4 Development of 20 microsatellite and 7 single nucleotide polymorphism markers
4.3 Results and discussion
4.3.1 Marker development
From 13 different BAC clones we generated approximately 390 kb genomic sequence data. In relation to the BAC clone insert size, we cloned on average 30 subclones per BAC and sequenced them from both ends with an average read length of 500 bp. A search for microsatellites within these sequences resulted in the development of 20 microsatellite markers, one to four on each of the processed BAC clones (Table 2). Most of the new microsatellites are (CA)n or (GT)n dinucleotide repeats (Table 2) and the number of observed alleles ranged between 2 and 11 (Table 3). Marker characteristics were determined by genotyping animals of six different cattle breeds and the average PIC value (45 %) for the 20 new markers (Table 3) was comparable to the PIC value of the 6 genotyped markers from the literature (52 %) (Table 4). The mendelian nature of these markers was confirmed by observing their inheritance through two or three generations of the genotyped cattle families.
There are several methods of developing microsatellite markers, but however, most of these methods have time-consuming laborious steps such as isolation of targeted clones by hybridization. Our results demonstrated that the method of random sequencing of subclones from before mapped BAC clones was effective for developing microsatellite markers in specific regions of interest. Additionally, we obtained about 30 kb raw sequences per BAC, which provide valuable resources for further marker development (SNP) or sequencing efforts. The selected 13 BAC clones are distributed over 3 Mb of the recently reported BTA1 BAC contig and finally, all new markers were exactly mapped by STS content mapping on the BAC contig (Chapter 3) in relation to already mapped markers (Figure 1).
Table 2 BAC clones, repeat motifs, primer sequences and annealing temperature of the new microsatellites.
Markersymbol BAC Repeat motif Primer sequences AT1 MS3B269 37H23 15xTG F CTTAATCTCTCTCCTTTATTG 53
R CATGGAAAGCAAAGGTGAC
MS4B269 37H23 11xCA + 6xCA F CAACTGAGCGACTCACAC 54
R TGACTGTACTGGGTGTTC
MS2B269 37H23 14xCA F CACAACTGACCACATACATGTG 58
R CCCCTCATTCACAATTGAAGG
MS1B269 37H23 16xGT F ATGAATCTGGGAGGTAAGTC 57
R TCGGGGAAATATACATACAC
MS2B101 80B9 10xCA F CTTATAACTGCTGCTGCTTG 55
R ATAGCTCATATGTTTATCTGTG
MS1B101 80B9 24xGT F GGACCATTCTTGAAAAGGAGTG 59
R CACAGAGTCAGACATGACTGAG
MS1B349 380B9 15xTG F GAGGGATTTGTGGAAAACTGTG 59
R TGGGGATCACAAAGTCAAACAC
MS1B331 218J17 15xAT F ACCCAGTGCAGCCAGAAATA 56
R CAACTGCAAAAAGGTGCTGA
MS1B360 543J10 7xGT + 3xGT F TACGGGGAAGCTGAGAACAC 59
R ATGGACAGAGAAGCCTGGAG
MS1B271 69E7 11xTA F CGACTAAGACTCGGCACTGTC 59
R ATCTGAGGCTGCTTTGAAGG
MS2B49 554P19 19xCA F CTGGAATGGCTTCTTCACAC 57
R TGGTCAATTTTCGTGTTGTG
MS1B34 351B8 21xCA + 2xCA F CATGAATACTTAACACTACTG 58
R CCTCTAATGTGGAATCCAG
MS1B267 21K5 17xTG F CCAGCCACAGTCTCTCAATC 58
R CCTGGTGGGATACAGTCCAT
MS1B104 249E18 8xTG + F TAGACATCAGCACAGTGACTTC 59 3xTG + 7xTG R TGTCTGATTGAAGCGACTTAGC
MS3B104 249E18 8xCA F GCTGTCAGTGAAGCCTTGTG 58
R ACTGTTTCCCCTTCCATCAAG
MS2B104 249E18 17xGT + F CTTCAGTGTTTTCTGGGAAAGG 58 4xGA + 6xGT R CAAAAGTCAGTCAGTCACTCAC
MS2B214 493P3 19xGT F CCTGTTGAAGTATTTGTGTG 58
R CTGGGGTTTGAGAAACAGTC
MS1B214 493P3 13xGT F AAGAGGGAGGGAAGTTGAGT 56
R ACTAGACCATTTTCACTCTG
MS1B189 553A8 16xCA F GCAGTGTGCAAAGCTTTGTG 59
R GTTTCCACCAGGAACTTCTCAG
MS1B42 199N3 23xCA F AAGGGTCATGAGCTTTTCAGTG 59
R AGCCCTTGGTACAATGAATACG
Table 3 Characteristics of 20 new bovine microsatellites in the polled region.
