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Open Access
DEVELOPMENT OF A DIVERSIFIED SET OF Triticum timopheevi BASED FERTILITY RESTORERS IN WHEAT
(Punjab Agricultural University, Ludhiana, 2018) Harmeet Singh; Srivastava, Puja
The study was based on 2100 backcross progenies derived from crosses of fertility restorers with 9 European winter wheat lines. BC1F6 progenies were evaluated at PAU, Ludhiana in 2017-18 for key traits of fertility restoration and anther extrusion, along with preliminary agronomic performance. On the scale of 1-9 for anther extrusion, about 75% progenies exhibited score of 7 and above as high anther extrusion winter wheat lines were used as parents. The range for fertility restoration varied from 12% - 80% in the backcross derivatives. Mean yield of the diversified restorer set was found to be higher than the R-parents. In the restorer evaluation trial, two top yielding lines were line no. 6 (R-1//AMORAK/R-19) and 78 (RS-534//HURLEY/R-4). A total of 26 restorer lines surpassed the highest yielding R-parent. The back cross derivatives involving Amorak as winter parent proved to be superior, followed by Exotic and Maxyl based restorers. R-1//AMORAK/R-19 came out to be best performing in terms of mean yield as well as fertility restoration, whereas R-1//AMORAK/R-1 and RW-17//EXOTIC/R-1, both proved to be best performing for pollen viability and anther size characters. RS-317//HURLEY/R-4 and RS-534//MAXYL/R-28 displayed superiority in terms of tiller number and thousand grain weight, respectively. In the preliminary evaluation of test hybrids, 11.1% F1s showed significant heterosis over commercial checks. The best hybrid from the restorer set was TH 32 (CMS BWL 3275 X R-4//HURLEY/R-1), exhibiting standard heterosis of 29.5%, 21.5%, 25.4% and 28.5%, over checks HD 2967, HD 3086, PBW 725 and PBW 723 respectively. In F2 populations derived from fully fertile F1s, significant association was observed between fertility restoring Rf8 gene with fertility restoration trait.
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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.
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Paddy straw, Growth, Lignocellulolytic, Laccase, Endoglucanase, Exoglucanase , -glucosidase, Pretreatment, Biogas, Delftia sp. PP4_S3, Coriolus versicolor MTCC 138
(Punjab Agricultural University, Ludhiana, 2018) Sandeep Kaur; Dhillon, Satwinder Kaur
Soybean harbours many anti-nutrients unsuitable for human digestive tract, among them “Kunitz Trypsin inhibitor (Kti)” is major anti-nutrient inhibiting activity of digestive proteases. Further, the crop is prone to devastating biotic stress i.e. yellow mosaic virus (YMV) prevalent in northern region of the country. Therefore, the present study was planned to identify YMV resistant soybean genotypes carrying null kti gene and cross was made between SL525 x NRC101 to develop YMV resistant and Kti free soybean genotypes. SL525 is YMV resistant but has Kti gene on the other hand NRC101 susceptible to YMV and lacks Kti gene referred to as ‘null kti’. A total of 45 F4 lines of the cross were screened for YMV resistance using infector row technique in kharif 2017. A total of 125 genotypes were selected and grown in kharif 2017 as F5 generation. Molecular markers (gene specific and SSR markers linked with kti locus) were used to screen plants for presence/absence of Kti allele. A total of 65 genotypes were Kti positive, 50 null kti and 10 genotypes were heterozygote. Phenotyping was done by measuring trypsin inhibitor activity (TIA). Consequently, the genotypes lacking Kti allele recorded reduced levels of TIA. Furthermore, YMV resistance of F5 genotypes was confirmed by molecular markers and phenotypic screening. Analysis of variance of F5 genotypes revealed that genetic variation for the morphological and quality traits was present in genotypes. Trypsin inhibitor activity was observed to be positively correlated with germination, days to maturity, plant height, grain yield and seed weight. Non significant direct effect of TIA was observed on grain yield whereas traits such as germination at 6 months after harvest, number of pods per plant, 100-seed weight, plant height, days to maturity showed significant positive direct effect on grain yield. The comparison of performance of null kti and kti genotypes revealed that null kti genotypes had low germination, delayed maturity, less grains and lower TIA values in comparison to kti genotypes whereas traits such as days to flowering, plant height, pods/plants, seed weight, oil content, protein content, fatty acid composition were unaffected by allele present. A total of 14 null kti genotypes had significantly higher grain yield than SL525 and these genotypes were resistant to yellow mosaic disease.
