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Allele mining for phospholipase D locus to improve the rice bran quality
(Punjab Agricultural University, Ludhiana, 2016) Amandeep Kaur; Kuldeep Singh
Rice (Oryza sativa L.) is the most important staple food for a large part of the world’s population. Rice bran which is a by-product after milling is rich in oil (12–23%), proteins (14–16%), vitamins and crude fiber (8–10%). Rice bran oil is typically an oleic–linoleic type fatty acid and qualifies for good quality edible oil. The primary limitation of rice bran as a source of oil is the presence of highly active lipolytic enzyme Phospholipase D alpha1 (PLDα1) that increases free fatty acids content in the oil and causes the ‘stale flavor’ in the product. The wild relatives of rice constitute a major gene pool for rice improvement. In this study, sequence based allele mining of the gene PLDα1 (6.28 kb) was taken up in wild species of rice to identify potentially novel alleles for bran quality improvement. Differences in expression patterns were also investigated for potential correlation with sequence variation. A representative subset of 86 accessions belonging to 16 wild Oryza species and eight O. sativa cultivars were selected for this purpose. Alignment of the assembled sequence contigs relative to the Nipponbare reference (Os01g0172400) gene revealed the presence of 2555 variations (SNPs and InDels). Highest polymorhism at PLDα1 locus have been detected in the O.officinalis species. Expression profiles in immature grains generated by exon-specific qRT-PCR indicate significant differences in transcript abundance within as well as between the selected accessions and different 3’and 5’ truncated PLDα1 mRNA. Cis-elements identified in the upstream sequences showed variations relative to the reference in low expressing wild species accessions. These results suggest that potential PLDα1 allelic variants are defined by differential regulation at the levels of transcription initiation and/or mRNA splicing. The wild species accessions with variations in protein structures and low expression, identified in the present study, can be further utilized in the breeding programs to transfer desirable allele into elite lines through marker assisted selection.
Open Access
Molecular mapping of drought and heat tolerance traits using interspecific cross in chickpea (Cicer arietinum L.)
(Punjab Agricultural University, Ludhiana, 2018) Kushwah, Ashutosh; Sarvjeet Singh
To understand the complex mechanisms of drought and heat stress tolerance, molecular mapping of RIL population through ddRAD-Seq has been undertaken. In this study, all the traits measured were significantly affected by drought stress environment except harvest index as well as by heat stress environment except hundred seed weight and harvest index. The pooled ANOVA for all the traits measured showed highly significant differences in irrigated, rainfed and latesown conditions for genotypic variance as well as for standard error variance. Correlation coefficient exhibited strong association of traits with yield under drought and heat stress conditions. Multiple linear regression analysis revealed the contribution of traits towards the total variation in yield under drought and heat stress conditions. Path analysis explained that biomass followed by harvest index were the major contributor to grain yield under non-stress as well as drought stress and heat stress environment at both locations (Ludhiana and Faridkot) with pooled condition. Principal component analysis also revealed the strong positive association among the traits. In genotypic analysis, a total of 1365 filtered polymorphic and functional SNPs loci were used for linkage map construction through ddRAD-Seq. The average linkage map distance was 4569.09 cM with an average interval of 6.79 cM between the markers. Five QTLs of days to germination (qdg-1), days to flowering initiation (qdfi-1), days to 50% flowering (qdff-1), days to 100% flowering (qdhf-1) and yield (qyld-1) identified using BLUPs under heat stress condition. A total of twenty eight QTLs for days to flowering initiation (qdfi-1 to qdfi-4), days to 50% flowering (qdff-1 to qdff-4), biomass (qbio-1 to qbio-4), yield (qyld-1 to qyld-3), days to 100% flowering (qdhf-1 to qdhf-2), harvest index (qhi-1 to qhi-2), relative leaf water content (qrlwc-1 to qrlwc-2), root-shoot ratio (qrsr-1 to qrsr-2), root dry weight (qrdw-1 to qrdw-2), hundred seed weight (qhsw-1), membrane permeability index (qmpi-1) and fresh root weight (qfrw-1) identified using BLUPs under drought stress condition. These SNPs loci need to be validated in different genetic background and use them in marker-assisted selection for developing superior varieties with enhanced drought and heat tolerance.
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Genetic and molecular assessment of breeding methods and mapping novel QTL in soybean (Glycine max L. Merrill)
(Punjab Agricultural University, Ludhiana, 2018) Khosla, Gaurav; Gill, B. S.
