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ThesisItem Open Access Physical and Genetic Mapping of chromosome 2AL of wheat (Triticum aestivum L.)(PAU, 2015) Jindal, Suruchi; Singh, KuldeepAmong 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.ThesisItem Restricted Cloning and characterization of heat shock protein gene(s) from Aegilops speltoides (Tausch) Gren. and their association with heat tolerance(PAU, 2015) Pratibha; Singh, KuldeepWheat 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.ThesisItem Open 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, KuldeepBread 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.ThesisItem Restricted Marker assisted transfer of novel powdery mildew resistance gene(s) from Triticum boeoticum (Boiss.) to hexaploid wheat Triticum aestivum (L.)(PAU, 2014) Elkot, Ahmed Fawzy Abdelnaby; Singh, KuldeepPowdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases in the world and development of resistant varieties is safest and most economical approach for containing this disease. Wild species are an important source for PM resistance genes. In a previous study, two PM resistance genes designated as PmTb7A and Pm1Tb were identified in T. boeoticum acc. Pau5088 and mapped on chromosome arm 7AL the two genes were approximately 48cM apart and two resistance genes analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were found to be linked to the PmTb7A and Pm1Tb, at a distance of 0.6cM and 6.0cM, respectively. In the present study the two genes were transferred from T. boeoticum to T. aestivum cvs PBW 343-IL and PBW 621using T. durum cv PBW114 as bridging species. As many as 12317 florets of F1 of the cross T. durum cv PBW114/T. boeoticum acc pau5088 were pollinated with T. aestivum cvs PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively of three-way F1. The resulting F1s of the cross T. durum/T. boeoticum//T. aestivum, were screened with marker flanking both the PM resistance genes PmTb7A and Pm1Tb (foreground selection) using SSR and RGA-STS markers and selected plants were backcrossed to generate BC1F1. Marker assisted selection was carried in both BC1F1 an BC2F1 generations with the objective of transferring these genes with minimum of the linkage drag. In BC2F1, out of total 121 BC2F1 plants of cross PBW114 / T. boeoticum acc. pau 5088//3*PBW343-IL, 41 had only PmTb7A linked markers, 13 had Pm1Tb linked markers, six had introgression for markers linked to both the genes and 61 plants did not show any introgression. Likewise, out of 93 BC2F1 plants derived from cross of PBW114/T. boeoticum//3*PBW621, 21 had only PmTb7A linked markers, 16 had Pm1Tb linked markers, 14 had introgression for markers linked to both the genes and 42 plants did not show any introgression. Out of more than 110 plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants having minimum of the alien introgression. Introgression in different BC2F1 plants varied from 15.4 - 62.9 per cent with minimum introgression in plants CBT76-4 which had PmTb7A only but not Pm1Tb and CBT101-3 which showed introgression for Pm1Tb but not for PmTb7A. Most of the selected plants had yield component traits similar or better than the recurrent parents. Cytological analysis showed that most plants have chromosome number ranging from 40-42. BC2F2 plants homozygous for the two genes have been identified and these will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien ntrogression. One of the PM resistance genes PmTb7A which is novel, maps in a region on 7AL where Sr22, a stem rust resistance gene effective against the race Ug99, also maps. Markers analysis with Sr22 linked gene showed introgression in 31 plants out of total 40 selected BC2F1plants. Thus in addition to PM, the line with PmTb7A should also be resistant to stem rust race Ug99. To the best of our knowledge, this is the first example of marker assisted transfer of a gene from wild species into cultivated wheat.ThesisItem Open Access Marker assisted transfer of novel powdery mildew resistance gene(s) from Triticum boeoticum (Boiss.) to hexaploid wheat Triticum aestivum (L.)