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20151
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Subject: Biotechnology × Author: Kaur, Parampreet × Start date: 2010 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Physical mapping of chromosome 2AL of hexaploid wheat and generation and mapping of EST based SNPs in Triticum monococcum
    (PAU, 2015) Kaur, Parampreet; Singh, Kuldeep
    Bread wheat has highly complex genome relative to any other food crop because of its gigantic genome size (17Gb), hexaploid nature and >80% of repetitive sequences. These biological features of bread wheat restricted the progress towards the goal of acquiring gold standard wheat genome sequence. International Wheat Genome Sequencing Consortium (IWGSC) has been working for generation of chromosome/chromosome-arm based whole genome shotgun sequences and BAC-by-BAC based sequences. India was entrusted with the responsibility to decode chromosome 2A of wheat and the present study aimed at the development of BAC based physical map of chromosome 2AL and gene based SNP markers for chromosome 2Aand their mapping onto 2A linkage map. BAC library of 2AL comprised of 76,800 clones and in the present study about 20,000 BACs were fingerprinted using SNaPshot™ technology. However for the generation of physical map of 2AL the fingerprint data of all the 76,800 BACs, fingerprinted by other members of our laboratory were analyzed as a unit. Of the 76,800 BACs fingerprinted, 57,733 clones were cleaned using Finger Print Background (FPB) removal software and screened for cross-contamination using GenoProfiler. Finally, 46,782 high quality fingerprints (9.7equivalents of 2AL) were used for contig assembly using two assembly programs. The FingerPrintedContigs (FPC) assembled 33,424 BAC clones into 2,450 contigs and 7,373 clones represented its Minimum Tiling Path. The assembly generated by another advanced algorithm, Linear Topology Contigs (LTC) assembled 30,334 BACs into 1,204 better ordered and longer contigs. Its MTP was defined by 7,854 clones which are being used for MTP sequencing for generating pseudomolecule. In a parallel experiment, draft sequence assembly of 2A generated using Roche 454 and Illumina shotgun sequencing data was used for in silico identification of genes corresponding to full-length cDNAs (FlcDNAs) available in public domains. Primers were designed from 429 genes and used for amplifying Triticum monococcum and T. boeoticum. The amplicons of about 1,000 bp size were fractionated in 0.8% agarose gel to identify polymorphic markers. The amplicons which did not show size polymorphisms were sequenced in both the parents to identify SNPs. Sequence based markers were identified for 146 primers, out of which 96 SNPs were genotyped using Fluidigm SNP genotyping assay. Linkage map was developed using 123 polymorphic primers (93 SNP based, 9 size based and 21 presence/absence based). Out of these, 85 markers were mapped to pre-existing 2A linkage map with a final map length of 549.6 cM and 23 markers mapped onto chromosome 1A with small number of markers mapped onto other chromosomes. These markers will be used for the anchoring of physical map of 2AL to its genetic map. Development of an anchored physical map will complete one aspect of the multi-phase sequencing strategy of IWGSC and will serve as India’s contribution towards the IWGSC initiative.