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Chaudhary Charan Singh Haryana Agricultural University, Hisar
Chaudhary Charan Singh Haryana Agricultural University popularly known as HAU, is one of Asia's biggest agricultural universities, located at Hisar in the Indian state of Haryana. It is named after India's seventh Prime Minister, Chaudhary Charan Singh. It is a leader in agricultural research in India and contributed significantly to Green Revolution and White Revolution in India in the 1960s and 70s. It has a very large campus and has several research centres throughout the state. It won the Indian Council of Agricultural Research's Award for the Best Institute in 1997. HAU was initially a campus of Punjab Agricultural University, Ludhiana. After the formation of Haryana in 1966, it became an autonomous institution on February 2, 1970 through a Presidential Ordinance, later ratified as Haryana and Punjab Agricultural Universities Act, 1970, passed by the Lok Sabha on March 29, 1970. A. L. Fletcher, the first Vice-Chancellor of the university, was instrumental in its initial growth.
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ThesisItem Open Access Molecular breeding for developing grain protein and micronutrient (Iron and Zinc) dense Indian bread wheat genotypes(CCSHAU, 2014) Tomar, Luxmi; Yadav, Neelam R.Marker assisted selection strategy was used to introgress the functional Gpc-B1 allele in Indian wheat varieties (WH711 and PBW343) from Lassik, a hard red spring wheat. Fifty two of the most widely accepted Indian varieties were screened for their GPC and micronutrient (Zinc and Iron) content. A narrow range of variability was observed in GPC (9.3-13.7%), zinc (25.8-54.1mg/kg) and iron (37.5-57.9mg/kg) content. H711 and PBW343 were selected and crossed with Lassik. F2 plants were screened for presence/ absence of functional Gpc-B1 and rust resistance genes Lr37/Yr17/Sr38 and Lr34/Yr18 using allele specific amplification (ASA) markers. Plants from F2 observed, on an average 26.4% increase in GPC, 37.5% in zinc and 64.8 % in iron content. The plants did not show any rust susceptibility under field conditions. F2 plants with minimum reduction in yield were chosen for back-crossing with their respective Indian parent. Plants obtained from subsequent back crosses i.e . BC1F1 and BC1F2, also had better quality attributes than the parents. The GPC increased on an average by 30.2% and twice zinc (90.2%) and iron (52.5%) as compared to the recurrent parent. Hybrid plants also had better agronomical properties and showed resistance to stripe rust under field conditions whereas parental genotypes faced significant yield loses. These plants showed complete rust resistance due to presence of Gpc-B1/Yr36 with the Lr37/Yr17/Sr38 and Lr34/Yr18 loci. This population has all the combined benefits of better nutritional quality, yield and disease resistance and could be used both in MAS as well as for transferring the Gpc-B1in other Indian varieties to minimize the linkage drag. Though introgression of Gpc-B1 was linked with a net reduction in yield, interestingly, some of these plants with functional Gpc-B1 allele had higher TKW than either of the parents emphasizing the extent of influence of GxE interaction could have on Gpc-B1 allele. A consensus between quality and quantity has to be reached while breeding for improving GPC in wheat.ThesisItem Open Access Phenotypic and molecular analysis for terminal heat stress tolerance in wheat (Triticum aestivum L.)(CCSHAU, 2013) Pritam Kumar; Dhillon, SantoshThe present investigation was conducted to study the phenotypic and molecular analysis of terminal heat stress tolerance in wheat (Triticum aestivum L.) in the Department of Molecular Biology & Biotechnology, CCS Haryana Agricultural University, Hisar, India. Two F2 mapping populations containing 90 plants each involving crosses between Hindi62 (heat tolerant) x WH711 (heat susceptible) and DBW16 (heat tolerant) x WH147 (heat susceptible) were developed. Seeds from individual F2 plants were harvested and sown in two environments, normal sown (30th November 2011) and late sown (26th December 2011) to get F3 mapping population. Heat stress was created