(Punjab Agricultural University, Ludhiana, 2014) Abdelnaby Elkot, Ahmed Fawzy; Kuldeep SinghPowdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases in the world and development of resistant varieties is safest and most economical approach for containing this disease. Wild species are an important source for PM resistance genes. In a previous study, two PM resistance genes designated as PmTb7A and Pm1Tb were identified in T. boeoticum acc. Pau5088 and mapped on chromosome arm 7AL the two genes were approximately 48cM apart and two resistance genes analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were found to be linked to the PmTb7A and Pm1Tb, at a distance of 0.6cM and 6.0cM, respectively. In the present study the two genes were transferred from T. boeoticum to T. aestivum cvs PBW 343-IL and PBW 621using T. durum cv PBW114 as bridging species. As many as 12317 florets of F1 of the cross T. durum cv PBW114/T. boeoticum acc pau5088 were pollinated with T. aestivum cvs PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively of three-way F1. The resulting F1s of the cross T. durum/T. boeoticum//T. aestivum, were screened with marker flanking both the PM resistance genes PmTb7A and Pm1Tb (foreground selection) using SSR and RGA-STS markers and selected plants were backcrossed to generate BC1F1. Marker assisted selection was carried in both BC1F1 an BC2F1 generations with the objective of transferring these genes with minimum of the linkage drag. In BC2F1, out of total 121 BC2F1 plants of cross PBW114 / T. boeoticum acc. pau 5088//3*PBW343-IL, 41 had only PmTb7A linked markers, 13 had Pm1Tb linked markers, six had introgression for markers linked to both the genes and 61 plants did not show any introgression. Likewise, out of 93 BC2F1 plants derived from cross of PBW114/T. boeoticum//3*PBW621, 21 had only PmTb7A linked markers, 16 had Pm1Tb linked markers, 14 had introgression for markers linked to both the genes and 42 plants did not show any introgression. Out of more than 110 plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants having minimum of the alien introgression. Introgression in different BC2F1 plants varied from 15.4 - 62.9 per cent with minimum introgression in plants CBT76-4 which had PmTb7A only but not Pm1Tb and CBT101-3 which showed introgression for Pm1Tb but not for PmTb7A. Most of the selected plants had yield component traits similar or better than the recurrent parents. Cytological analysis showed that most plants have chromosome number ranging from 40-42. BC2F2 plants homozygous for the two genes have been identified and these will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. One of the PM resistance genes PmTb7A which is novel, maps in a region on 7AL where Sr22, a stem rust resistance gene effective against the race Ug99, also maps. Markers analysis with Sr22 linked gene showed introgression in 31 plants out of total 40 selected BC2F1plants. Thus in addition to PM, the line with PmTb7A should also be resistant to stem rust race Ug99. To the best of our knowledge, this is the first example of marker assisted transfer of a gene from wild species into cultivated wheat.ThesisItem Restricted Association mapping for grain micronutrient density, grain size and grain weight in Aegilops tauschii species(Punjab Agricultural University, Ludhiana, 2016) Arora, Sanu; Chunneja, ParveenBread wheat is the most important cereal being consumed worldwide. However, due to expanding world‟s population size, the problem of nutritional deficiencies is increasingly affecting the health of people with predominant cereal-based diet. This poses the futuristic demands of both increasing the yield and producing nutritionally enriched wheats on the wheat breeding programmes. However, the hexaploid wheat exhibits limited genetic diversity, which can be broadened by exploiting the wild relatives of wheat by introgression of novel genes/alleles. Aegilops tauschii, the diploid D-genome progenitor of hexaploid wheat, can be used to increase the genetic diversity both for improving grain yield and nutritional quality of bread wheat. To identify the new sources of improving grain yield and nutritional quality, we performed genome wide association study (GWAS) using a collection of 190 Ae. tauschii accessions. The Ae. tauschii germplasm was assessed for grain micronutrients concentration (Fe, Zn, Cu, Mn), grain size (length and width) and 50-grain weight. Quantitative distribution and low to medium heritabilities were observed for