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Open Access
Inheritance studies and mapping of yellow mosaic disease resistance in an interspecific cross of mungbean (Vigna radiata (L.) Wilczek) and urdbean (Vigna mungo (L.) Hepper)
(Punjab Agricultural University, Ludhiana, 2020) Saini, Pawan; Bains, T. S.
Yellow mosaic disease (YMD) is the most vicious viral disease causing more than 85 percent economic losses. Among Vigna species, ricebean (V. umbellata) and urdbean (V. mungo) are known to possess high level and stable resistance to YMD. In the present investigation, an interspecific cross between mungbean cv. ML 267 x urdbean cv. Mash 114 was made for inheritance studies and mapping of YMD resistance. A total of 190 were made, of which eight successfully reached pod development stage with low crossability rate of 4.21 percent. The interspecific cross (ML 267 x Mash 114) recorded lower (16.67 percent) F1 seed germination.The hybrid lethality and hybrid survivability were observed as 60 and 40 percent in F1 generation respectively. The F1 hybrid plants exhibited intermediate phenotype between the parental genotypes with respect to growth habit, leaf colour, stem colour and maturity. The true interspecific hybrids were also confirmed with molecular markers. The F2 population derived from this cross was phenotyped for YMD resistance and segregation ratio revealed presence of two genes with duplicate gene action with chi square value of 7.35 at 0.05 level of significance. Further, a set of newly designed mungbean whole genome sequence (WGS) based SSR markers were used for validation of on different Vigna accessions. The SSR markers showed 100 percent transferability on 25 different accessions of 13 Vigna species. The obtained polymorphic information content value (PIC), effective multiplex ratio (EMR), marker index (MI) and resolving power (RP) suggested their usefulness in different genetic studies. Different Vigna accessions were also phenotyped for YMD resistance and V. glabrescence identified as the most promising YMD resistance source. Out of 250 WGS based SSR markers, 144 markers showed polymorphism between ML 267 and Mash 114 with 57.60 percent markers were polymorphic. These polymorphic markers along with 32 Vigna species specific markers were utilized for bulked segregant analysis (BSA) Out of 144, only one marker SSR 121 showed polymorphism between YMD resistant and susceptible bulk. Further, the SSR 121 marker showed co-segregation in F2 population with YMD resistance. The marker SSR 121 was found to be located at 6 cM distance from YMD resistance locus. The findings of study paved the way for further validation of SSR 121 and its utilization in marker assisted breeding programme in mungbean.
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Mapping QTL for heat tolerance components and development of SNP markers in wheat (Triticum aestivum L.)
(Punjab Agricultural University, Ludhiana, 2020) Walia, Puneet; Saini, Johar Singh
Bread wheat (Triticum aestivum L. em. Thell) is an important staple cereal consumed across the globe. To meet the food requirements of the growing population, wheat production must increase while addressing the upcoming challenges of which heat stress is increasingly causing a concern as the global temperature is expected to increase. A meagre increase beyond the optimum from anthesis to grain filling can result in significant yield losses. The main aim of this study was to find QTLs for heat tolerance in wheat using Genotyping by Sequencing (GBS) and thus identifying Single Nucleotide Polymorphism (SNP) for the same. Around 196 doubled haploid(DH) lines derived from a cross between Sunstar (heat tolerant) and PBW 343 (heat susceptible) were evaluated under timely sown(normal), late sown and very late sown conditions (heat stress) at two locations (Ludhiana & Hisar) for two seasons. Days to flowering (DF), days to maturity (DM), grain filling duration (GFD), plant height (PH), spike length (SL), number of spikelets/main spike (NSMS), Grain yield (GY),grain weight/main spike (GWMS), grain number/main spike (NGMS) and hundred grain weight (HGW) were recorded for three treatments and two years at two locations. Heat susceptibility index (HSI) of these traits were used as phenotypic data for QTL identification. A total of 16 QTLs were identified on chromosome 2A, 4A, 7A, 1B, 3B, 1D, 3D, 5D & 6D. The QTLs were detected for DF (1), DM (1), GFD (5), PH (2), NSMS (3), Grain yield (1) and NGMS (3). Maximum number of QTLs were found on chromosome 3B, followed by 4A, 1B & 1D.