by late sowing the crop. Data was recorded on individual plant basis for both the crosses in respect of characters viz. membrane thermostability index, chlorophyll stability index, grain yield, kernels/spike, thousand kernel weight (Test weight), days to heading, days to maturity, plant height, number of tillers/plant, biomass/plant, harvest index, grain filling rate and grain filling duration.103 SSR, 25 RAPD and 2 known gene primers were used in molecular marker analysis for terminal heat stress tolerance. For QTL identification data was analysed by WinQTL-Cartographer. In cross Hindi62 x WH711 three QTL were detected for chlorophyll index at map positions, 135.7 (1B), 184.6 (2A) and 36.4 (6B) by SSR marker WMC 416, Xbarc188, Xgwm526, WMC170, Xgwm219, WMC398respectively. Six QTL were detected for membrane thermostability index at map positions, 118.7 (1B), 58.2 (2A), 137.2 (2A), 176.7 (2A), 62.0 (5A) and 11.7 (6B) by SSR marker WMC416, Xbarc188, WMC407, WMC642, Xgwm356, Xgwm95, Xgwm526, WMC170, Xgwm443, WMC475, Xgwm219 and WMC398. For yield per plant 6 QTLs were detected at map position103.4 (1B), 51.6 (2A), 130.6 (2A), 178.9 (2A), 55.4 (5A), 22.6 (6B). For grain filling duration one QTL was detected at chromosome no. 2A at a map position 171.9 and for grain filling rate 2 QTLs were detected on chromosome 2A and 6B at map position of 66.0 and 76.3 respectively. Similarly in cross DBW16 x WH147, 4 QTLs were detected for chlorophyll index on chromosome 1B, 2A, 6B and 6B respectively. Five QTLs were for membrane thermostability index on chromosome 1A, 1B, 2A, 3A and 6B.One QTL was identified for yield per plant and one for grain filling rate at chromosome 2A and 4A respectively. Five QTLs were identified for grain filling duration at a map position 113.5 (2A), 31.5 (2A), 88.5 (3A), 31.0 (5A) and 29.0(5B). These QTLs may be used for further improvement of the traits they represent. HSP16.9 and HSP 101b primers showed clear distinction between thermotolerant and thermosensitive genotypes and are suggested as potential DNA markers for improving thermotolerance in wheat using marker-assisted selection.ThesisItem Open Access Marker assisted selection for introgression of bacterial blight (BB) resistance genes in rice (Oryza sativa L.)(CCSHAU, 2013) Malik, Rekha; Boora, Khazan SinghThe present investigation was undertaken to introgress the major BB resistance genes (Xa21, xa13 and xa5) into BB susceptible rice variety CSR-30, from BB resistant donor varieties Pusa Basmati -1460 (having two BB resistance genes Xa21 and xa13) and IRBB-60 (having three BB resistance genes Xa21, xa13 and xa5) through marker assisted selection. Crosses were made between CSR-30 x Pusa Basmati -1460 and CSR-30 x IRBB-60. Foreground selection was carried out in F1 plants of both the crosses using specific STS markers pTA248, RG136 and RG556 linked to Xa21, xa13 and xa5, genes respectively. In cross CSR-30 x Pusa Basmati -1460, four F1 seeds were harvested. Foreground selection was carried out in which two plants were found to have both the BB resistance genes Xa21and xa13. These F1 plants were grown in the net house and selfed to get F2 seeds. In cross CSR-30 x IRBB-60, 6 F1 seeds were harvested. Foreground selection was carried out and three plants were found to have three resistance genes Xa21, xa13 and xa- 5. Backcrosses of these positive F1 plants were made with CSR-30 and the BC1F1 seeds of this cross harvested. These BC1F1 seeds were selfed to produce BC1F2 seeds. In foreground selection, 10/250 BC1F2 plants were found to have all the three resistance genes Xa21, xa13 and xa-5. For background selection, 300 SSR primers were used to identify the polymorphism between parental genotypes Pusa Basmati-1460 and CSR-30 and between IRBB-60 and CSR-30, out of which 72 and 104 SSR markers produced polymorphic alleles between the parents Pusa Basmati-1460 and CSR-30 and parents IRBB- 60 and CSR-30, respectively. Cluster tree analysis revealed that the three gene pyramided BC1F2 rice genotypes and the recipient parent CSR-30 fell in one group with two major sub-groups and the donor parent IRBB-60 remained as a separate group. The percentage recovery of recurrent parent genome in the three-gene