Two hundred and seventy five lines from seven different crosses of soybean derived from four different breeding methods viz. pedigree method (PM), single pod descent with selection (SPDS), single pod descent (SPD) and bulk method (BM) were used to assess the efficiency of breeding methods. Based on different yield contributing traits, bulk method was the best method in terms of mean of all lines over two years followed by pedigree method. When the yield of the highest yielding line from each method within each cross was compared with the best check mean, it was observed that the bulk method had the highest yielding line in three out of seven crosses whereas in four crosses it was ranked second. This performance was closely followed by pedigree method. Molecular assessment of the breeding methods was done based on the dissimilarity coefficient among lines within a breeding method using SSR primers. Based on the dissimilarity coefficients, number of unique lines in each breeding method were calculated. SPD method had the highest per cent of unique lines among all the methods in all the crosses. PM and BM had the lowest number of unique lines. Comparison of lines derived from bi-parental and multi-parental crosses revealed that biparental crosses having distantly related parents generated high yielding lines as compared to the multiparental crosses as well as the biparental crosses with less diverse parents. For mapping for YMD resistance genes and seed weight QTL, F2 plants from the cross SL 958 x AGS 456 were used. Segregation ratios in F2, BC1F1 and BC1F2 populations suggested that YMD resistance is controlled by two genes in inhibitory gene action. A total of 269 SSR markers were used for polymorphic studies and the identified polymorphic primers were used for genotyping studies. Bulk segregant analysis was performed with 114 polymorphic markers. Out of 114 markers, 13 markers showed polymorphism in resistant and susceptible bulks. Nine markers were present on C2 linkage group whereas three were present on D1b linkage group. Three major QTL were identified on LG C2 explaining phenotypic variation of 17, 12 and 27 per cent for YMD resistance. One minor QTL was also identified on LG D1b. For seed weight, one major QTL was identified on LG M with Sat_244 and Satt175 as flanking markers having an estimated 19.0 per cent phenotypic variance. Two minor QTLs were also identified on LG D1b.
Open Access
Assessment of genetic diversity of shoot fly-resistant sources and transfer QTLs associated with shoot fly resistance into forage sorghums (Sorghum bicolor L.)
(Punjab Agricultural University, Ludhiana, 2018) Arora, Naveen; Sohu, R.S.
The present study consisted of two different sets of genotypes evaluated for variable reaction to shoot fly under field conditions. The first set consisted of a diverse collection of 53 different germplasm lines and the second set consisted of 30 genotypes including 10 cytoplasmic male sterile lines, along with their respective maintainers and 10 restorer lines. On the basis of morphological evaluation under field conditions, the genotypes varied significantly for all the traits under study. ICSA/B 487, ICSA/B 14037, ICSA/B 467, ICSV 93046 and ICSV 700 exhibited high glossiness, pigmented leaf sheath, lower number of eggs and suffered lesser incidence of deadhearts and exhibited resistant reaction to shoot fly while ICSA/B 458, ICSV 25337 and ICSV 25292 exhibited moderate resistance to the shoot fly damage and ICSV- 25280, 25292, 25303, 25316, BTx 623, SSV 74 and Swarna were susceptible to shoot fly. In the germplasm lines significant variation was found for most of the traits except the leaf sheath pigmentation, number of plants with shoot fly eggs and number of shoot fly eggs per plant. Significant positive and negative correlations were found among the important traits for shoot fly resistance like glossy score, leaf sheath pigmentation, shoot fly deadhearts, total number of eggs, number of plants with eggs and trichome density. The inheritance studies based on Line X Tester analysis, revealed that shoot fly resistance is a complex trait and crosses involving resistant parents yielded resistant lines. The lines ICSA 458, ICSA 467, ICSA 487, ICSA 14024 and ICSA 14037 and testers ICSV 700, ICSV 93046, S-35, ICSV 25292 and ICSV 25337 had significant SCA and GCA effects for the traits important for shoot fly resistance. The estimation of biochemical components, revealed that the genotypes exhibiting moderate (ICSA 84, ICSB 52, ICSB 14024) and high (ICSA 467, and ICSB 487) resistance to shoot fly recorded lesser content of total soluble sugars, proteins, higher content of tannins and there was no significant association found for phenol content. From the HPLC analysis of methanol extracts of leaf samples a total of 25 different peaks with 12 known compounds were recorded. Diversity analysis based on GBS based SNP markers grouped the germplasm and parental lines into three major clusters consisting of resistant, moderately resistant and susceptible genotypes. Population structure was analysed using principle component analysis. The first three PC were sufficient enough to explain maximum diversity within population structure. For Genome Wide Association Studies, first three PCs were used as a covariate for which, significant association of SNPs were found for glossy score on chromosome 3, 9, 10 and for percent plants with shoot fly eggs on chromosome 2. For leaf sheath pigmentation, SNPs on all the chromosomes were significantly associated. Similarly, multiple SNPs were associated for percent tiller with shoot fly deadhearts on chromosomes 2,3,4,6,7 and 9. SNPs for plant vigour score were significantly associated with chromosomes 1,2,3 and 6.
