGCTAGACGTATGCA CTCACAAATGTACAATGTTGCCCACCATCTCTCTCTCTCCTTACCTTCCCTTTACccgAC GTGTGTGTTCTCAGTACGAT 15 KM120 (complete) GAAATCCAGTTGCCCTCATTTCCTCTTCCTCCCCATGGAGACCAGACCCATGGGCGGATG GATGGATGCATGAATGATGGATAGATGGATGGCGGATGGATGGACGATGGATGAATGGTG GATGGATGGATAGATGACGGCTGGATGGATGCACGCATGGACGGATGATGGATGGAAGAT 20 GGATGATGGATGATGGATGGATGATGGATGGATGATGATGCATGTATGGATGGATGATGG ATGGATGGGTGATGGATGAAGAATTGACGATGGGTGGATGGATGAATTGATGAGAGGATG GATGGATGGATGGGTTGATGGGTAAGTGGATAGATGGG KM121 (incomplete) 25 GTGGCTGGTGGGTTAGCTGACTAGCTAGCTGTCTGGTGTTTGTCTGGCTGGCTGACTGGC TGTTTGTCTGGCTGGCAGTTTGTCTGGCTGGCTGGCTGGcTGtCTGGCTGGTTGGCTGTC TGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGGCTGTCTTTCTGTCTGTCTGGCTAGC TGGTTGGCTGTCTAGCTGGCTGGTTGGCTGGCTGTGTGGCTGGTTGGCTGTCTGTCTGTC TGTCTGTCTGGCTGCCTGGCTGTCTGTCTGTCTGTCTGTCTGTCTGGCTGCCTGGCTGTC 30 TTTCTGTCTGTCTGGctaGotGGtTGGCTATCTCCCTTCTGCTAGCAAGGCCTTAAATCA CTAGTGAATTCgcGGccGcCTGCAGGTCGACCATAtggGAGAGCTCCCAACGCGTTggAT GCatagCTTGAGTATTCTATAGTGtcaCCTAAATagcTTGgCGTAATCATGGTcaTAGCT GTTT 35 KM123 (complete) AAATATATCGATAGATAGACAGATAGATAGATAGATTGaTAGGtaGATTGATTGATAGAT AGATAGATaGATAGATAGATTGATAgANcGatAGATAGATAGATaGataGATAGATTGGT AGATTGATTGaTTGATTGATTGAAAGATAGATAG 40 KM124 (incomplete) CCTGGCGTGCTGCGATTCATGGGGTCGCAAAGAATCAGACATGACTGAGCGAAAGAACTG AACTGAACTGAACTGAGTGGTTGGATGGCTGAATGGATGGATGGGtaGtTGGGTGGATAG GTGAGTGGGTGAGTGGATGGATAGAGAGATGGATGGCTGATTTACTAATTCTGGTTGCTA TAGCCTCCACTTCTAGAAGCAGAAATATGAACAGAAATCCTGTTTTCTGAATACTTTTAG 45 ACATATAAGAAGCAGGAATCTGTAAACCAGGATGTTCCTATGAGAGTCCTAGGCTGTTTT GCACATCCAAAGAGGTTTTGATACTTcaGAGAAGGCTCCAAACTTCGGATGccAATGTAA AGGAAACCCACCGAGGTTCACTTATAGcttGTTCACACAGATGTAAAGCCAGCTTTGATT TTCCCTAAAATCCTGcATGTTTTGCCACTGCTTCGAGGATTTTAGGAGAAGCTACCCTAA