pyramided BC1F2 genotypes ranged from 44.2% to 78.9% using GGT software. The pyramided lines exhibited very high level of resistance to bacterial blight disease when artificially inoculated with Xanthomonas oryzae strain isolated from the BB infected fields of RRS, Kaul, CCSHAU, Hisar. The agro-morphological traits of the three-gene pyramided BC1F2 genotypes were found to be either superior or on par with the recurrent parent CSR-30. Genotype G-3 showed the best agronomic features with maximum recovery of recurrent parent genome (RPG) 78.9% followed by G-169 with RPG 76.2% followed by line G-7 with RPG 72.5%. The plants having maximum recurrent parent genome were backcrossed with the recurrent parent CSR-30 and BC2F2 seeds were harvested. It is suggested that positive F2 seeds of cross CSR-30 x Pusa Basmati -1460 should be backcrossed with CSR-30 for further work. However, in cross CSR-30 x IRBB-60, BC2F2 genotypes should be further Evaluated for agronomic traits. This work demonstrates the successful application of MAS for targeted introgression of multiple resistance genes into premium quality rice variety CSR-30.ThesisItem Open Access Development ofRhizobia with improved symbiotic properties for diverse legume crops(CCSHAU, 2014) Ikbal; Sikka, Virendra K.Present investigation was undertaken to develop efficient rhizobial strains and to over ride host specific symbiotic nodulation behavior in the process of symbiotic infection in diverse legume crops. Such promiscuous Rhizobium strains with improved efficiency to fix nitrogen might add higher amount of nitrogen to the plants. Towards these efforts 295 rhizobial strains were isolated from nodules of legumes chickpea, pea and lentil grown in different fields. These isolates were then analyzed for their cellulose and pectin utilizing ability by growing them on CMC and pectin media. Best growing strains on CMC medium were selected for gamma irradiation to create genetic variations. These gamma irradiated mutant isolates were screened for their enhanced cellulose and pectin utilizing ability. These mutants were compared with their parents for enhanced cellulose and pectin utilization ability. It was found that percent increase in growth on CMC media over parents was in the range of 8.82 to 422.22%. Rhizobial mutants having contrasting antibiotic profile were electrofused for obtaining cross infective rhizobia. Total five fusants each from the rhizobial cross combination E1, E2, E3, E4 and E5 which had their resistance pattern confirmed were obtainedwith the help of electroporation. Symbiotic infection behaviour and cross infectivity of electrofusants, mutants and wild type rhizobial isolates were analysed on chickpea, pea and lentil plants through plant infection test. It was found that doses of gamma-rays on biofertilizer appeared significant effect on number of nodules, plant weight and shoot N content. Greater number of nodules and promiscuous behavior was observed in mutants followed by electrofusants as compare to their parental rhizobial isolates.Mutant rhizobial strains improved shoot nitrogen content up to 50% inchickpea,45% in pea plant whereas the same trend was also observed on lentil plants. The developed Rhizobium strains with improved symbiotic association and ability to infect across strict specificity for host legumes would be of great help for the farming community at large. Representative strains were selected for the molecular analysis based on PCR amplification. As a nodulation genes marker, nodA, nodC, nodD, nodE, nodV and nodW genes were selected, which are common nod genes essential for nodulation in all rhizobial species investigated so far and these are also determinants of host range. The comparison of the nod genes between symbiotic strains and host plant groups taken together indicated that lateral transfer of Sym genes and genetic rearrangements are involved in the genetic modification.Apparently the Nod factors of the strains are quite variable. The PCR amplification of these factors determined that these factors were considerably altered through mutation and rearrangements were taken place by electro-poration.ThesisItem Open Access Molecular mapping of quantitative trait loci for yield, yield components and terminal heat tolerance in bread wheat (Triticum aestivum L.)