Open Access
Marker assisted development of wheat lines with enhanced nutritional quality
(Punjab Agricultural University, Ludhiana, 2018) Kuldeep Kaur; Sohu, V. S.
The aim of present study was the genetic enhancement of grain micronutrient and yellow pigment content in high yielding wheat lines. The study was based on BC1F2 and BC2F2 introgression lines developed from crossing advanced backcross lines of T. durum with grain zinc QTL introgressed from T. monococcum and T. boeoticum, with high yielding varieties of North Western Plain Zone i.e. PBW621, HD2967 and WH1105 and two gene pyramided version of PBW343 (carrying rust resistance genes Yr10, Yr15, Lr24, and Lr28) as recurrent parent. A total of 210 BC1F3 and 210 BC2F3 progenies from different cross combinations were evaluated for grain zinc concentration, yellow pigment content and agronomic traits. Eighteen highly promising BC1F3 progenies (>80ppm zinc concentration) and 15 BC2F3 progenies (>75ppm zinc concentration) were identified and advanced at Keylong as ear to progeny rows. A total of 42 BC1F5 and 43 BC2F5 derivatives were evaluated in a square lattice design and analysis of variance revealed significant variation among the progenies for grain zinc, yellow pigment as well as agronomic and quality traits studied. The BC1F5 progenies from recipient parent WH1105 and tetraploid donor BF13 were recorded with highest grain zinc (64.1-67.9ppm) and iron concentration (29.8-36.0ppm). The highest grain yellow pigment content (5.35 ppm) was observed for the cross involving PBW703 as recipient and BF 21 as tetraploid donor parent. The markers for the previously mapped grain zinc QTL which was identified at diploid level in the donors, were found to be non polymorphic. Monitoring of backcross derivatives with the marker YP7A-2 linked to yellow pigment gene Psy-A1, revealed nine lines positive for high yellow pigment allele (Psy-A1c) indicating introgression from T. durum. To test the effect of environment on grain zinc concentration, eight high zinc BC1F5 lines and ten BC2F5 lines were evaluated in a randomized block design at two locations i.e. Ludhiana and Gurdaspur. The pooled analysis of variation revealed location effect to be significant for grain iron and zinc concentration. Furthermore, BF22/PBW698//PBW698 derivatives were crossed with BWL3560 (carrying Gpc-B1) with the aim of conglomerating enhanced protein content, zinc and yellow pigment content within the same wheat background. A total of 1068 F2 single plants were phenotypically analysed for grain iron and zinc concentration. High zinc lines were shortlisted and 192 F3 lines were sown in square lattice design. Molecular marker analysis of F3 progenies for Gpc-B1 gene (Xucw108) revealed 129 F3 lines to be homozygous positive. Correlation studies revealed that the improvement for four nutritional quality traits (grain protein, zinc, iron and yellow pigment content) was constrained and yield/yield components might have to be compromised.
Open Access
Pyramiding of QTLs for grain yield under water stress and genes for brown planthopper resistance through marker assisted backcross breeding in indica rice (Oryza sativa L.)
(Punjab Agricultural University, Ludhiana, 2018) Raghavendra N R; Mangat, G.S.