AGACTATGACATTTTTCCCCCTTTGTTTCTAATCATACTAGGAAGCACTGATTTACTTTC

WO 2006/089366 PCT/AU2006/000240 -40 GTAGAGACTTGGCGATGCTTCAAGTTTGCCCACCCCCATGGATCTACAAAGTGCAGATGG CAGAGCAgGAGTAAAAACGAGACAGAaa KM125 (complete) 5 GACACAGACCGTGATCTTcAGAAGCCTGAAGGACACACTGGAAATTTGAGCCGGAGGGA AGGAATGAGCGGACTGTCTTCCCCTCCCCTCCGCAGAATGacCTTAAAAGAGAAAAGGAA AAAAGAAAGGAAGGAAGGaaGGGGGAGAAAGAAACAGAAGAAAGAAAGACAGAGGAGGAG GGCGCAAGAGAAAGAGAAAGGCAGGAAAGAAGGGCGGGAAGGAGGAAGGAAGGAAGGAAG AAAAGGAGAGATACAAAGAAATCAGTTCCTCTTGG 10 KM126 (complete) TTATGTTGCGTCAGAGZAGCATTAGATGGCTAGCTAATGGTTGGATGGATGGATGGCTAG ATGGATGGATGACTAGATAGATGGATGGATGACTAGATGGATGGaTGaccAGATGGATGG ATGGCTAGATGGATGAATGGCTAGATGGATGAATGGCTAGATGGATGGCTGGCTAGATGG 15 ATGGATGGCGAGGTGGATGGATGGATAGCTAGATGGACAGATGGATGACTAATGTTTGGT TGGCTAGGTGGATGGAGTGAAAAAGATTTTTTGTGATC KM127 (complete) GGAGAGTGcaTCACGGAACAACGCGAAgTCTTGTGACTGTTAATGGTGGGAGGGACAGTG 20 GAGGGTTGAgACAGACAGACAGAGACACGGAgAGACAGACAGAgacagagAGAGAGAGAC AGACACAGAGAGACAGAGAGGcaGAGACAGAgAGCCAGACAGAGACAGAGAGACGGAgAC AGACAGAGACAGACAGAGACAGGGAAAGACACACAGAGAGAGACCCAgAGAGACAGACCG GGNTCTAGCCCAGCACGTGTCTGCaCCTGcTGTCCCCAGAGGTAGGAGCACAGGGaTcCT GGcAGTCGTCAGCCCcTCTTCGCACGGGaacctcgcgcGcaCCATCTTCCCTCCTCACGG 25 GTGG KM128 (complete) GATCCTTCTCATAAGGTGCAgAcAGt /gCCACACGGGACACACTCCCTGGg/cTCTCTCT TCCTTCCTTCCTTCCTTCCATCCTTCCTTCTTTCCATCCTTCCCCCTTCCcTgCTTCCTC 30 CTTCCATCCTTCCTTCTTTAATCCTTCCCTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCT CCTCCCATCCTTTCTTCCTTCCGTTACGCTATCCTCCACGAGTGTTCCTTAGCATCCTCT GAAGGAGACCATCCTGGATTCTCCAAAAAAAAAGGAGGGTGTTCCTTGGAGTGTGCTCTT CACATCTTGCTGATGGGATGATCGTGGATGTCaTTCTCCAGCTGCCGCCGCTGCTGTTTG TTCTCATCTGGtGtGGGTACGTCGTGt aGtGtGtCAGATGGGAGTCTGAT 35 KM129 (complete) CCTCTGCTCTTCCAAGCTAACATTTGCTCCAGGGTCACCCATTGGTTGTCAAGACTGGGT CTTCTCCCTTTCCCAACACAGATAGACAGACAGACAGACAGACACACACACACACACATA CACACAGACACACAAACACAGACACACACACACTCTCCCCCTCAGGTGGAGACAGGAACT 40 GGAACTGGAAGAAGGGTTCTGGAATCCCTGCCAGTTGAGATTATGTTGCCTTTCTGTTAG AGGTGATGTTGAGATCTGGTAGCATTTGGAAAGCAGTAGAGGGTGTTGATGGGCTCCCCA GGTGGCACTAGGGGTAAAGAACCTGCCTGctAATGCAGGAGAAGAAGTAAGAGATTTTGG TTCAATCCCTGGGTTGGGAAGATCCCCTGGAGAAGGCAATGGCACCCCACTCCAGTACTC TTGCCTGGAAAATCCCATGGATGGAGGAGCCTGGTGGGCTGCAGTCCATGGGGTCGCTGG 45 GAGTTGGACACGACTCAGTAACTTACTTTCACTTTTCACTTTCATGCATTGGAGAAGGCA ATGGCAACCCACTCCAGTGTTCTTGCCTGGAGAATCCCAGGGACGGCGGAGCCTGGTGGG CTGCCATCTATGGGGTCACACAGAGTCGGACACGACTGAAGCGACTTAGCAGCAGCAGCA GCATCaAGTTTAATATCAACACTTGG WO 2006/089366 PCT/AU2006/000240 -41 KM130 (complete) ATCAGGc/ gGGGGAGGGACGGGGCTCCg/ aTGAaAGAGAGAGACAGAGACAGACAGACAG ACaGACAGACAGACAGACAGACAGACAGACAGACAGACAGAGAGTGAGAGAGAGAGAGAG AAGGTTACAGTACTGGAATGACGCAGAAACCGTCAAAGAGATGATGAAAAGAAGTGCAAT 5 TGCAGGTAAACAGAGATGAGGAAGAGAAGATAAGAAGAGAGAATGAGAAAGAAAGATAC AAATACAGACAAATACAAAAATAGATAGATAGACAGATAGATAGATAGATAGATATGATC KM131 (complete) GATCAAACATCTCGTACTGGAGGCATTATGGACAATGAAgGAGCGAGGAACAATGACgtG 10 CAAAGAAAACTAAAACTTACTGACAGACAGACAAACAGGCAGACAGACAGACAGACAGAC ATACAAACAGACAGACAGATAGACAGATAGACAGACAGACCGGCATAGTCAAAGGGATTT CATCTTCTGGACAATAAAGCTTACATAAAA KM132 (complete) 15 CCTTCGCTTACTGCTTACTGTTTTTGTGCCAATGGCAAGTAAGCAAGCATTAGACAGCAA GGGTCACCTGTCCTTCCCCAGTAGACCCAGAGCTGGGCACAGGAAGCTGtTAATTAGTA TTGTTGGAAAGAAAGAAGGPAGGAGGAAGGAAGTATGGAGGGAGGGAGAGAGGGAGGAA GGAAAGGAGGGCAAGGAGAGGCAAGGAGGAG TAGGGGCAGGGCATGGCTTCC CTGTGCAGCCAGTTTGGCAAAGTCATGCTGTGTTTTCACATCTCTCATGCACCTTTCTTT 20 CCTCATTTTTTTTCATCCTACTTTTATCcagtccttcaGCAGCTTACACATTCAGAGCAA ACGAATT KM133 (complete) GGATGGAGGAGTGGAACAGTGAATGGACAGCAGCCGAGAGAGAGAGGAGCAGCTGGAGAT 25 GGCGGACGGATGGATGGGCGGGTGGATGGATGGGTGGATGGATGGATGGGCGGATGGATG AATGGGTGGATGGATTAATGGATGGATGGATGGATTAATGGGTGGATGGATGGATTAATG GGTGGATGGATGGATGAATGGGTGGATGGATTAATGGATGGATGGGTGGATTAATGGGTG GATGGATAAATTAATGGGTGGATGGATGGATTAATGGATGGATGGGTGGATTAAkTGGGTG GATGGATGGATGAATGGGCGGATGGNNGNNTGGGCGGATGGATGAATGGGCGGATGGATT 30 AGTGGGTGGATGGATAGACAGTGNGTGAATGTGTGAAAGGATGG KM134 (complete) cCCAGGACACCTTGGAAGAGGAAATGGGAGGAGGGAGCGGTGGGAATAGGTACACCGGGG GCTCCAGCaTTTCCGAAGAAGAGGATAGGAAGTGGGGTAAAGGGGATGGGAAACTTGTCT 35 AGAAGATGCCTTTGCCCGGCAAACACGGATTCACAAAGACTGTATACTGAGGATGCTGG TCTTGGAGAAGCAGCTGGAAGGGATAAGGCTGCGGCGGAGGGGGACAGAGTCCATGCCTG ATTGGACAAATGGATGAATGCGTGGATGGATGGATGGATGGACGGACTAAGTGAGTGAgt GGTTGCGGGAACCTCAGACGTTCCCAAgttGGAGCAGCGCGCCCGGCAGGGGT 40 Example 2 - Locating Microsatellites in Bovine DNA Results A number of repeat elements were located in bovine DNA sequences. The repeat motif is highlighted in blue. From these located sequences, a number of primer WO 2006/089366 PCT/AU2006/000240 -42 sets were developed (highlighted in red, bold, itaficised and underlined, and shown at the end of the sequences). SEQ 2A AGGGAGAGGAGGCTCCGCTAAGCTCACAAGGAATGAGTGTGTGGAAGGGCCGATGGTCAGGCGTGGGCTT 5 TGGGAAGTGCCCCCCTCCCCGAAGATTTCAACCCTGGAGGGAAATCGGAGCTCAGTGACTGGCCTTCCTT GGCCAGGGGAGCAGAGCGCAGGCTGAACACGGACCCTGTGGCATTTGGATCCAACCAGGGACAAGTTCAC AGTTCCTCAATAAACTCGTGAACAGCACTTAATGTGTGTACGACACAGCTGGATCAGGAGTCGGGTCCAT