Open Access
Analysing Brassica rapa genome by comparing natural accessions and the derivatives extracted from B. juncea allotetraploid
(Punjab Agricultural University, Ludhiana, 2020) Snehdeep Kaur; Gurpreet Kaur
Present studies were conceived to undertake a comprehensive characterization of the phenotypic and molecular genetic diversities available in a large germplasm set comprising 195 fixed genotypes of Brassica rapa (AA; 2n=20). These included derived B. rapa lines extracted from B. juncea (AABB; 2n=36). DArTseq platform was used to sequence genomic DNA samples to aid SNP discovery. Field experiments were conducted during 2017-18 and 2018-19 over two sowing dates each at the forms of Punjab Agricultural University, Ludhiana. Morphological characterizations involved 18 traits representing flowering, crop architecture, yield components and seed quality. Molecular diversity analysis revealed that derived B. rapa clustered separately from the natural B. rapa. Natural B. rapa, itself formed two major groups with several subgroups representing variations among various subspecies of oleifera forms. Analysis of variance for phenotypic traits indicated significance of genotypic and genotype x environment interactions. Coefficient of variations was high for flowering, plant height, secondary branches and yield per plant. CV was lowest for pod length. Genome wide association studies were conducted using GAPIT 3 software. MLMM and Blink were preferred algorithms. We identified a large number of MTA’s involving target traits. Many of these were exclusive for DR. Annotation helped to predict candidate genes in the genomic region associated with the significant SNPs. Fifteen candidate genes were predicted for flowering traits. These genes are involved in the photoperiodic, gibberellic acid signaling pathways that control the expression of flowering genes. Several genes regulating flower development, maintenance of floral organ identity, regulation of timing of transition from vegetative to reproductive phase were also identified. For yield related traits we predicted genes involved in the growth and development, maintenance of shoot apical meristem, development of lateral organs, branching, auxin growth factors and leaf morphogenesis on A09. Two very important genes, IKU2 and LACS8 were identified for seed size. Candidate genes predicted for seed quality traits included genes involved in regulation of seed storage and reserves such as oil, protein and GSL. In conclusions, our studies have helped to catalogue molecular-genetic and phenotypic diversities present in B. rapa. These also established derived B. rapa as a genetic resource distinct from natural B. rapa. Many candidates were predicted for regulating key economic traits in the crop. Identified loci can be used in future marker-assisted selection (MAS) after due validation.
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Identification of quantitative trait loci (QTLs) for β-glucan in oat (Avena sativa L.)
(Punjab Agricultural University, Ludhiana, 2020) Gagandeep Kaur; Kapoor, Rahul
Characterization of germplasm resources is needed to facilitate its use in plant breeding and in research. In the present study, morphological traits and molecular marker methods were used to assess the genetic diversity among 96 oat genotypes. Analysis of variance showed a significant variation among all the genotypes for morphological traits. Based on squared Euclidean distance, all genotypes were divided into 10 different clusters. The best genotypes for dual purpose were IC 372493, EC 537815, P 7 295932, EC 537856, IC 372529, IC 37248, EC 246179, EC 537834, EC 209524, EC 528888, EC 537924, IC 372478, EC 246200. Ninety two SSR markers were used for diversity analysis, these revealed high level of polymorphism, detecting 250 alleles. Diversity analysis was evaluated using DARwin. The dissimilarity values ranged from 0.33 to 0.79 signifying a moderate degree of dissimilarity among the A. sativa accessions evaluated. As our major goal in this research project was to find the QTLs for β glucan content, so we needed the contrasting parents for β glucan content in that case. The best genotypes for higher β glucan content were OL 10, EC 237851, EC246158, EC 528874, EC 372463. The F2:4 mapping population consisting of 91 lines was developed from the cross between OL10 (5.8 %) and OL11 (0.8 %). On phenotypic evaluation, the values of β glucan content in the population ranged from 1.05% to 6.08%. Out of 231 markers, 25 were found to be polymorphic between the two parents and were used to genotype the mapping population. Using MAPDISTO with LOD score of 3.0 and recombination fraction of 0.3, four linkage groups were generated. QTL cartographer with single marker analysis (SMA) and composite interval mapping (CIM) were used to identify the QTLs associated with beta glucan content. QTL region was mapped on LG 1 and with LOD score 3.7, explaining the phenotypic variance of 31% and Second QTL region was mapped on LG 4 with LOD score of 4.3 and explaining the phenotypic variance of 33%. Further, fine mapping of putative QTL will facilitate the eventual identification of genes contributing to β-glucan content.