The Punjab Agricultural University, Ludhiana during Kharif 2013 released PR121, a high yielding, bacterial blight (Xa4+xa13+Xa21) resistant rice variety of indica rice. Due to short duration, high yield, less biomass and excellent milling characteristics, PR 121 became the most popular variety among the farmers and millers in the state and occupied more than 7.0 lakh hectares area during Kharif 2017. However it is susceptible to brown plant hopper (BPH). In view of increasing incidence of BPH and depleting water resources, the present study aimed at pyramiding QTLs (qDTY1.1 and qDTY3.1) for grain yield under water stress and genes (Bph20 and Bph21) for BPH resistance into this popular variety through marker assisted backcross breeding (MAS). Introgression line IR96321-1447-561-B-1 (from Apo and Nagina 22) and Oryza minuta alien introgression line IR71033-121-15-B, were used as donors for drought and BPH tolerance respectively. Marker assisted foreground selection was carried out using the SSR markers RM3825, RM5794 and RM520, RM16030 linked to the drought QTLs and RM8231, RM16556 and RM28561, RM28571 linked to BPH resistance. A set of 59 BC1F3 progenies carrying different combinations of drought QTLs/BPH genes along with parents were evaluated under reproductive stage drought stress in rain-out shelter and non-stress conditions for various agro morphological traits. In general, days to 50% flowering were delayed and plant height was reduced under stress conditions. Four lines homozygous for both the drought QTLs yielded at par with recurrent parent under stress conditions. One of the BC1F3 line viz., PAU 7182-1645 possessing both drought QTLs had higher yield potential than PR 121 under non-stress and stress conditions. However, it was comparable with the donor parent under stress conditions. Lines with either of the two QTLs did not show any yield advantage under stress over PR121. The BPH resistance of lines with Bph20 and Bph21 was not comparable to the donor parent. The disagreement between marker genotypes and the phenotypic evaluation could probably be due to incomplete linkage between the selected markers and the target genes or due to the effect of genetic background. The lines carrying different combinations of QTLs/genes can be useful as improved genetic stocks of rice, especially to develop cultivars for the future.
Open Access
Identification of genomic regions associated with seed size in lentil (Lens culinaris Medik.)
(Punjab Agricultural University, Ludhiana, 2016) Sood, Ankita; Sandhu, Surinder K.
Lentil (Lens culinaris Medik subsp. culinaris) is an autogamous diploid (2n=2x=14), cool season food legume crop cultivated globally. Genomic resources in lentil are limited in comparison with other food legumes, primarily due to large genome size and lack of genetic variation. The present study was aimed to evaluate a set of recombinant inbred lines (RILs) derived from L9-12 × FLIP 2004-7L under irrigated and rainfed conditions and to identify the genomic regions associated with seed size related traits in lentil. The parent L9-12 was local lentil drought tolerant cultivar with small seeds whereas; FLIP 2004-7L was an advanced breeding drought susceptible line from ICARDA with bold seeds. Analysis of variance revealed highly significant differences among the RILs for the morphological, yield and its related traits, root and shoot traits. Based on the overall mean performance, RILs viz., RIL-14, 17, 28, 33 and 71 were found to be significantly superior lines for all the studied traits. Proline content and membrane permeability index were up regulated among RILs due to stress under rainfed conditions over irrigated conditions. Out of 770 markers screened, 62 markers were found to be polymorphic between the parents. The Lens SSR markers generated more polymorphism followed by ITAP (Intron-Targeted Amplified Polymorphic Sequence) markers and markers from LcSSR (Lens culinaris SSRs) series. Of the 62 polymorphic (31 Lens SSR, 27 LcSSR and 04 ITAP) markers, 61 were mapped in three linkage groups (LGs), varying in length between 13.60 to 254.80 cM and covering 346.70 cM with an average marker spacing of 7.49 cM. Number of markers per linkage group varied from 3 to 52. The study reported assigning of 12 novel markers on the linkage map. A total of 17 quantitative trait loci (QTLs) were identified for most of the yield and its related traits under study. These identified QTLs for various traits were present in five genomic regions (GR) viz., GR-A, GRB, GR-C, GR-D and GR-E. Among these, a compact genomic region GR-C spanned by markers SSR19 and GLLC106 harbored four major QTLs for number of pods per plant, 100seed weight, seed diameter and seed yield within a map distance of 184.5-190.6 cM (6.1 cM) on LGI. This region may serve as the potential target for utilization in yield enhancement of lentil. Further research work towards the fine mapping of this key genomic region will open a window to develop molecular markers suitable for marker assisted selection and to identify the novel genes governing these traits.
Open Access
Genetic analysis and molecular marker tagging of stripe rust resistance genes in wheat lines PBW 621, PBW 636 and HD 2967
(2016) Rupinder Pal Singh; Bains, N.S.