No. No.
Markersymbol genotype alleles smallest largest Hetobs1
Hetexp1
PIC1
MS3B269 498 5 144 156 0.57 0.57 0.55 MS4B269 224 6 224 236 0.37 0.38 0.32 MS2B269 167 5 171 179 0.42 0.51 0.41 MS1B269 609 8 92 104 0.55 0.72 0.70 MS2B101 327 3 220 230 0.26 0.25 0.25 MS1B101 342 10 246 274 0.42 0.49 0.45 MS1B349 633 8 80 98 0.67 0.71 0.65 MS1B331 442 14 147 171 0.75 0.73 0.74 MS1B360 91 2 178 184 0.18 0.22 0.20 MS1B271 223 3 116 120 0.40 0.49 0.40 MS2B49 39 4 143 153 0.36 0.31 0.29 MS1B34 523 10 126 148 0.27 0.27 0.29 MS1B267 24 2 152 154 0.08 0.08 0.08 MS1B104 286 6 169 185 0.32 0.33 0.30 MS3B104 44 4 163 169 0.43 0.43 0.39 MS2B104 73 9 153 175 0.71 0.72 0.67 MS2B214 287 7 156 176 0.65 0.63 0.55 MS1B214 103 4 192 200 0.48 0.49 0.45 MS1B189 323 10 204 234 0.71 0.72 0.68 MS1B42 493 12 153 171 0.68 0.69 0.71
1Annotations to Table 2, 3 and 4
AT annealing temperature Hetobs observed heterozygosity Hetexp expected heterozygosity PIC polymorphism information content
Table 4 Characteristics of additionally genotyped bovine microsatellites in the polled region.
Markersymbol No.
genotypes No.
alleles smallest largest Het obs1
Het exp1
PIC1 IFNAR1 472 2 252 292 0.18 0.18 0.16 BM6438 691 6 256 268 0.64 0.64 0.59 SOD1MICRO2 444 8 136 178 0.64 0.64 0.64 TGLA49 423 9 109 127 0.56 0.56 0.57 ARO24 288 3 139 149 0.48 0.48 0.41 ARO9 405 12 111 139 0.76 0.78 0.74
4.3.2 Single nucleotide polymorphism development
In total 9 BAC clones were chosen for the SNP development, from these BAC clones we generated shotgun genomic sequence data. In relation to the BAC clone insert size, we get approximately 198 sequences per BAC, sequenced them from both ends with an average read length of 800 bp. Only the HSA21 homologous subclone sequences were used to identify variations within these sequences. The sequences were PCR amplified from 8 animals of the German Fleckvieh breed. If there were SNPs in these 8 cattle, the heterozygote bulls of the families with recombination animals were genotyped with this SNP. If one or more of the bulls heterozygous for the SNP, selected cattle of the family were genotyped with the SNP.
So seven SNPs were developed (Table 5). The search for SNPs was performed to assist the microsatellite markers in this region and further in hope for scale down the region of polled.
Only in four of the selected nine BAC clones informative SNPs could be detected. But the detected SNPs are not able to narrow the polled region down. The selected 9 BAC clones are distributed over the 1 Mb polled region of the recently reported BTA1 BAC contig and finally, all new SNPs were exactly mapped on the bovine BAC contig (Figure 1). During the SNP search we performed mutation analysis about approximately 138,204 bp of the genomic sequence. In these sequences we could not detect the responsible mutation or mutations for the polled phenotype. In conclusion, these genomic regions could be excluded for the localization of the polled gene.
4.3.3 Linkage and haplotype analysis
To link our study to previous studies, we selected all 6 known polled linked polymorphic markers (IFNAR1, BM6438, SOD1MICRO2, TGLA49, ARO24, ARO9) from the published contig map (Chapter 3) and genotyped them in addition to 20 new developed markers (Table 2). In total 26 BTA1q12 markers ordered on the BAC contig map were genotyped.