(CCSHAU, 2014) Bishnoi, Shewane; Chowdhury, V.K.A total of 103 F10 RILs of bread wheat derived from HUW-510X WH-730 were evaluated for yield, yield components and tolerance to terminal heat stress in normal and late sown field experiments during the Rabi season of 2010-11 and 2011-12 using an alpha design in two replicates. The traits evaluated were : days to heading, days to anthesis, grain development rate, grain filling duration, number of tillers /1/2m row-length, number of kernel per spike, number of spikelet per spike, spikelength, days to physiological maturity, thousand grain weight and yield/1/2 m row. HSI of the traits was used to assess tolerance to heat stress. Highly significant genotypic differences were observed for all traits. Gene dispersion between the parents caused transgressive segregants for all the traits. Selective genotyping QTL method was used to screen 300 SSRs in a subpopulation of 37 RILs. 21 linkage groups were formed using 99 polymorphic markers. SMA detected 171 QTLs at 5%, 38 at 1% and 8 at 0.1% level .CIM detected 37 QTLs (31 for yield and related traits and 6 for HSI). Some QTLs were present in coupling and repulsion phase, explaining genetic basis of positive and negative association respectively between traits. Favourable QTLs for a number of traits and for heat tolerance (HSI) were present in WH-730 parent useful to counteract the terminal heat stress. Some QTLs were detected controlling more than one trait. This may be due to linked QTLs or pleiotropic effect of a single QTL. Favourable allele from WH-730 QTLs for HSI TGW and HSI DPM in the interval barc137-gwm140 on 1B may be used to counteract the terminal heat stress.ThesisItem Open Access Pyramiding of bacterial blight resistance genes in rice (Oryza sativa L.) using marker assisted selection(CCSHAU, 2014) Reddy, P. Vishnuvardhan; Boora, Khazan SinghRice (Oryza sativa) is one of the major dietary staple food in the world. Bacterial blight (BB), one of the most serious threats to the rice crop in irrigated and rainfed areas of the world, is caused by the bacterium, Xanthomonas oryzae pv. oryzae (Xoo). The present work was committed to pyramid major BB resistance genes (Xa21, xa13 and xa5) into susceptible rice varieties HKR-47 and HKR-127, from BB resistant donor variety IRBB-60 through marker assisted selection. Crosses were made between HKR-47 × IRBB-60 and HKR-127 × IRBB-60. Twenty four seeds of former and seven F1 seeds of latter were harvested respectively. Foreground selection was carried out in the F1 plants using specific STS markers pTA248, RG136 and RG556 linked to Xa21, xa13 and xa5 genes, respectively. A total of three and two plants from both the crosses were found to have all the three resistance genes Xa21, xa13 and xa5.These positive F1 plants were backcrossed with respective recurring parents HKR-47and HKR-127. Seventy three and eighty one BC1F1 seeds of these crosses were harvested. Foreground selection of BC1F1 and BC2F1 was carried out to confirm the presence of three BB resistant genes using specific markers. Plants having three genes were selected for background selection. Three hundred SSR markers representing whole of the genome were screened with the parental genotypes. Finally, the plants having maximum genome recovery was analyzed by NTSYS and GGT softwares. The backcross population of HKR-47 and HKR-127 showed varied recovery of recurrent parent genome i.e. from 81% to 88 % (NTSYS) with HKR-47. Plants C-22 and C-23 had the maximum recurrent parent (HKR-47) genome contribution of 86.4 and 83.3 (GGT). In case of cross HKR-127, it ranged from 55% to 88% (NTYSYS). The plants D-15 and D-19 had the maximum recurrent parent (HKR-127) genome contribution of 83.3%. BC2F1 plants were selfed and BC2F2 seeds were harvested. Foreground selection was carried out in these BC2F2 plants of both the crosses as well as in BC3F1 plants. It was found that yield and agro-morphological traits of three-gene pyramided BC3F1 plants were similar to recurrent parents HKR-47 and HKR-127. BC3F2 seeds were harvested separately. The present work demonstrates the successful