Parents and derivatives crosses PBW 621 X HD 2967, PBW 621 X PBW 343, HD 2967 X PBW 343 and PBW 636 X PBW 343 involving stripe rust resistant lines and highly susceptible cultivar PBW 343 were studied. Characterization of parental lines namely against 78S84 pathotype highlighted gradual and continuous shift in resistance over four seasons (2011-12 to 2014-15) depicting the increasing aggressiveness pathotype. F2 and F3 analysis of cross PBW 621 X HD 2967 revealed the additive nature of the genes involved as also the fact that each parent is contributing one gene towards resistance. Crosses PBW 621 X PBW 343 and HD 2967 X PBW 343 supported that each parent contributes one gene towards resistance. Contrary to the expectations, highly susceptible parent PBW 343 also contributed a resistance gene in the R X S crosses. Over these years, PBW 636 showed diminishing resistance levels against 78S84 pathotype, ultimately succumbing completely. Cross PBW 621 X HD 2967 was chosen for molecular marker tagging as a clear genetic basis of resistance was evident from the segregation pattern. Out of total 327 SSR primers used, 95 polymorphic primers were put to use in bulked segregant analysis of 20 individuals in each pool. Primers wmc 235 and wmc 75 showed single parent alleles in the bulks. HD 2967 allele of primer wmc235 was found only in resistant bulk while PBW 621 allele of primer wmc75 was also found only in susceptible bulk. A fresh set of 26 extreme individuals selected from F3 based on their stripe rust score were subjected to marker analysis with the two identified primers. The number of homozygous progenies for the two extreme categories was equal as per the genetic expectations. Thus there is a good indication that the two genes are not linked. Additional markers need to be used to map these genes. Evaluation of high yielding resistant genotypes (F6) derived from cross PBW 621 X HD 2967 was conducted in 2013-14and 2014-15. Promising lines were identified for use in the breeding programme. Two of these lines viz; BWL 4438 (PBW 709) and BWL 4443 (PBW 724) were promoted to national co-ordinate trials. An experiment was conducted to refine pre-regeneration chromosome doubling in wheat DH production using alternate antimicrotubular compounds APM and Trifluralin was scrutinised. APM 250 μm enhanced embryo formation frequency to 24.15 per cent and Trifluralin 250 μm was significant in increasing plant regeneration frequency to 57.69 per cent as compared to 18.20 per cent and 41.42 per cent in control (colchicine 0.2%) treatment. Combination of APM and Trifluralin at (125 μm each) enhanced both embryo formation frequency (21.50 per cent) and plant regeneration frequency (52.24 per cent).
Open Access
Genotyping alien introgression lines of Brassica juncea (L.) Coss. and mapping of gene(s) for resistance to Sclerotinia sclerotiorum (Lib.) de Bary
(Punjab Agricultural University, Ludhiana, 2016) Rana, Kusum; Banga, S.S.
Two sets of Brassica juncea introgression lines (ILs) carrying introgressed chromosome fragments from two wild Brassicaceae species, Brassica fruticulosa and Erucastrum cardaminoides were investigated for their resistance responses to stem from inoculation with Sclerotinia sclerotiorum, the world wide pathogen of crop brassicas. Molecular characterization of ILs was also undertaken using SSR and candidate gene based markers. The percentage of donor genome in the B. juncea-B. fruticulosa ILs varied from 2.1 (IL-283) to 30.2% (IL-248). A total of 22 significant marker trait associations were detected across two crop seasons. Of these, 14 marker loci were A-genome specific; eight were B-genome specific. Five were common across the seasons. Significantly associated markers were SB4817, SJ4933, SJ3640I, SJ0644, SB1871, nia-m043, nia-m138, cnu-m482, cnu-m298, cnu-m245 and cnu-m254. These introgression lines were also genotyped using 60 gene-specific markers developed from the sequence information of the candidate genes associated with resistance to Sclerotinia stem rot. Five significant marker trait associations (MTAs) were identified. Out of these, MTA involving ABI2-1 was consistent across the seasons and the pooled data. The phenotypic variance explained ranged from 21-30 percent. MTAs involving ABI1-3_1, also steady over seasons, accounted for 14 to 26 percent of the phenotypic variation. A second set of 96 B. juncea- E. cardaminoides introgression lines (ILs) were also genotyped with SSR markers and phenotyped for their resistance to Sclerotinia stem rot as well. Association mapping allowed the identification of six marker loci (based on A genome markers) influencing the resistance to Sclerotinia stem rot resistance. Of these, three were detected in both seasons as well as pooled data. In case of B genome based markers, six associations were observed in season I, 5 in S II and 5 in pooled over both seasons. These studies not only provided important new insights into the introgressed resistance but also opens the way for novel engineering of new B. juncea varieties that express this key resistance to this worldwide devastating pathogen of mustard crops

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