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CAPS markers conversion and linkage analysis A random selection of 24 SNPs was made from the CcRAD2 dataset in order to validate the SNP calls by conversion to a CAPS format. These assays were then used to genotype the globe artichoke × cultivated cardoon mapping population members . Primer pairs were designed for testcross SNP loci expected to segregate only within cultivated cardoon (Table 2). Successful amplification was obtained for all the assays, and 19 out of the 24 segregated consistently with the predicted 1:1 ratio (Table 2). Three of the assays produced not readable patterns of segregation and were discarded, while other two showed no evidence of any restriction cleavage, suggesting either a false SNP call (e.g. assembly of paralogs, sequencing error) or failure in the assay (e.g. selective amplification of one allele). Among the 19 CAPS loci retained, none showed a significant level of segregation distortion (c2 ≤ c2 a = 0.1); 17 loci were distributed over ten cultivated cardoon linkage groups, one (SNP site 5548-175) was associated to a previously linked pairs of markers and thereby generated a new LG (Alt_22), and CAPS 14600-111 was linked to the previously unmapped microsatellite locus CyEM-134 (Figure 6). CAPS loci 5983-127 and 20149-154 were most tightly linked with one another (1.3 cM on LG Alt_1b+16). The inclusion of these 17 loci generated only minor changes in locus order; some rearrangements were induced in Alt_4 (CELMS-42, Δ10.0 cM), Alt_8 (CyEM_48, Δ10.8 cM and CyEM_286, Δ 11.2 cM) and Alt_9 (e39/m50-240, Δ19.4 cM). The mapping exercise confirmed that the RAD-derived SNP markers are suitable for genotyping purposes. Conclusion In crop species where the number of markers available to date is limiting, the use of high throughput sequencing to generate large numbers of genetically informative assays can make a valuable and rapid contribution to linkage mapping, and its major downstream application, markerassisted selection. RAD tag sequencing based on the Illumina platform has proven to be a highly reliable and costeffective means of SNP discovery. We were able to identify thousands of putative SNP markers in this way, and the majority of a random sample of 24 was fully validated through conversion to CAPS assays and linkage analysis. Furthermore, the reduction in template complexity generated by the RAD approach greatly facilitates its implementation in mapping-by-sequencing approaches. A large proportion of the methylation present in DNA occurs in the form of CpG dinucleotides, and there is little evidence for negative selection against these in the many genomes which have been analysed to date . Acquiring genome-wide sequence has given a glimpse of the genome complexity present in C. cardunculus. Even though the RAD tags represent only a sample of the genome as a whole, it was clear that there exists a relationship between the frequency of CpG dinucleotides and the level of sequence repetitiveness, consistent with the known role played by methylation in controlling genome expansion due to transposable element activity . |
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fjtony163
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2楼2013-03-28 16:47:27
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