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Molecular mapping of leaf rust and stripe rust resistance genes transferred from Aegilops speltoides to hexaploid wheat
(Punjab Agricultural University, Ludhiana, 2020) Angoth, Sravan Kumar; Chhuneja, Parveen
Leaf rust and stripe rust are most serious diseases of wheat worldwide. Utilization of resistance cultivars is an effective and economical method to reduce losses from devastating diseases. Aegilops speltoides with SS genome, has been reported to be a valuable source of resistance genes for leaf rust, stripe rust and stem rust. In present study, inheritance studies and molecular mapping of leaf rust and stripe rust resistance genes were undertaken. Stripe and leaf rust resistant accession of Ae. speltoides was crossed with T. durum cv. Aconchi89. F1 was treated with colchicine and an amphiploid was developed which was selfed 2-3 times to develop stable amphiploid. Aconchi89-Ae. speltoides acc. TA1784 amphiploid was crossed and backcrossed with PBW550 and stable BC2F4 introgression lines were developed. Disease resistant introgression line P33-15 was further crossed with PBW550 for mapping population. In F2 population, 161 plants were segregated in ratio of 3:1 for strip rust and in F2:3 populations, plant were segregated in 1:2:1 ratio for resistant and susceptible for leaf rust at adult plant stage. F3 population genotyping was done by dd-RAD-seq, involves cleaving DNA with two restriction enzymes Sph1 and Mluc1 and by using dDocent pipeline, TASSEL V5.0 raw data was processed for processing steps. SNP were identified between parental genotypes P33-15 and PBW550 and 2118 total SNPs were found be polymorphic which were used for linkage map construction. The chromosomes 1D having minimum and chromosome 3B having maximum number of SNPs. Complete linkage map encompassed a total linkage distance of 17875 cM with a SNP density of 0.12 SNPs/cM. With only 569 SNPs the D genome showed least coverage of 4468.48 cM with a density of 0.127 SNPs/cM. A-genome mapped 660 SNPs while B-genome mapped 824 SNPs. Linkage map was further used for mapping genes/QTL for stripe rust and leaf rust resistance using ICI mapping using BIP function. The phenotypic data of the F2 population and genotypic data of the F3 population were used for mapping the stripe rust resistance loci. The QTL mapping was conducted with LOD threshold of 4.0. Ae. speltoides leaf rust resistance gene was mapped on terminal end of short arm of chromosome 7B at a distance of 26cM from nearest SNP marker S7B_733831709. Overall, one gene each for leaf rust and stripe rust resistance have been introgressed from wild progenitor species Ae. speltoides into hexaploid wheat. A SNP based linkage map consisting of 2118 SNP markers has been developed. A major QTL for stripe rust resistance has been mapped on chromosome 3B and a major gene for leaf rust resistance has been mapped on wheat chromosome 7B. Linked markers can be used for transferring these genes to other wheat backgrounds.
Open Access
INTRODUCTION OF GLYOXALASE I GENE FOR SALT TOLERANCE INTO RICE (Oryza sativa L.) VARIETIES ‘PR118’ AND ‘KITAAKE’ THROUGH PARTICLE BOMBARDMENT
(PAU Ludhiana, 2013) Saroj Kumar Sah; Ajinder Kaur
The present investigation dealing with introduction of glyoxalase I gene for salt tolerance into rice (Oryza sativa L.) through particle bombardment was carried out using two varieties PR118 (indica) and Kitaake (japonica). Calli were induced from both the varieties on MS medium supplemented with 2,4-D (3.0 mgL-1) + BAP (0.25 mgL-1) + proline (600 mgL-1) + maltose (40 gL-1) + phytagel (3 gL-1). Embryogenic calli were subcultured on shoot regeneration MS medium supplemented with BAP (4.0 mgL-1) + NAA (0.2 mgL-1) + sucrose (30 gL-1) + phytagel (2 gL-1) + agar (8 gL-1). An attempt was made to introduce GlyI gene into both the varieties. Mature seed-derived embryogenic calli were used as explants for all the transformation experiments. Success was achieved in both the varieties. Using GlyI gene, in PR118, out of 2600 calli bombarded, 249 putative transgenic plants were regenerated on medium containing hygromycin (30 mgL-1). Likewise, in Kitaake, out of 615 calli bombarded, 469 putative transgenic plants were regenerated on medium containing hygromycin (30 mgL-1). Among the 249 regenerants of PR118, 13 plants were PCR positive and in case of Kitaake, among 615 plants 41 showed PCR positive results. Further, these plants were grown to maturity in transgenic glass house. Presence of copy number of transgene was done by Real Time PCR. In total, a set of 10 PCR positive plants (5 of PR118 and 5 of Kitaake) 10 samples were analyzed, among them sample number four has single copy gene and rest has multiple copy ranging from 2-14 copies. To conclude tissue culture base line has been established in two varieties PR118 and Kitaake. Using this baseline, a total of 54 PCR positive transgenic plants were developed. The protocol developed here is genotype independent and is suitable for japonica as well as indica varieties.