CCTAGTGGGGCTTAGAGTCCAGTGACACTAAGTCTCAGCAATA AA TCCarrCACTArrT6CTCCTT CTCAATTGCTGTCTATCTCTCTCTTTTTCTCCTCTCTCCCCTGATCCACCCACCCACCCACCCACCCATC 10 CATCCACCCACCCACCCACCCACCCACCCATCCATCCACCCATCCACCTACCCATCCATCCACCCACCCA CCCATCCATTTTTCCATCCATCCACCCACCCGTT CACCCACCCaTTA TC-GSTC aT TGC CCTCTGTGACTCTCCCCGGCCCCCCAAGCCCTCTGAGACCTGCAGCCTGGTCTCGGCCCCCCACCCTCAG GGACAGCAGCAGGGCAGACAGGTTTCTCTCCCATCTCAGGAGCTGCCATGTCCAGCTGATTGCTGAGGCC AAATTCAAGGAATTAGCCTGGGTTCTTCTGCGCCTCACACCTCATATTAATCCACTAGAAGTTTCTATCA 15 CACTTCAGAACTGTTCCAAACGTTCCTAGTTCTCTCCGCCGCTCCTCTGACACCCCAGCCCTCACCACAC Bos1 9F: 5' AATCCACTCACCTGTACCTG 3' Bos19R: 5' AGAAGACCAGACGGGATAAG 3' SEQ 2B GGAATCTGCAGCCTTCTTCCAGGAGTGATGAAGGTGAGGAAACAGGGCCTCAGGAGCCCAGGGAATCCAG 20 CTTGGGAGAGTTTCCCAGGGTGATTTTCTGGGTTGGTTGGTTTGTTTTGGTTGGAAACGGGAAAAGCTAG ATCTGTGCAGAACCCACTTfA6IEIEGST gIZ2CAGAGCTCCGTGTCATGGGAGTAACTGTCT GCAGACAGGCTTCTCTCCTCAGTGCACCAACACAAGCCCACTGCTTGATATCTCAACACATAGAGGGGTG GGTGGAGGGGTGGAAGGGTGGGTGGATGGATGGGTGGGTGGATGGATGGATGGATGGATGGGTGGATGAA TGGATGGGTGGGTGGATGGGTGGGTGGGTGGGTGGGTGGGTAGATGAATGGATGGGTGGGTGGATGGGTG 25 GGTGGGTGGGTGGGTAGATGAATGGATGGATGAATGGATGGGTGGGTGGATGGGTGGATGGATGGATGGA TGGGTGAATGGATGGATGGGTGGATGGATGGGTGGGTGGGTGAATGGATGGGTGGGTGGACAGATGGATG GATGGATGGGTGGATGGATATATGGATGGGTATGGATGCATGGGTAGATGGATGGACCACTGAATATTCT CAGGACCCAGAAjGCITTAAGTTAATCAGATACATGAGAAAATTATAATGCTTCAAGGTGCCAATATTT CAACACTCCAAGTAACACAATGATTCAGCCCAAATCCTCAATATTACTTTAAGGAATGACACTCATGAGT 30 GAGATGTGAGAGTTTTCAGAAGGTTGCAGGCATTGACATTTTTTGGTCCCGAATGACACTGACTCTGCCT Bos 17F: 5'TTTTCCAAGGCTTGATTCTA3' Bos17R: 5'AGTGAGCGTCAGAGAGAAAG3' SEQ 2C CCACACAGATCCCAACTCT ThCAACrT T&TTTTCCTGTCCCACTTTGCTCTAAGGAACTTCAA 35 GAAGCAAAGGCAAAGCATCAGCTCAAGAACATTTGACTATCCATCCATCTGTGCATCCACCTGTCCACCC ATTCATCCACCCATCCCTACCCATCCATCTACCCATCCACCCACCCACTCATTCCCATTAATCCATCTAT CCATCCATCCATCCTCATCCATCCATCTGTCCACCCATCCATCCATCCATCCATCCACTCACTCATTCCC ATTCATACATCTATCCACCCACCCATCCATCTGTCCATCCATCCATCCACCTACCCACCCATCCATCTGT CCATCCATCCATCCATCCACCCACCCATCCACTCAACGTGTCCATTAACCATCTTCTATGTGTAAGGCAT 40 TTTGCTTGTTTTGTGAGGACAGATCAAAGGAAATCAAGTTATTGTTTCTATTCAAGAGAGATTTAAACTT GAAGGGAAGATTGAAGCAGAAGGGGGAACAGGAGAAAGATGGAGATGATATATATAAATATAAGACACAT AGAAACCCTACCAGGTCATAAATACATCECAA AACICAICAICTCCCCACAAACCACTTCCTTTT CCAGCCTTCCTCACGTGGCCGTCGTCCCACAGCTGTCTTCACGTAGCCTTTCACTGTATCCATCTCCTGT CCACCTCTATTGTTGTCAGTTATGCATTTGCCCACTACCTGAGGAGGACTGTACCTTAAACCTGGCATCT 45 GATGGCAGATCTGGTTCCTAGTCACCTCCTCATCCCTGGAGATGACTCCAGTTTTCAGAGGGAAGGACAC