SNP BACnameFamilySNPF-PrimerR-Primerbp RPCI42 76J4POL01852, 54GAACATCCATG/AAAGTGGTGACGCCTCTTCTTTCCAGTTGTGCATCTGGCATAAGTCCCAAC1281 RPCI42 76J4POL01771ATTGTTTTCCA/GGAACAATTGCTATCAGAACCCAAGGGAAATCGTCTGTCGGCTGTTTACTGG585 RPCI42 21K5POL00552AACAAGCACGC/TGCACACACACGCAACATTTCACTGATGGAGTCTGTCATCTCTTCTCACAGATGG722 RPCI42 21K5POL01152CCGGAGAGTGC/TGCTGAACAGCGCTTGACCTTCTCCCTTTGTGGGCTCAGGGGTCTTTCTGAG749 RPCI42 564N14POL01671TATTAATTTTC/ATCCACATTTTTCTCATTCTCTTCATCAATGGTAAGGGTTCAAATGCCCATG617 RPCI42 543J10POL00254,71CTGTTTGAAGC/TTGAACAAGATCCCCCATATCTCTTACTTTTGGGTCTCTTGAACTCAGTTCTCTC518 RPCI42 543J10POL00612,26,52,54AGTTTTGGGCG/ATTCTGAATGCGCCTCTCGATTTCCTTCCTTCCAGGAGCTAGGCTTCAATTGC720 Annotation Annealingtemperature for all SNPs is 58°C.
Table 5 Single nucleotide polymorphisms within four BACs of 1 Mb polled region.
Table 6 Error probabilities (pZmeans, pLOD) for pairwise non-parametric linkage analysis of the microsatellite markers and the polled phenotype in 30 paternal half sib families.
Markersymbol Zmean pvalue LOD pvalue
MS3B269 17.91 <0.000001 14.17 <0.000001 MS4B269 18.05 <0.000001 14.28 <0.000001 MS2B269 18.20 <0.000001 14.38 <0.000001 MS1B269 18.34 <0.000001 14.46 <0.000001 MS2B101 18.37 <0.000001 14.53 <0.000001 MS1B101 18.40 <0.000001 14.59 <0.000001 MS1B349 18.42 <0.000001 14.65 <0.000001 MS1B331 18.52 <0.000001 14.76 <0.000001 MS1B360 18.70 <0.000001 15.01 <0.000001 MS1B271 18.83 <0.000001 15.35 <0.000001 IFNAR1 18.84 <0.000001 15.39 <0.000001 MS2B49 18.85 <0.000001 15.43 <0.000001 MS1B34 18.87 <0.000001 15.50 <0.000001 MS1B267 18.90 <0.000001 15.57 <0.000001 BM6438 18.94 <0.000001 15.67 <0.000001 MS1B104 18.87 <0.000001 15.67 <0.000001 MS3B104 18.81 <0.000001 15.67 <0.000001 MS2B104 18.74 <0.000001 15.66 <0.000001 MS2B214 18.53 <0.000001 15.59 <0.000001 MS1B214 18.11 <0.000001 15.43 <0.000001 MS1B189 17.70 <0.000001 15.24 <0.000001 MS1B42 17.65 <0.000001 15.21 <0.000001 SOD1MICRO2 17.20 <0.000001 14.94 <0.000001 TGLA49 17.17 <0.000001 14.94 <0.000001 ARO24 16.95 <0.000001 14.66 <0.000001 ARO9 16.72 <0.000001 13.71 <0.000001
The marker order was ascertained by PCR inside the contig. In the first step, we genotyped the founder sires for all 26 markers and secondly, the genotyping of each corresponding family was done only for those markers, where the sire has been shown heterozygous before.
The performed multipoint linkage analysis revealed a highly significant pairwise linkage between all 26 tested markers and the polled phenotype (Table 6). Multipoint analysis assuming a fixed marker order corresponding to the established contig map could not order the polled locus with respect to the 26 markers because of the low number of recombinants.