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MOLECULAR PROFILING OF INTROGRESSION LINES DERIVED FROM CROSSES OF O. sativa (L.) X O. longistaminata (A.CHEV. &ROEHR.)
(PAU Ludhiana, 2012) Nguyen Le Van; Kuldeep Singh
Rice (Oryza sativa L.) is staple food for more than a third of the world’s population, and we need to produce 30% more rice to meet the demand in next 25 years. Utilization of wild species for enhancing productivity is one of the several approaches proposed for meeting food requirements. In this study we evaluated a number of BC2F6 introgression lines (ILs) generated from the crosses between O. sativa cv PR114 X O. longistaminata acc IRGC 104301 and using DNA markers identified regions introgressed from the wild species. A set of 45 ILs and three checks were evaluated in a complete randomized block design with three replications for two years. Data were recorded on 11 agronomic traits. Several ILs showed significant increase or decrease over the recurrent parent in yield components like plant height (PH), days to flowering (DF), tiller number (TN), panicle length (PL), spikelet per panicle (SPP), spikelet fertility (SF), grain length (GL), grain width (GW), thousand grains weight (TGW) and plot yield (PY) compared to the recurrent parent PR114. Ten ILs showed significant increase in SPP during both the years over the recurrent parent and the increase ranged from 9.3 – 41.0 per cent. Likewise, five ILs showed significant increase in grain length ranging from 4.95 – 10.1 per cent, two ILs showed significant increase (16.0%) in TGW, and nine ILs showed significant increase (6.4-24.1%) in grain width. Parent polymorphism between PR114 and donor species O. longistaminta was done using a set of 366 SSR markers spanning all the 12 linkage groups and 92 polymorphic SSRs were used for characterizing the ILs. The alien segments introgressed in each of the ILs varied from 1.09% to 13.04%. Alleles which showed positive effect from O. longistaminata were observed in chromosomal regions associated with DF, PL, SPP, GL and GW traits. Alleles associated with negative effects were also observed for PH, TN, SF, PY and grain size traits. The ILs showing significant increase for one or more traits have been identified and these could be used for mapping QTL introgressed for these traits, using bi-parental crosses.
Open Access
MARKER ASSISTED INTROGRESSION OF THE opaque2 (o2) GENE INTO ELITE MAIZE (Zea mays L.) INBRED LINES
(PAU Ludhiana, 2012) Ravneet Kaur; Yogesh, Vikal
Maize (Zea mays L.) is deficient in the essential amino acids, lysine and tryptophan. The low nutritive value of maize endosperm protein is genetically corrected in Quality Protein Maize (QPM), which contains the opaque 2 (o2) gene along with numerous modifiers for kernel hardness. Two normal inbred lines viz. LM12 and LM13 were targeted for conversion into high quality protein versions using gene based SSR markers located within o2 using two generation marker-based backcross breeding program. DMR7 and CML165 were used as the QPM donor parents. In BC2F1 foreground selection for o2 gene was done using SSR marker phi057 in cross 1 (LM12/DMR7//2*LM12) and with SSR marker umc1066 in cross 2 (LM13/CML165//2*LM13). Out of the total 188 BC2F1 plants of cross 1, 87 heterozygous plants were obtained and similarly in cross 2, 114 heterozygous individuals were obtained out of 206 plants. The BC2F1 plants having o2 allele in heterozygous form were further screened using o2 gene flanking SSR marker and the plants either single or double recombinants, were identified. Whole genome background selection on the single or double recombinants using 104 SSR markers identified three plants with 83.7 to 91.0% recurrent parent genome content in each cross and were selfed to generate BC2F2 population. The three BC2F2 families were subjected to foreground selection and phenotypic selection for kernel modification. Fifty six plants in cross 1 and thirty nine plants in cross 2 having o2 allele in homozygous condition were selfed to generate BC2F3 progenies. The kernels from BC2F2 ears were segregated for hardness of endosperm and showed different levels of modification. In BC2F2 kernels the tryptophan concentration ranged from 0.56% to 0.97% for cross 1 whereas for cross 2 it ranged from 0.58% to 0.91%. 50% opaque BC2F3 lines were evaluated for agronomic traits for selection of single plant progenies to generate BC2F4 progenies.