TTCTCAAGGCCTTGGTTTATGCTGAAAACCACTCTTTTAAAAAAAAAAAAAACAACCACTTTTTATTTTG

WO 2006/089366 PCT/AU2006/000240 -43 TATTGGAGTATAGCCGATTAACAAATGTGATAGTTTCAGGTGAACAGTGAAGGGACTCAGTCATACAAGT ATCCATTCTTCCTCAAACTCCCCTCTGCCACGAGCCAGCGTGAGCCAGCGTGAGGAACTCCGCCCGTGGC AAAGGTCGTGAGGAAGGAGGCTCGGCATACAAAAAGGCGGGATCGAACCTCAGGAGTCCCCCTGGAAATT CTCGAGCATCTACCCCCAAAACCAGAGTCTGCCTACTTTACTGCTTTGTGTTCTCACCTACACCTCTGAC 5 TTTATGGGGGGCGGGGCGCGAGAGACATCAATAACCTCAGATAGGCAGATGACACCACCCTTATGGCAGA AAGTGAA BOS3F: 5'TTCCAACCTCTGTTTTCCTA3' BOS3R: 5'AGATGATGAGTTTGGTTTGG3' SEQ2D 10 TTCTCTTCTCGTACGTAGGTATTCTGGTCACACACAGAAGTTAAAGATCTAGAGAGAGGCATGTGGTTAG GAGAATTGGTTATTGCAGAGCGAGGCAGAGCTGAGTTTGCAGTCCAGCTCTGTAGCCTCACCTGTATACT CTCAGTTAATCCATAGCCTCTCAGTTTTCCCAGCAATAAAAGAGCTAGAATAGTCCTGCTTTCCCCATAG CATTGTCATAAG&4A TAAACSATS A GACAAGTGCTTAGCTTAGGGCTTACATGTTATTATAG TTGTTATGTCTTTTCTTCCTTCTTCCTTTCCTCTCTCCCTCCCTCCCGACTTCTTTTCTCTCTTTTTTCT 15 TCCTTCCTGCTCTTTTCTTTCTCTCTTTGTTCCCTTCCTTCTTCCTTTCTTTCCTTTCTTCTTTCTCTTT CTTTTCTTTCCTTCTTTCCTTCCTTTCATTTCCAACTGCTGCTTTGCCCATCTCGCTAACATCTTCTGAG AIGACfACAMGAGAICAAAGAGGAATATTCAGAATAAAAAGCGTCACTCTCCATTGGCCTTTGAAG CCCAGGGACAACCATGACGTCACATCTCATCTTCCTCTCCGAATAGAGAAGATTCAAGTGGCCCAATGCT TTCAGATGGGACGGCAGTGGCGTTAGCATGAGAAACCGGTTAAGGAGAGGTGTGAAGCTCTTCTGTGTTA 20 GAGACCGTCCCCGATCTGGCCGTCAGCTGCCTTTGGCCTCCTTGTCCTCTGCTTTCTCTCACGAGCTGGC Bos23F: 5'GAATAAACGAAATGCGAGTC3' Bos23R: 5'GTGATCTCTTTGTGGTCCAT3' Example 3 - Location of DNA Microsatellites in Sheep DNA Using Information From Cattle Repeat Regions 25 Materials/Methods Primers were designed from cattle genomic sequences which contained a suitable repeat motif. These primers were designed using the software program Primer 3. As an example, DNA from sheep was PCR amplified using primers BOS3F: 5' TTCCAACCTCTGTTTTCCTA 3' and BOS3R: AGATGATGAGTTTGGTTTGG 30 under the following PCR conditions: 950C 5 minutes 35 cycles of 94 0 C 30 seconds 52 0 C 30 seconds 72 0 C 30 seconds WO 2006/089366 PCT/AU2006/000240 -44 one cycle of 72 0 C 10 minutes.. PCR was carried out with a final volume of 10 ul, containing: 1 ul of DNA template and 9 ul of PCR master mix containing all four dNTP's, MgCh2, forward and 5 reverse primers and PlatinumTaq Polymerasem (Gibco). The PCR master mix was made up as 10 ml volumes containing 20 ul of 100 mM dCTP, dGTP, dTTP and dATP (Bmankein), 300 ul of 50 mM MgC2 (Gibco), 100 ul of 20 mg/ ml BSA (Gibco) and 8280 ul ultra pure water (Biotech). To 100ul of master mix, 200ng of each primer (forward and reverse) and 2pg of IRD 800 10 labelled forward primer was added. 5 units of Taq (Invitrogen) was added to each 100ul of master mix. The PCR fragments were then subcloned into pGEM Teasy (Promega), transformed into E. coli by electroporation or a similar methodology. The DNA sequence determined on an ABI 3730 DNA sequencer. The DNA sequence 15 obtained was then aligned with the region defined by the PCR primers from >gil672398911gblAAFC02218335.11 Bos taurus Con233460, whole genome shotgun sequence. New primers BOS3.4RF: 5'AAgCAAAATgCCTTACACAT3' and BOS3.4RR: 5'AgCATCAgCTCAAgAACATT3', designed to align with conserved DNA regions 20 identified between sheep and cattle, were used for PCR. One primer was labelled with an infrared dye (IRD800) although any fluorescent or radioactive label can be substituted. Sheep and cattle DNA was PCR amplified and analysed on a LiCor DNA fragment analyser. Results 25 The sheep DNA region was sequenced, giving the following: >Sheep clone 4 from Bos 3. GAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTAGATGATGA GTTTGGTTTGGGATGTTTTTATGACCGGGTAGGGTCTCTATGTGTCTTATATTTATATAT ATATCATCTCCATCTTTCTCCTGTTCCCCCTTCTGCTTCAATCTTCCCTTCAATTTAAGC 30 CTCTCTTGAAACAATAACTTGATTTCCTTTGATCTGTCCTAACTAAACAAGCAAAATGCC TTACACATAGAAGATGGTTAATGGACATTTGTTGAGTGGATGGGTGGGTGGACGGATGGA TGGATGAATGGATGGATCGATGGATGGGTGGATGAATGGATGGATGGGTGGATGAATGGA