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Inheritance and mapping of Ascochyta blight resistance in chickpea (Cicer arietinum L.)
(Punjab Agricultural University, Ludhiana, 2020) Shruti; Dhillon, S.K.
To identify new sources of resistance to Ascochyta blight, a total of 100 genotypes including 9 checks were screened over a period of two years using field screening technique and cut-twig screening technique. Amongst these, 17 genotypes viz. FLIP 09294, GL 15007, GL 15010, GL 15011, GL 15012, GL 15014, GL 15016 GL 15019, GL 15020, GL 15021, GL 15031, GL 15032, GL 15033, GL 15036, GL 15043, GL 15044 and GL 15050 showed moderate resistance to Ascochyta blight during both the years of testing. Analysis of variance revealed significant differences amongst the 100 genotypes for almost all the yield and its attributing traits. On the basis of D2 analysis, these 100 genotypes including 9 checks were grouped into 11 clusters. Inter-cluster distance amongst these 11 clusters varied from 19.15 (cluster VII and cluster III) to 600.61 (cluster XI and IX) while intra-cluster distance varied from 0.00 (cluster IV and cluster IX) to 17.97 (cluster VIII). Harvest Index (56.46%), seed yield (8.59%), ascochyta blight score (6.28%), followed by biomass (5.03%), days to maturity (4.69%) and days to initiation of flowering (4.53%) contributed maximum towards genetic divergence. Selection for divergent parents based on these characters will be useful for developing potential hybrids and will increase the scope of isolating desirable recombinants in segregating generations. Concludingly, genotypes such as ICCV 15117, GL 15046, GL 15008, GL 15010, GL 15015, ICCV 15109, ICCV 15116 and ICCV 15311, FLIP 0962 will serve as suitable candidates for hybridisation or future breeding programmes. In order to understand the inheritance of Ascochyta blight resistance against the isolate 8 of race 6(3968) 5 F1s and 362 F2 plants derived from the cross PBG 7 x HC 5 were used and confirmation was done in F3 generation with pooled data. The study on genetics of resistance to Ascochyta blight in the cross PBG7 x HC 5 indicated dominance of susceptibility in F1, and segregation ratio of 15 (S): 1 (R) with χ2 value of 0.58 obtained in F2 indicated digenic recessive control to AB resistance in this cross. ddRAD-Seq of 75 RILs of this cross was undertaken to map the Ascochyta blight resistance in the cross PBG 7 x HC 5. A total of 2833 filtered polymorphic loci were used for linkage map construction through ddRAD-Seq. The maximum number of markers were mapped on CaLG04 (1227), while the minimum number of markers were on CaLG08 (75). One major QTL was identified for AB resistance on LG04 at 121 cM flanked by rs_NC_021163_1_3584639 and rs_NC_021163_1_7078480 at 115.97cM and 124.03 cM respectively. This QTL was characterised by high LOD (29.179) and high PVE% (29.66%). The SNP loci found to be linked with AB resistance in this study needs to be validated in different genetic background of other population to be used in marker-assisted selection.
Open Access
Genome wide association mapping for heat tolerance in maize (Zea mays L.)
(Punjab Agricultural University, Ludhiana, 2019) Longmei, Ningthaipuilu; Gill, Gurjit Kaur
The present investigation entitled “Genome wide association mapping for heat tolerance in maize (Zea mays L.)” was carried out at Punjab Agricultural University, Ludhiana during spring, 2016 and 2017 under optimal and heat stress conditions. Substantial significant variability was observed in the association mapping panel for heat associated traits under both the conditions. These traits were significantly affected by high temperature stress. Under heat stress, anthesis-silking interval (ASI) prolonged and some genotypes had no silk emergence. Average grain yield (3.46 t/ha) was low compared to that under optimal condition. Broad-sense heritability ranged from low to high in both conditions. The traits viz., days to 50% anthesis (AD) and silking (SD) and anthesis-silking interval (ASI) had significantly negative association with grain yield while plant and ear height and ear position showed significant positive correlation with grain yield. In 2016, 2017 and pooled over both years, total 159, 154 and 130 SNPs, respectively, were found associated with these traits under optimal condition. However, under heat stress, total 182, 109 and 71 SNPs were found associated with these traits during 2016, 2017 and pooled condition, respectively. Out of these, 7 and 9, SNPs was found associated with more than one trait under optimal condition while 23 and 11, SNPs under heat stress condition in 2016 and 2017, respectively. Similarly, total 122 and 123 haplotype blocks were associated with these traits individually while 20 and 6 haplotype blocks with more than one trait under optimal and heat stress conditions, respectively. Eighty one DH lines were found promising in both the conditions and were found associated with favorable alleles of significant SNPs and haplotype blocks for heat associated traits.