grain phenotypes. Ae. tauschii was found to have two to three fold more variation for these phenotypic traits compared to hexaploid wheat cultivars. The accessions were genotyped using SSR markers and SNP markers through genotyping by sequencing (GBS) approach. A total of 59,830 SNPs were mined from the GBS reads of 190 Ae. tauschii accessions and after filtering for more than 30% missing data and minimum allele frequencies of 0.05, a total of 24,567 SNPs were obtained. These filtered GBS SNP tags were aligned to wheat POPSEQ data for assigning the genetic positions. A total of 11489 SNPs mapped to seven Ae. tauschii chromosomes. Genetic diversity was assessed using STRUCTURE software and by conducting phylogeny as well as principal component analysis (PCA). These analysis revealed presence of two distinct lineages (lineage 1 and lineage 2) in Ae. tauschii germplasm. The two lineages broadly corresponded to ssp. tauschii and ssp. strangulata. Both the lineages differed significantly for grain micronutrient concentrations (Fe, Cu and Mn), grain width and grain weight. High mean values for grain micronutrients concentration (Fe, Zn and Cu) and grain weight was observed for lineage 2 (except for Mn concentration). The majority of the accessions identified for higher grain micronutrients from lineage 2 had their origin from Azerbaijan and Iran. Since Ae. tauschii depicted strong structure, GWAS was conducted separately for the two lineages. Linkage disequilibrium across whole Ae. tauschii genome was assessed and observed to be widespread with LD decay to 50% at 9.7 cM and 2.8 cM for lineage 1 and 2, respectively. GWAS was conducted for grain phenotypes using 78 (non-redundant) accessions of lineage 2. Three, six and two significant marker trait associations were found for grain length, width and 50-grain weight, respectively with –log10 (P) >3 with most of the associations located on chromosomes 5D and 7D. For grain Fe concentration only one significant association was observed for –log10 (P) >3 and for grain Zn concentration, SNP marker associations were detected at –log10 (P) 2.5. The study demonstrated the existence of high genetic variation for grain micronutrient concentration and grain size in Ae. tauschii germplasm and utility of GWAS to identify new loci that will be useful to diversify the D-genome of hexaploid wheat for improving both the grain yield and nutritional quality.ThesisItem Restricted Genomics of domestication related traits of Brassica juncea (L.) coss(Punjab Agricultural University, Ludhiana, 2016) Simarjeet Kaur; Banga, S. S.Present studies involved genetic and transcript analysis to explore directional selection and polyploidy in Brassica juncea, and its progenitor species, B. rapa and B. nigra. Patterns of population differentiation were investigated by genotyping a global collection of 235 germplasm lines with Sequence Tagged Site markers. Flowering loci (FLC and CO in B. rapa; FLC and LFY in B. juncea) appeared to be under directional selection. Transcript analysis using Tajima’s D test confirmed positive selection for FRI and FT in B. rapa and for FLC and LFY in B. juncea. Most flowering homoeologs did not contribute equally to the transcript pool of allotetraploid B. juncea. The expression bias seemed to be modulated by the background cytoplasm. MADS-box genes were investigated using sequence information curated from transcriptome sequencing data from three test species. These seemed to be evolving independently at the DNA sequence level. These genes also exhibited highly variable expression patterns in allotetraploid B. juncea in comparison to its progenitors. AGL18, AG, SHP1, SHP2, SEP1 and MAF5 appeared to be under positive selection in B. rapa. Same was true for AGL6, AGL16 and SHP2 genes in B. nigra. MADS-box genes carried highly conserved gene phylogeny. Impact of directional selection was investigated on size, metabolite composition and calorific value of the seeds. AP2 and ARF2 were identified as major targets of selection in both B. rapa and B. juncea. DA1 and CYP78A5 also showed evidences of positive selection. Directional selection was stronger for seed-size as compared to correlated oil content. Positive selection on seed-size is likely to have played a significant role in structuring regional variation in the germplasm.