Open Access
CYTOGENETIC CHARACTERIZATION AND MOLECULAR TAGGING OF LEAF RUST RESISTANCE GENE TRANSFERRED FROM Aegilops peregrina INTO HEXAPLOID WHEAT
(PAU Ludhiana, 2012) Deepika Narang; Satinder Kaur
Leaf rust is the most damaging disease of wheat worldwide. Wild germplasm of wheat is known to be a rich source of different useful genes. Ae. peregrina acc. pau 3519, a non-progenitor tetraploid species with UUSS genome, was found to be an excellent source of resistance for various diseases. In the present investigation, WL711-Ae. peregrina introgression lines (ILs) were characterized through Genomic in situ hybridization and no hybridization signal was detected in any of the IL. F2 population derived from the cross of introgression line 973 with WL711, was tested at seedling stage and segregated in a ratio of 191 resistant:63 susceptible plants with 2=0.0052(3:1). At the adult plant stage, the population segregated into 185 resistant: 65 susceptible with 2=0.0053(3:1). The progeny testing in F3 confirmed the transfer of a single gene for leaf rust resistance. Molecular characterization of the IL973 with the SSR markers indicated Ae. peregrina specific introgression on homoeologous groups 1, 2, 5 and 7. Bulked segregant analysis was done using 79 polymorphic markers and one of the SSR marker Xcfd50, detected IL973 specific allele in resistant bulk. This marker has already been reported to be linked with known gene Lr58 from Ae. triuncialis on long arm of chromosome 2B. STS marker Xncw-Lr58 amplified on whole F2 population and it was mapped at a distance of 1.7 cM from leaf rust resistance gene using computer software MapDisto. Nullitetrasomic analysis confirmed the introgression of Ae. peregrina leaf rust resistance gene (LrAP) on the wheat chromosome 2D. LrAP gene studied during the present investigation is either a new gene or an allele of the already known gene Lr58 will be established through allelic tests in subsequent studies.
Open Access
INTRODUCTION AND EXPRESSION OF ANTIFUNGAL CHITINASE AND β-1, 3-GLUCANASE GENES IN ARBOREUM COTTON TO INDUCE RESISTANCE AGAINST Fusarium oxysporum
(PAU Ludhiana, 2013) Aradhna Sonik; Jagdeep Singh, Sandhu
In the present study, immature embryos of diploid cotton (Gossypium arboreum L. cv. RG8) were transformed with three recombinant plasmids viz., H1Z, H2Z and pBI121 containing -1, 3-glucanase, chitinase and GUS gene cassettes respectively, under the control of CaMV 35S promoter and NOS terminator, with an objective to induce resistance against Fusarium oxysporum. The immature embryos were excised from 18-day old cotton bolls obtained during early, mid and late flowering seasons of the diploid cotton plants, cultured on germination medium (GM) [basal MS medium containing proline (560mg/l), BAP (2mg/l), GA3 (40mg/l), activated charcoal (0.20%), omnatax (500ppm) and casein hydrolysate (500mg/l)] followed by incubation under dark at 28±2oC. After five days of culture establishment, the germinating embryos were transferred under 16 hr(s) photoperiod during which they elongated into shoots and developed true leaves. Root multiplication was induced by placing elongated shoots on root induction medium (1/2 strength MS medium fortified with 0.20% activated charcoal). The plantlet formation efficiency of the embryos cultured during the mid flowering season was found to be significantly higher (37.67%) compared to the embryos excised during late (32.80%) flowering season and at par with the efficiency of embryos excised during the early season (36.28%). The three recombinant plasmids–H1Z, H2Z and pBI121, after being confirmed for the presence of gene cassettes by PCR and restriction digestion were introduced in the immature embryos using particle bombardment. The integration of gene cassettes was analysed in transient histochemical GUS assay and PCR, 48 hr(s) post bombardment. The results revealed 91.6% bombarded embryos expressing GUS gene, whereas 66.6% embryos had the co-integration of H1Z and H2Z plasmids containing -1, 3-glucanase and chitinase coding region respectively. The bombarded embryos were cultured on media GM supplemented with 210mg/l ampicillin for five selection cycles of 20 days each. After five selection cycles, 12 plantlets survived and the plantlet formation efficiency of 0.65% was obtained, which was significantly higher than the efficiency of non-bombarded embryos (0%). The PCR analysis, 100 days post bombardment, showed co-integration of -1, 3-glucanase and chitinase genes in eight plantlets with a transformation efficiency of 0.43%, while the remaining 4 plantlets showed integration of either of the genes. These putative transgenic plantlets are currently growing in the glass-house.
Open 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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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.
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, 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.

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