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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Introgression of cotton leaf curl disease resistance from Gossypium armourianum to G. hirsutum
(Punjab Agricultural University, Ludhiana, 2018) Bhupinder Singh; Rathore, Pankaj
The present investigation was undertaken to evaluate the performance of interspecific F1 hybrid and backcross derivatives of G. hirsutum and G. armourianum cross for response to cotton leaf curl disease as well as to identify the alien segments in the backcross derivatives. Experimental plant material consisted of G. armourianum Acc. PAU 1, G. hirsutum cv. F 1861 and LH 1556, their F1, BC1F1, BC2F1 derivatives. The interspecific F1hybrid between G. hirsutum and G. armourianum was found to be completely resistant to CLCuD. Two of the four BC1F1 plants were observed to be CLCuD resistant. Out of 84 BC2F1 plants, 43 were observed to be resistant, whereas 41 plants were susceptible to CLCuD. Total genomic DNA of the experimental plant materials was amplified employing virus specific primer pairs. Presence of virus DNA was detected in the American cotton parents as well as CLCuD susceptible backcross derivatives. Of the 100 SSR markers used to detect polymorphism in the parental lines, 52 were polymorphic and were used to identify alien segments in CLCuD resistant BC1F1 Plant 2. Eight markers were amplified in this plant demonstrating introgression from the G. armourianum parent. These primers were used to genotype 84 BC2F1plants. The range of G. armourianum specific alleles in the BC2F1plants was observed to be 38.09 through 75.0%. The introgression of G. armourianum specific chromosomal segments in the BC2F1 plants as revealed by SSR markers was independent of the CLCuD response suggesting that none of the eight primers may be linked to CLCuD resistance. Markers TMB 1271 and NAU 6316 exhibited segregation distortion and were transmitted in higher frequency than expected in the BC2F1 progeny.
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Evaluation of an intra-arboreum recombinant inbred line population
(Punjab Agricultural University, Ludhiana, 2018) Lakhvir Kaur; Pathak, Dharminder
The present investigation was undertaken to evaluate the performance of intra-arboreum recombinant inbred lines (RILs) for various quantitative traits and to unravel molecular diversity among a subset of RILs. These two sets including 62 (Set I) and 34 (Set II) RILs along with parent LD 902 and standard check LD 949 were evaluated in a simple lattice design at three locations (Ludhiana, Bathinda, and Faridkot) during kharif 2016. Mean squares for genotypes, locations, and genotype x environment interaction were significant for all the characters except for genotype mean squares for elongation percentage and location mean squares for fibre strength in Set I and location mean squares for micronaire and fibre strength in Set II. The results suggested existence of substantial genetic variability in the experimental plant material, considerable differences among the locations, and profound effect on the expression of seed cotton yield, its components and fibre quality traits. Three RILs namely 431, 241, and 217 in Set I and RIL 105 in Set II significantly out yielded parent LD 902. RIL 105 of Set II appeared to be promising as it recorded numerically higher seed cotton yield than that of LD 949. It also recorded higher values for some of the yield component traits such as number of bolls plant-1, sympods plant-1, and boll weight. Twenty seven RILs of Set I registered significantly higher boll weight than that of LD 949. Ten RILs of Set I and five RILs of Set II possessed at least 18 g/tex fibre strength. Likewise, many transgressive segregants were identified for various traits. Based on SSR diversity analysis of 64 genotypes, RILs were broadly divided into three main clusters. No specific pattern for grouping of genotypes was observed. Substantial variation as revealed by dissimilarity coefficient (0.03 – 0.46) has been observed among different pairs of desi cotton recombinant inbred lines. Further, 29 pairs of RILs recorded at least 0.4 value of the genetic dissimilarity coefficient. The present study has provided a useful insight into performance of desi cotton RILs for various economic traits over locations as well as shed light on the molecular diversity/similarity among the test genotypes.
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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.