pyramiding of bacterial blight resistant genes Xa21, xa13 and xa5 into HKR-47 and HKR-127 rice varieties which further can be continued for field evaluation, disease reaction and yield parameters.ThesisItem Open Access Genetic and molecular analysis of segregating population(s) derived from crosses between the mineral-rich and high-yielding indica rice genotypes(CCSHAU, 2012) Jitender Kumar; Jain, R. K.Molecular markers can greatly improve the efficacy of rice breeding programs to improve the mineral (iron and zinc) content and bioavailability. Two F2 populations (PAU201 x Palman 579 and HKR47 x BR 4-10) and 8 BC 1 plants (PAU201 x Palman 579/PAU201) derived from crosses between iron rich (Palman 579 and BR 4-10) and high yielding indica rice varieties (PAU201 and HKR47) were evaluated for various agronomic traits and mineral (Fe and Zn) content. Both the segregating F 2 populations displayed large variation for plant height (55.8-102.3 and 77.5-110.2 cm), effective number of tillers per plant (2-20 and 3-20), panicle length (15.9-25.4 and 16.3-32.03), grain yield per plant (1.93-41.53 and 3.32-59.06 g), 1000 grain weight (11.57-29.15 and 16.31-34.43 g), grain length/breadth ratio (2.90-5.10 and 2.70-4.41), Fe content (18.6 to 475.4 µg/g and 7.3 to 146.5 µg/g) and Zn content (4.4 to 157.4 µg/g and 12.5 to 92.9 µg/g), respectively. Variation for Fe (69.4 to 86.1 µg/g) and Zn (21.5 to 95.6 µg/g) contents was also observed in BC 1 plants. One of the PAU201 x Palman 579 F 2 plants, had exceptionally higher iron (475.4 µg/g) and zinc (157.4 µg/g) contents in comparison to the donor parent, Palman 579 (Fe-382.36 µg/g, Zn-21.6 µg/g). Transgressive segregation for grain iron and/or zinc contents was noticed in F 2 individuals of both the crosses. Grain iron content showed significant positive correlation (r=0.523 and 0.299) with grain zinc content in both the F 2 populations indicating the feasibility of improving Fe and Zn levels simultaneously in rice grain. As many as 76/100 SSRs, which showed polymorphism among the two parental rice genotypes, were used to map the QTLs associated with mineral content in grains using 50 PAU201 x Palman 579 F 2 plants displaying the entire range of variation in Fe and Zn contents. A total of 171 alleles were identified in 50 PAU201 x Palman 579 F 2 plants and 14 new recombinant alleles (different that those in parent rice varieties) were identified. NTSYS-pc based two dimensional PCA analysis showed scattering of the F 2 population between the two distinct parental genotypes. Composite interval mapping (CIM) analysis by WinQTL cartographer 2.5 revealed a total of eleven QTLs for mineral content in grains on chromosome 2, 3, 7, 10 and 12. Eight QTLs for Fe content were mapped on chromosome 2 (qFE1, qFE2 and qFE3 at map positions 54.4, 138.6, 159.2 cM espectively), chromosome 3 (qFE4 at map position 54.6cM), chromosome 7 (qFE5 at map position 31.3 cM), chromosome 10 (qFE6 and qFE7 at map positions 25.9, 108.0 cM respectively) and chromosome 12 (qFE8 at map position 60.3 cM). In addition, three QTLs (qZN1, qZN2 and qZN3) for Zn content were identified on chromosome 2 (qZN1) and 10 (qZN2 and qZN3) with the map positions of 65.5, 23.8 and 115.9 cM respectively.ThesisItem Open Access Exploring synteny and functions of pearl millet (Pennisetum glaucum (L.) R. Br.) drought tolerance QTL region across rice and perennial ryegrass (Lolium perenne L.)(CCSHAU, 2014) Richa; Yadav, R.C.Previous genetic mapping studies in pearl millet [Pennisetum glaucum L. (Br.)] have identified and validated a major QTL of drought tolerance (DT) on linkage group (LG) 2 that enhances yield under drought stress by more than 32%. Recently, this QTL has been fine mapped to a 3 cM region using a high resolution cross and 14 candidate genes based markers were mapped in the fine mapped region as SNP and CISP markers. In the present study, the synteny and functions of these candidate genes across rice, sorghum and ryegrass were explored. Although a broad conservation of gene linkage (macrosynteny) has been reported previously in grass genomes, our results indicate that extensive translocations and duplication events in these grass genomes has disrupted regional