TGGATGGATGGGTGGGTGGATAGATGTATGAATGGGAATGAGTGAGTGGATGGATGGATG

WO 2006/089366 PCT/AU2006/000240 -45 GATGGATGGATGGATTGGAAGGGGTGAGTGGATGGGTGGATGGATGGATGGGTGGGAGGG GATGGATGGGTGGATAGGTGGATGGACGGGCAGGGATGGCTGGATAAATGGGTGGACAGT TACATGCACGOATGGATGCAGAGTCAAATGTTCTTGAGCTGATGCTTTGCCTTTCATTCT TGAAGTTCCTTAGAACAAAGTGTGACAGGCTAGGAAAACAGAGGTTGGAAAATCGAATTC 5 CGCGGCGCCATGGCGCGCGCAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCG TATTACAATT CACT Example 4 - Identification of microsatellites in Alpaca by screening a DNA library Whilst this is an example of screening a DNA library, the skilled person would 10 understand that similar techniques could be used to screen BACs, YACs , P1 Bacteriophages, Lambda bacteriophage or cosmid libraries Materials/Methods 1. Genomic DNA Digestion 20 pg of genomic alpaca DNA was digested to completion with an excess (5 U/pg 15 DNA) of Haelll enzyme overnight at 37 'C. using the following; 1Oul alpaca DNA (20ug) 23 pl water 12 pl Haelll (8 UlpI) (Promega, California, USA) 5 pl buffer C (Promega, California, USA) 20 An aliquot was run on a 1% low melting point gel with a 100 bp ladder. The digest was then extracted once with equal volumes of phenol/chloroform. The DNA was precipitated with 2 x volume isopropanol overnight at 4 *C and then washed in 200 pl of 70% ethanol. The pellet was dried well and resuspended in 20 pl of 25 distilled water. 2. DNA Size selection Loading buffer (10 pl) was added to the sample, which was then heated for 10 min at 60 *C. The entire sample was loaded while still warm and the digest was run WO 2006/089366 PCT/AU2006/000240 -46 overnight on a large gel tray with broad tooth combs, using a 2% low-melting point agarose gel, with a 100 bp ladder on either side of the DNA. The 100-500 bp fragments were excised from the gel using a sharp sterile scalpel blade and the gel plug was then incubated overnight at -70 *C to disrupt the agarose 5 architecture The sample was centrifuged at 14000 rpm for 20 min and the supernatant was removed to another tube, DNA was eluted from the supernatant by precipitating overnight at -20 *C in double the volume of isopropanol. The sample was centrifuged again at 14000 rpm for 20 min and washed twice in 70% ethanol to 10 reveal a white pellet of DNA. This pellet was then dried in a 60 *C oven for 5 min and resusupended in 20 pl of TE. A 3 pl aliquot was electrophoresed on a gel with DNA standards and a size ladder to determine the quality and concentration of the digest. The rest was stored at -20 C. 3. Preparation of digested plasmid pUC1 8 vector 15 Digestion of I pg of pUC18 supercoiled vector (1 pl) with Smal. Vector (1 pg/pl) I P1 10 x RE digest buffer E I pl Small enzyme (I U/pl) 5 pl sterile water 3 pl 20 The digest was incubated at 37 0 C for 30 min, then the restiction enzyme was inactivated by heating the reaction to 65 0 C for 15 min. This plasmid was further treated with Shrimp alkaline phosphatase (Promega) under manufacturer's conditions. 25 4. Ligation of Plasmid and Insert DNA The ligation was set up as follows: WO 2006/089366 PCT/AU2006/000240 -47 Vector (Smal digested/Alk Phos pUC18) 1 pl (250 ng) Digested DNA Insert 7 pl (53 ng) 10 x Ligase buffer (Promega)(with ATP) 1 pI T4 DNA ligase (Promega)(2.5 U/pL) 1 p1 5 Total Volume 10 p1 The ligation was incubated at 160C for 1-4 h. Reactions can be used immediately, or stored at - 200C until required. The ligated DNA was again precipitated with 4 x volume of ice -cold isopropanol at -80 0 C for 30 min and then centrifuged at 11000 10 x g for 10 min at 4 0 C. The supernatant was discarded and the pellet was washed twice with 70% ethanol. After air drying, the pellet was resuspended in 10 p sterile water and transformed immediately. 