Open Access
Disruption and delineation of the Brassica fruticulosa introgressions in Brassica juncea (l.) coss
(Punjab Agricultural University, Ludhiana, 2019) Agrawal, Neha; Banga, S. S.
Three B. juncea - B. fruticulosa introgression lines (ILs) AD-64, AD-101 and AD-104, carrying resistance against mustard aphid, were investigated using cytogenetic and genome sequence analysis to physically map the introgressed chromatin fragments into B. juncea genome. For the cytogenetic characterization of lines, four sets of massive oligo probes were developed for the first time in Brassicas. These probes allowed identification of all chromosomes of recipient B. juncea. Subsequently, these probes were used together with B. fruticulosa genomic probe to physically map introgressed segments using modified genomic in situ protocol. Multicolour in situ analysis led to identification of introgressed segments on chromosomes A05, B02, B03 and B04 in the distal regions in ILs AD 104 and AD-64. IL-101 revealed the introgressions on the same set of chromosomes with an additional introgressed segment on chromosome A01 at sub terminal to intercalary position. Introgressions on A01, B02, B03 and B04 were validated through bioinformatic analysis of whole genome sequences from these ILs, B. juncea and B. fruticulosa. We also attempted to disrupt these introgressions through gamma irradiation. A carefully selected set of 35 M3 progenies (out of 160 M3, developed following gamma irradiations (200 kR) were phenotypically characterized for aphid resistance and agronomic traits. These progenies showed varied levels of resistance to mustard aphid under screen house conditions coupled with early flowering, short stature and improved seed and pod size. Bioinformatic analysis confirmed post irradiation, breakdown of large alien chromosome fragments in several progenies. Phylogenetic analysis of 35 M3 lines along with their parental lines and three B. juncea cultivars allowed identification of many progenies that became genetically closer to cultivated B. juncea as compared to the parental ILs. Few of these were resistant to mustard aphid as well. Progenies N-31, N-36, N-38, N-78, N-65 of IL AD-64 were considered most important in that respect.
Open Access
Genetic and physiological basis of nitrogen uptake and utilization in wheat (Triticum aestivum L.)
(Punjab Agricultural University, Ludhiana, 2019) Mohd. Shamshad; Sharma, Achla
Nitrogen (N) plays the crucial role for plant growth, and plant canopy photosynthesis drives grain yield and grain quality. However, excessive input of nitrogen ultimately increases the production cost and pollutes the environment. For combating these environmental and health issues, breeders need to improve the nitrogen uptake and utilization efficiency in elite agronomic backgrounds. Therefore, the present investigation was planned to assess genetic and physiological basis of nitrogen uptake and utilization efficiency in structured mapping population derived from the cross between PBW677 and PBW 703. The study also dissected the location of genes/QTLs governing nitrogen uptake and utilization efficiency in the wheat genome using high through-put sequencing (35K Axome® Breeder array) methods. A total of 296 wheat genotypes were sown under full nitrogen (Nf), half nitrogen (Nh) and zero nitrogen (Nz) conditions in alpha lattice design in the experimental area of wheat section, Department of Plant Breeding and Genetics, PAU, Ludhiana during rabi season of 2016-17 and 2017-18. Analysis of variance indicated a larger amount of genetic variability for components traits i.e., morpho-physiological traits, nitrogen uptake (NUpE), utilization efficiency (NUtE), grain protein content, biological yield and grain yield. The spikelets per spike and tiller number were found to have the highest heritability and genetic advance whereas straw N percentage in above ground parts was found to be least heritable. Half N regime in comparison to full nitrogen regime was more effective in discriminating the genotypes for various nitrogen uptake and utilization efficiency component traits. Grain yield, biological yield, grain N yield, total N yield, straw N yield, NUpE, NUtE and NHI has strong correlation