synteny. It is clearly evident from our results that DT-QTL region on pearl millet LG 2 shows synteny to >1 linkage groups in rice, sorghum and ryegrass. Most immediate practical application of this work would be future identification of tightly linked markers for marker-assisted selection and map-based isolation of candidate genes in rice, sorghum and ryegrass. The expression patterns of these candidate genes underpinning drought tolerance QTL were also investigated under different water stress treatments at flowering and post-flowering stages. Six candidate genes, protein phosphatase 1 regulatory subunit SDS22, Zn finger CCCH type transcription factor, ubiquitin conjugating enzyme, serine threonine protein kinase, chlorophyll a/b binding protein and dipeptidyl peptidae IV showed either differential expression or fold change among genotypes, H 77/833-2 (drought susceptible), PRLT 2/89-33 (drought tolerant) and DT-QTL NIL ICMR01029 under drought stress conditions which suggests the role of these putative candidate genes under drought stress in pearl millet. The candidate gene strategy shows promising results for bridging the gap between quantitative genetics and molecular genetics approaches to study complex traits like drought tolerance. Once the genes are validated, we can develop CGs-based “functional markers” to enhance the speed, precision and efficiency of introgression of this QTL into farmer-preferred cultivars. Further, metabolome profiling of the pearl millet genotypes contrasting for drought tolerance has also been done by both FIE-MS and GC-MS to see the compositional differences in metabolites under control and drought stress conditions. Compositional differences among the genotypes in different environment conditions may play a key role for the selection of the genotypes for further use in breeding programmes.ThesisItem Open Access Cloning and Expression Analysis of HSP 101 Gene In Bread Wheat (Triticum aestivum L.Em.Thell.)(CCSHAU, 2014) Singal, Manjjri; Dhillon, SantoshThe four genotypes/varieties of wheat i.e. two heat tolerant (WH730 and WH 1021) and two heat sensitive (WH 147 and WH 1021) varieties were raised in field under normal and late sown conditions and data on morpho-phsiological traits like canopy temperature depression, grain yield, 100 seed weight and heat susceptibility index were evaluated to assess the effect of high temperature on these traits. High temperature negatively affected all these traits. The affect was more in thermosensitive varieties as compared to thermotolerant varieties. The order of thermotolerance based on heat susceptibility index (HSI) was: WH 730 (0.42) > WH 1021 (0.68) > WH 711 (1.19) > WH 147 (1.53). Expression analysis of HSP101transcripts (HSP101, HSP101band HSP101c) were studied by growing the above four varieties in controlled conditions where artificial heat stress at different temperatures (25º, 31ºC, 34ºC, 37ºC and 40ºC) were given to plants at the seedling stage. All heat shock transcripts (HSP101, 101b & 101c) were found to be expressed at all heat shock treatments and were found to be maximum at 40ºC. In all heat shock transcripts (HSP101, 101b & 101c) WH730 showed the maximum level of gene expression and WH 147 showed minimum level of gene expression. Among all the three HSP transcripts, (HSP101, HSP101b and HSP101c) expression level of HSP101 was found to be maximum in all the four varieties. Partial cDNA sequences of HSP101 (1066 bp), HSP101b (1077 bp) and HSP101c (1249 bp) were cloned and sequenced from WH 730 (heat tolerant) and WH 147 (heat sensitive) genotypes. Phylogram generated using gene sequences of HSP101 isoforms (HSP101, HSP101b and HSP101c ) of WH 730 and WH 147, gene sequences of HSP101 isoforms of Triticum aestivum and other crop plants (Triticum turgidum, Zea mays, and Oryza sativa) available in Genbank, demonstrated that all the sequences are descendents of a common ancestor. Sequences of HSP101 gene and sequences of HSP101b gene were found to be closest in comparison to sequences of HSP101c gene. WH 730 showed maximum thermotolerance while WH 147 was least thermotolerant, based on overall picture of morpho-phsiological traits and gene expression analysis studies.