5. Bacterial Transformations Twenty pi of the culture of electrocompetent E. coli, (Invitrogen) thawed on ice 15 was transferred to a sterile 1.5 mi microfuge tube. The cuvettes for electroporation were also placed on ice for chilling. Two pl of the ligation reaction was added, mixed and stood on ice for 1 min. The mix was then transferred to the pre-chilled cuvette and electroporated using a pulse of 1.8 kV, 25 pF, and 200 ohms. Successful electroporation was indicated by time constants in the range of 4.2-4.6 20 msec. Immediately after electroporation, I ml of ice-cold SOC media was added to the cuvette, mixed gently, transferred to a sterile 10 ml centrifuge tube and incubated on ice for 1 hour with gentle shaking. Following incubation, 100 pi of the transformation mix was plated out on LB Ampicillin (100 pg/ml) plates containing 1 mM IPTG, 1mM X-gal. After the liquid 25 was absorbed the plate was inverted and incubated at 37 0 C overnight. 6. Screening the plasmid library WO 2006/089366 PCT/AU2006/000240 -48 Hybond N+ nylon membranes were carefully laid over the plates and marked with a needle in three positions to preserve orientation. After I min, membranes were gently lifted from the plate using forceps, placed colony side up on filter paper and dried for approximately 10 min at 60 "C. The plates were incubated at 4 'C until 5 required. The dried membranes were placed in 20% SDS for 10 min to lyse the cells, then rinsed and soaked in transfer buffer for approximately 20 min. Membranes were removed from the transfer buffer, soaked twice for 10 min each in 1 M Tris-HCI, pH 8.0, before being dried for 1 h and either used immediately or placed between filter papers and stored at room temperature until required. 10 7. Radiolabelling the (CAAA)5 oligonucleotide The oligonucleotide (CAAA)5 (100ng) was radiolabellied using polynucleotide kinase and gamma32P ATP. 8. Hvbridising the probe, washing and autoradiography of membranes The membrane was then placed in a glass bottle and prehybridised for I h with 20 15 ml of hybridisation buffer. The membrane was unfurled when it was placed in a rotating hybridisation oven (Hybaid) and the rotisserie was activated. Following prehybridisation, the buffer was removed, 10 ml of fresh hybridisation buffer containing the probe was added, and the bottle incubated over night at 45*C. The annealing temperature of the hybridisation experiment is dependent on the 20 melting temperature of the particular probe used. The membranes were removed from the bottles and placed in a plastic container in a shaking waterbath. Membranes were washed twice with 2 x SSCI 1% SDS at 45 *C for 15 min, followed by one wash with 1 x SSC/1% SDS at 45 0C and lastly with I x SSC/0.1% SDS at 45 0C for 10 min. Washes were repeated up to three 25 times until the blank was at background count level. Following washing, membranes were rinsed in 2 x SSC, heat sealed in a plastic bag, and exposed to x-ray film (Hyperfilm -MP, Amersham). Positive colonies WO 2006/089366 PCT/AU2006/000240 -49 were picked with a sterile wire and inoculated into 6 ml of LB broth with 50 pg1ml kanamycin and grown overnight on a shaking incubator at 37 *C. Results The Alpaca DNA detected using the above method was sequenced to determine 5 the repeat region. The sequence obtained is shown below. >Alpaca 1.2 microsatellite (CAAA)n repeat motif ATCTCTGCCTGCAAGCTATGGTGGAAGGGAAAGTGGTGAGAGCCCCTTTTCTCTCTCTCAATTTAGATTAGC 10 AGGAAAAACTATTTGTGGGGCTTGTTCCTTGGAUAACAACTCTTGGGGATTTTTTTCCTGCCAGA ATGGT CACTGCTTTTCCTTCTTTCTCTCTCTCCCTTTCTCCCTTTCTCCCTTTCTCCCTTTCTCTCTTTCTCTCT CTTTCTCTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTCTTTCTTCTTTCCTTTCTTTTCUTC TTCCTTCTTCCTTTCTTTCTTTCTTCTTTCTCCCTCCCTCCCTCCCTCCCTTCCTCTCTTTCTCTCTTTCT TCTTTCTTTTTGTCASTGAGGAAGAAGAACCATAGGACAGAAGGGAGGGAATGGGCTCTGCTATTTGAGCCA 15 GTCTCACAGACTGGTGACTTAATGGCTCTCACAGGACAAATATCTATTG

