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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.
Open Access
Production of doubled haploids in rice (Oryza sativa L.) through anther culture and wide crosses and their molecular characterization
(Punjab Agricultural University, Ludhiana, 2019) Saundarya Kumari; Gill, R.S.
The doubled haploidy is one of the fundamental tools of plant breeding which provides the fastest way to accelerate plant breeding and develop homozygous lines in the shortest possible time. The F4/F5 population of japonica x indica derived cross (Dubraj x IR 75862-206-2-8-3-B.B.B.) was used for the production of doubled haploids through anther culture for two crop seasons. Approximately 74,834 and 88,100 anthers from 87 and 21 lines respectively were cultured during kharif 2016 and 2017 crop seasons. Standard N6 medium supplemented with 2, 4-D (2.0 mgL-1) + BAP (0.5 mgL-1) +Sucrose (3 %) was used. Callus induction frequencies (CIF) varying from 0.07405 to 8.235 and 0 to 8.835 respectively were obtained during kharif 2016 and 2017 and upon transfer of 2-3 mm calli on regeneration media with varying compositions the regeneration frequencies varying from 0 to 100 and 0-35 per cent respectively were obtained. Delayed callus response initiating 90 days after culturing was observed during both the culturing seasons. All the regenerated shoots were albinos and no green plantlets could be obtained, which may be due to genotypic specificity leading to delayed callusing. The parental lines also had poor response towards anther culturability with CIF of 0.00, 0.020 and 0.064, 0.04 for Dubraj and IR 75862-206-2-8-3-B.B.B. respectively during kharif 2016 and 2017. Anther culture of a different set of material belonging to 41 crosses combinations of Basmati and non basmati genotypes during kharif 2018 though in general, gave low callus induction frequencies but high frequency of plant regeneration varying from 2.56 to 100 per cent was observed. Contrary to previous seasons results the callus initiation started 30 days after culturing and about 16 putative doubled haploid plants were generated. Studies on identification of genomic regions associated with anther culture response by using twenty two polymorphic SSR markers revealed random distribution of indica and japonica type alleles in callus as well as callus responsive and callus non-responsive lines. From the present set, no marker could clearly differentiate its association with calli and callus responding lines. RM228 and RM23662 markers had heterozygous alleles in the callus indicating their origin from the callus wall. Studies on wide crosses of rice x maize and rice x pearl millet indicated strong pre-fertilization barrier. Pollen tube of maize failed to penetrate the stigma of rice even up to 48 hours of observations. The pollen tube of pearl millet penetrated the stigma in some samples of rice but only one pollen tube of pearl millet out of 400 microscopic fields was found to reach near the micropylar end at 24 hours of cross-pollination. Hence, pearl millet could be a better candidate for further study of wide crosses of rice for induction of haploids. Pseudo seed formation was observed in the wide crosses of rice due to application of 2, 4-D in low frequencies. Further, studies on media manipulation, identification of genes/QTLs for anther culturability, overcoming barrier of wide crosses in rice x pearl millet and exploration of new alternate techniques like use of CENH3, OSMATL and BBM1 are suggested.
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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.

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