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20153
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Subject: Biotechnology × End date: 2015 × Start date: 2015 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Physical and Genetic Mapping of chromosome 2AL of wheat (Triticum aestivum L.)
    (PAU, 2015) Jindal, Suruchi; Singh, Kuldeep
    Among crop plants, hexaploid wheat has one of the largest genome, being 17000 Mbp. The largest genome coupled with polyploidy nature and very high level of repeat sequence makes sequencing of hexaploid wheat very complex. Several studies, coordinated by IWGSC (International Wheat Genome Sequencing Consortium) are in progress with the aim of obtaining and characterizing the wheat genome. The IWGSC has produced a draft sequence of hexaploid wheat genome by sequencing chromosome arms that were isolated from double ditelosomic stocks of Chinese Spring by flow sorting. Under IWGSC, India has the mandate for generating Bacterial Artificial Chromosome based physical map and whole genome sequencing of chromosome 2A and and PAU has been given the responsibility for physical mapping and sample sequencing of chromosome 2AL. BAC library comprising 76,800 clones for the long arm of 2A with an average insert size of 120kb and 16X coverage was generated from DNA of chromosome arms purified by flow cytometry. Using HICF (High Information Content Fingerprinting) we have fingerprinted 76,800 clones in total as group, out of which 20,000 clones were fingerprinted for this thesis. Automated assembly of high quality fingerprints was performed to generate physical map for 2AL using FPC (Fingerprinting Contig) and LTC (Linear Topolgy Contig) software for the generation of Minimum tiling Path (MTP). FPC and LTC generated 2450 comprising 5804 clones and 1204 contigs comprising 7854 clones respectively. Whole genome shotgun sequence for the chromosome 2AL was also generated using Illumina GAII, Hiseq2000 (paired end) and 454 Roche platform. Both the platforms generated combined reads of 4, 50,120,605 for the long arm. De novo hybrid assembly resulted into 425,821 contigs for 2AL covering 63% of arm. Size based markers were generated from assembled chromosome data. SSR mining was done on the assembled data which resulted in identification of more than 3000 usable SSRs for 2AL using MISA tool. About 501 di-, tri-, and tetra- nucleotide SSR markers were identified, with one marker from each contig for genetic mapping. Insertion Site Based Polymorphism markers (ISBPs) were also predicted from the assembled data using ISBPFINDER.pl. A total of 2, 16,414 ISBPs have been predicted out of which 12,706 can be used as markers and 50 ISBPs were selected randomly for mapping. Parental polymorphism was done on Triticum monococcum and Triticum boeoticum using ABI 3730XL genotyping system and agarose gel system for SSRs and ISBPs respectively. 225 SSR markers and 6 ISBP markers were found to be polymorphic, out of which 95 loci (including SSR and ISBP markers) were used to enrich the genetic map of 2A using the RIL population derived from the cross between Triticum monococcum and Triticum boeoticum and 39 markers were mapped on 2AL while remaining markers mapped on other linkage groups.
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    ThesisItemRestricted
    Cloning and characterization of heat shock protein gene(s) from Aegilops speltoides (Tausch) Gren. and their association with heat tolerance
    (PAU, 2015) Pratibha; Singh, Kuldeep
    Wheat is staple food for half of the world population. Wheat yields are increasing at 0.9% annually, which is much less than the requisite increase to meet its demand in 2050. Wheat production is often limited by continual or terminal heat stress and not much is known about the mechanisms conferring olerance to heat stress. Heat shock proteins are known to play an essential role in preventing deleterious effects of high temperature and in many plant species HSP101 has a central role in heat stress survival. Aegilops speltoides, a close relative of B genome of bread wheat has been observed to confer tolerance to terminal heat stress. The present study, therefore, aimed at characterization of heat shock protein gene HSP101 in Ae. speltoides and comparing it with other species. The Ae. speltoides and other wild and cultivated wheat genotypes were analyzed for chlorophyll content at various growth stages until maturity. Ae .speltoides, per se, had significantly higher chlorophyll content at all the growth stages, even when the temperature was above 35°C. Coding sequences of HSP101C of T. aestivum were used to design the primers for studying expression of HSP101 at varying day/night temperature regimes. Expression analysis of HSP101C gene through Quantitative RT-PCR revealed differences in their induction in wild and cultivated wheat genotypes. Two Aegilops speltoides accessions pau3583 and pau3809 showed high level of expression of HSP101C gene at higher temperature compared to bread wheat, suggesting that it might be playing a role in conferring heat tolerance. Coding sequence of HSP101C gene of T. aestivum was used to identify the whole gene sequence in T. durum and Ae.speltoides genome databases. Overlapping primers were designed to amplify the whole gene from Ae. speltoides, Ae. tauschii, T. monococcum, T. durum and T. aestivum. Amplification was successful for all the fragments in all the species, however, clean sequence could be obtained in only one accession of Ae. speltoides acc pau3583. The HSP101C gene of Ae. speltoides acc. pau3583, designated as AsHsp101Cpau3583 is 4133 bp long with 2667 bp of coding sequence encoding an ORF of 888 amino acids. The AsHSP101C-pau3583 gene sequence contains more than 50 SNPs compared to AsHSP101C-TGAC. In silico comparative analysis of sequence of HSP101C of T. aestivum, Ae. speltoides, Ae. speltoides acc. pau3583, T. durum cv cappelli, T. durum cv strongfield, T. monococcum, Ae. tauschii and T. urartu HSP101C protein showed that multiple conserved domains (AAA, AAA+2, ClpB, ClpN, ClpD domains) are present. All ClpB/HSP100 genes in wheat share conserved nucleotide-binding domains. There appears to be HSP101C protein (encoded by Aegilops speltoides pau3583) that are variably homologous to proteins encoded by above wheat species throughout the entire amino acid sequence. The above eight wheat species Hsp101C gene show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. The protein models of HSP101C in all eight wheat species provides high information for the ATP-binding motifs within the nucleotide binding domains (NBD) which are specific for the chaperone activity and knowledge about the mutagenic sites. These findings are important for further dissection of the molecular mechanisms underlying the stress response and for understanding the functions of the HSP100 fami ly members. The sequence information could also be used designing markers for precise transfer AsHSP101C-pau3583 gene into hexaploid wheat and test its role in heat tolerance.
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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.