Claims (27)

1. A method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: (a) contacting under highly stringent conditions a nucleic acid probe capable of 5 hybridizing with a nucleotide sequence flanking said element; and (b) detecting the complex formed between the probe and the target nucleic acid wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein 10 the repeating nucleotide element is not CAGG or GGAT.

2. The method of claim 1 wherein the repeat elements are formed of repeating nucleotide sequences of at least 5 nucleotides.

3. The method of claim 1 wherein the repeat elements are formed of repeating nucleotide sequences of at least 6 nucleotides. 15

4. The method of claim 1 wherein the probe is selected from the group described in the results section of any one of Examples 1, 2 or 3.

5. A method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting under highly stringent conditions a nucleic acid probe capable of 20 hybridizing with a nucleotide sequence flanking said element; and b) detecting the complex formed between the probe and the target nucleic acid - 51 wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT and wherein the target ruminant nucleic acid sequence is selected from the group of DNA 5 sequences in the clones described in the results section of any one of Examples 1, 2, 3 or 4.

6. A method for detecting a plurality of repeat elements in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting under highly stringent conditions a plurality of nucleic acid 10 probes capable of hybridizing with nucleotide sequences flanking said elements; and b) detecting the complexes formed between the probes and the target nucleic acid wherein the repeat elements are formed of repeating nucleotide sequences of 15 at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT.

7. The method of claim 6 wherein the detection of a plurality of repeat elements is carried out simultaneously.

8. A nucleic acid probe selected from the group consisting of the probes as 20 described in the results section of any one of Examples 1, 2 or 3.

9. A method for detecting a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting under highly stringent conditions a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; and 25 b) detecting the complex formed between the probe and the target nucleic acid using DNA amplification - 52 wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT.

10. The method of claim 9 wherein the repeat elements are formed of repeating 5 nucleotide sequences of at least 5 nucleotides.

11. The method of claim 9 wherein the repeat elements are formed of repeating nucleotide sequences of at least 6 nucleotides.

12. The method of claim 9 wherein the probe is selected from the group described in the results section of any one of Examples 1, 2 or 3. 10

13.A method for characterising a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting under highly stringent conditions a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; b) extending the complexes formed between the probe and the target nucleic 15 acid and amplifying the sequence containing the repeat element; and c) characterising the repeat element using the amplification products wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT. 20

14. The method of claim 13 wherein the repeat element is characterised according to the number of repeats of at least 5 nucleotides.

15.The method of claim 13 wherein the repeat element is characterised according to the number of repeats of at least 6 nucleotides. -53

16.A method for characterising a repeat element in a target ruminant nucleic acid sequence, the method comprising the steps of: a) contacting under highly stringent conditions a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; 5 b) extending the complexes formed between the probe and the target nucleic acid and amplifying the sequence containing the repeat element; and c) characterising the repeat element using the amplification products by contacting said amplification products with a chip comprising at least one probe selected from the group consisting of the probes described in the 10 results section of any one of Examples 1, 2 or 3 wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT.

17. A chip comprising at least one probe selected from the group consisting of the 15 probes that are described in the results section of any one of Examples 1, 2 or 3 and complements thereof.

18.A method of detecting an association between a genotype and a phenotype in a ruminant using a repeat element in a target ruminant nucleic acid, the method comprising the steps of: 20 a) contacting under highly stringent conditions a nucleic acid probe capable of hybridizing with a nucleotide sequence flanking said element; b) extending the complexes formed between the probe and the target nucleic acid and amplifying the sequence containing the repeat element; c) characterising the repeat element using the amplification products; -54 d) determining the frequency of the repeat element in a trait positive population of ruminants; e) determining the frequency of the repeat element in a control population of ruminants; and 5 f) determining whether a statistically significant association exists between said genotype and said phenotype wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT. 10

19.The method of claim 18 wherein the ruminant control population is a trait negative population, or a random population.

20.The method of claim 18 or 19 wherein the method is applied to a pooled biological sample derived from each of said populations

21.The method of claim 18 or 19 wherein the method is performed separately on 15 biological samples derived from each individual in said population or a sub sample thereof.

22.A kit for detecting a repeat element in a target ruminant nucleic acid sequence, the kit comprising: a) a nucleic acid probe capable of hybridizing under highly stringent 20 conditions with a nucleotide sequence flanking said element; and b) means for detecting the complex formed between the probe and the target nucleic acid wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein 25 the repeating nucleotide element is not CAGG or GGAT. - 55

23. The kit of claim 22 wherein said kit contains a plurality of probes selected from the group consisting of the probes described in the results section of any one of Examples 1, 2 or 3.

24.The kit of claims 22 or 23 wherein the probe is labelled with a detectable 5 molecule.

25.The kit of any one of claims 22 to 24 wherein the probe is immobilized on a substrate.

26.A method for identifying a repeat element in a ruminant nucleic acid sample, the method comprising the steps of: 10 a) contacting under highly stringent conditions a nucleic acid probe or a plurality of nucleic acid probes, designed to hybridise to repeat elements with at least 3 repeats, with the sample; and b) detecting the hybrid complex formed between the probe and nucleic acid sample 15 wherein the repeat elements are formed of repeating nucleotide sequences of at least 4 nucleotides selected from any one of Tables 1, 2, 3 or 4 and wherein the repeating nucleotide element is not CAGG or GGAT.

27. The method of claim 26 wherein the probe is capable of hybridising to 3 to 10 repeats of a repeat element selected from the repeat elements listed in any 20 one of Tables 1, 2, 3 or 4 wherein the repeating nucleotide element is not CAGG or GGAT.