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Genetic variation for key enzymes involved in nitrogen uptake in American cotton
(Punjab Agricultural University, Ludhiana, 2018) Manjeet Kaur; Paramjit Singh
The present investigation was undertaken to evaluate genetic variation for nitrogen uptake enzymes and to analyze the impact of varied nitrogen levels (0%, 50% and 100% N of RDF) on seed cotton yield, lint yield, and fibre properties of forty nine Gossypium hirsutum genotypes. The enzymes involved in N uptake (nitrate and nitrite reductase) were assayed for Bathinda, at pre-anthesis and post-anthesis stages for each genotype at each nitrogen levels along with chlorophyll and nitrogen content in leaves. The field evaluation of the plant material was undertaken in partially balanced lattice design (7 x 7) with two replications at Ludhiana and Bathinda. Mean squares for genotypes, locations, nitrogen level and genotype x environment, genotype x nitrogen level, genotype x location x interaction were significant for all the characters except for genotype mean squares for fibre fineness. The results suggested existence of substantial genetic variability in the experimental plant material, considerable differences among the locations, and profound effect on the expression of seed cotton yield, its components and fibre quality traits. Genotypes namely LH 900 and F 846 appeared to be promising as it recorded numerically higher enzyme activity at low nitrogen level (0% and 50% N) as compared to recommended dose of nitrogen. Higher enzyme activity of these genotypes at low N dose probably indicates their higher uptake as well as utilization efficiency over other genotypes It also recorded higher values for the yield as well as fibre component traits such as seed cotton yield plant-1, lint yield plant-1, boll weight, ginning out turn, 2.5% span length, and fibre strength. NR enzyme had positive and significant effect on NIR, N content, chlorophyll content, seed cotton yield and lint yield per plant. NIR enzyme showed positive significant effect on chlorophyll content. N content had positive effect on chlorophyll content and seed cotton yield. F846 and LH900 overall performed well as these genotypes were among the best for enzyme activity as well as for yield traits. These genotypes hold future potential for developing new cultivars with improved NUE.
Open Access
Marker assisted consolidation of low polyphenol oxidase and rust resistance genes in high grain protein bread wheat lines
(Punjab Agricultural University, Ludhiana, 2017) Gill, Manpartik S.; Bhagat, Indu
Low polyphenol content in wheat grain is a desirable quality trait, conferring bright, amber appearance to products including chapattis. The present study is based on two donor lines (09014/23 and Clear White 515- GpC) each possessing two major recessive QTL’s for low PPO, along with rust resistance genes (i.e. Yr5, Yr15, Yr36, Lr34 and Lr37) and high grain protein gene GpC-B1. The recipient lines BWL 5189 and BWL 5190 represent versions of superior chapatti quality variety PBW 550 carrying introgression of Yr15, Yr36, Lr34 and GpC-B1. The four all possible crosses were performed at main location (2015-16) and F1’s were grown at the PAU offseason research station during summer of 2016. F2 from four crosses were raised at main season location (PAU, Ludhiana) using a strategy for early planting with the objective of obtaining two generations within the season. Seedlings germinated in propagation trays at 14-18 ̊ C were transplanted in mid October and flowered near mid December. One population of 208 plants derived from a single F1 plant from cross, 09014/23 x BWL 5189 was chosen for marker analysis. Marker PPO33, PPO29 and Wmc175 for low PPO genes i.e. PpO- A1 (on chromosome 2A), PpO- D1 (on chromosome 2D) and Yr5 (on chromosome 2B) were used in the study. The marker profiling resulted in identification of 38 plants carrying desirable alleles for three genes, either in homozygous or heterozygous condition. Out of these 38, 2 were found to be homozygous for all the three target alleles. This population along with other F2 populations were carried forward using a rapid generation advanced (RGA) strategy involving, detached tiller culture, followed by harvesting and drying of immature caryopsis with or without cold treatment. Rapid generation advance system was refined further and caryopsis harvested at 16 Days after pollination (DAP) gave desirable combination of high germination with saving of time. It was also observed that cold treatment for 2 days prior to germination was not needed. The 2 homozygous progenies had PPO score of 1.8 and 2.0 as compared to 4.2 (out of 10) in the recipient parent BWL 5189 and 1.2 for 09014/23. The protein content of these were significantly higher (13.31% and 13.14%) than the grand parental line PBW 550 (12.3%) while their grain hardness was 10.69 Kg and 12.12 Kg as compared to 12.26 Kg of PBW 550 and 8.05 of 09014/23. The PPO allele profiling was extended to a set of 87 wheat varieties to see genetic changes that had taken place at this locus as we move from landraces to modern day cultivars.

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