with NUE whereas N percentage in shoot has no impact, thereby identifying the selection of traits to be used for screening genotypes. Principal component analysis revealed that the PCs contribution were more for grain yield, tiller number, total N yield, biological yield, straw yield, harvest index, NHI, NUtE grain protein content and GNC, So selection for these traits can be helpful for selecting N use efficient physiology. PCA also showed that the NUE, NHI and NUtE were markedly higher when there was no nitrogen apply (Nz regime). Further investigation on roots revealed the importance of root length, root diameter and root shoot ratio for NUpE, indicating the improvement for NUE. Few lines having most consistent performance under low N conditions were also identified and tested under local trials for preliminary yield evaluation. Four lines viz. BWL 6289, BWL 6838, BWL 6300 and BWL 6293 had high yield in local yield trials. These lines may be exploited for improving nitrogen use efficiency in wheat breeding programs. Inclusive composite interval mapping based on 35K SNPs array detected a total of 294 QTLs of which 105 QTL under full N, 93 under half N and 96 under zero N for nitrogen uptake, utilization efficiency and others agronomic traits were identified. The highest QTLs were found on D genome, with 90 QTLs (30.6%); 100 QTLs (34%) and 104 QTLs (35.4%) on A, B and D genomes respectively. The proportion of phenotypic variance ranged from 3.02 to 19.31 percent with LOD score 2.5 to 21.24. The number of QTLs ranged from 2 to 24 for the studied traits. These QTLs were mapped on different clusters. The identified QTLs will be further validated in other populations for their use in marker assisted selection for wheat improvement.
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Pyramiding bacterial blight resistance genes (Xa21, Xa38) in maintainer and cytoplasmic male sterile line of rice (Oryza sativa L.) through marker assisted selection
(Punjab Agricultural University, Ludhiana, 2019) Lego, Nancy; Thind, K.S.
Hybrid rice has great potential in certain districts of Punjab. However, most of the hybrids are susceptible to bacterial blight (BB). In the present study BB resistance genes (Xa21 and Xa38) have been pyramided through marker assisted selection (MAS) into PCMS 16A/B which has good outcrossing ability. An elite breeding line, PAU 5942-1-1-1-1-1-1 with good agronomic and quality characters and possessing Xa21 and Xa38 genes was used as donor parent. Marker assisted foreground selection was carried out using the gene specific markers pTA248 for Xa21 and Os04g53050-1 for Xa38 and background selection was done using 64 polymorphic SSR markers between donor and recipient parent. The recipient parent genome (RPG) recovery ranged from 80.5 to 92.9% in BC2F1 plants. A set of 254 BC2F2 progenies carrying different combinations of Xa21/Xa38 genes along with parents were screened for BB resistance using pathotype X of BB prevalent in Punjab. Based on the RPG% and BB screening, 11 BC2F2 plants were found to be desirable. Agronomically superior lines of improved PCMS 16B line are being converted into CMS line through backcrossing. The improved BB resistant CMS lines can be further used in developing hybrid varieties resistant against all the 10 pathotypes of BB in Punjab. Further, a set of 109 testcross hybrids were generated using 79 diverse rice genotypes and 21 Wild abortive (WA) based-CMS lines to identify new maintainers and restorers from the pool of locally adapted germplasm for the development of component lines in a hybrid breeding program. Molecular markers DRRM-RF3-10 and DRCG-RF4-14 were used to screen for the presence of restorer genes Rf-3 and Rf-4 in these 79 genotypes. Among these genotypes, 56% carried Rf4Rf4/rf3rf3, 10% carried Rf3Rf3/rf4rf4, 10% carried Rf3Rf3/Rf4Rf4 and 24% carried rf3rf3/rf4rf4 allelic combination. Rf-4 was found to be predominant than Rf-3. Based of spikelet fertility 17 maintainers, 17 partial maintainers, 18 partial restorers and 37 restorers were identified. The molecular and testcross results revealed differential fertility behaviour of genotypes with different WA based-CMS lines.

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