The number of paternal recombination events observed in our families was 19, no double-recombinants occurred. In the individual families we observed zero up to three recombinations in one family. Therefore, all 19 recombinant haplotypes were taken together
to describe the polled gene region based on the BAC contig map. Only the families with recombinations were taken for the SNP search. The most likely localization of the polled locus situates it between MS1B349 and BM6438 (Figure 2).
The non-recombinant interval corresponds to an approximately 1 Mb interval of the BAC contig where already 13 genes have been mapped in cattle (Chapter 3). The corresponding chromosome segment in humans contains 18 annotated genes or loci (Build 34 vers.1, NCBI, http://www.ncbi.nlm.nih.gov/). Finally, until sequencing of this bovine chromosome segment will allow a subsequent mutation analysis, the presented fine mapping efforts provides for the first time a set of highly polymorphic flanking markers for marker-assisted selection of the polled phenotype.
244 B6
167 I16
182 B8
400 B6 400 D6
206 B24
328 M7
566 F20
80 B9
132 D12
37 H23
494 B13 540 F4
31 K20
543 J23
380 C19383 K23 63 O12 496 H4 394 A5 386 F4 352 O20 44 B5 292 J15 234 N12 506 K17 23 E5 506 K15 311 D23 320 O18 26 I16 180 G7
323 G5
301 M9 447 G24
316 N2
196 M18
292 J17
420 A17
199 N3
266 O23
46 I17
213 N17
553 A8
314 I19
493 P3 320 F13 293 I14 68 K7 249 E18 271 E18 470 N12 217 G23 518 G6
569 F23 161 B10
420 E6 76 J4 351 B8 219 G21 21 K598 P9
554 P19
52 K19
487A22
69 E7
552B21 241 F8
332 I5
564 N14
200 A7
543 J10
79 M3 346 B6 509P 22
534 N15 376 M15 538 E7
368 A9 242 D1 520 B16
370 F8 79 N19 204 M10
372 L18 221 H19 380 B9 218 J7
374 D19
T7 end Sp6 end 51 I7 420 O24
382 D7
BTA 1 + _HSA 21 NCBI build 34 version 1
100 kb 100 kb Figure 1Contig map of the bovine polled region (Chapter 3). New developed microsatellites and SNPs are shown in the first line. Previously mapped polled linked microsatellites are represented in the second line. The arrow indicates the suggested polled gene region from the haplotype analysis.
Breed DH DH DH DH DH CH FV FV FV PI SH SH DH PI DH FV FV FV FV Family 51 55 52 57 57 61 03 04 12 73 83 83 57 71 54 14 14 19 77 Individual 85 371 243 60 79 18 63 41 83 1063 22 36 8 3211 55 118 272 15 600
Marker
MS3B269 154 156 154 154 152 152 156 152 156 152
MS4B269
MS2B269 173 175 175
MS1B269 98 100 100 92 100 92 90 98 98 100 92 100 98 98 92 98
MS2B101
MS1B101 250 250 266 250
MS1B349 90 92 60 90 94 90 94 92 90 90 94 94 90
MS1B331 155 157 157 157 155 163 163 157 147 155 155 MS1B360
POL006 3 2 3
POL002 4 2 4
MS1B271 118 116
IFNAR1
MS2B49 145 149
POL011 2
MS1B34 140 MS1B267
POL017
POL018 2 3 2
BM6438 268 268 258 268 256 258 258 258 258 258 258 258
MS1B104
MS3B104 163
MS2B104 161 161 157
MS2B214 162 170 170 168 168
MS1B214
MS1B189 214 224 228 222
MS1B42 163 163 163 161 161 165 171 161 165 165 161 171 161 161 153 153
SOD1MICRO2 154 142 154 154 142 148 142 142 148
TGLA49 115 123 115 115 111 109 109
ARO24 145 149 149 149
ARO9 119 119 121 129 133 121 131 129 129
Figure 2 List of recombinants present in the whole set of cattle families, arrayed according to the position of the recombination event. Alleles at the informative markers are represented by the allele size. The dark grey segments indicate the segment containing the polled gene, the light grey segments indicate cases where the exact position of the recombination cannot be determined because of marker non-informativeness. A horizontal linee displays the position of the recombination for each individual. The haplotype analysis suggests the position of the polled gene in the segment indicated by the bold typed markers.
5 Development of a reliable marker based test for polledness in different