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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
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.
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Heterosis and combining ability in relation to genetic diversity in sunflower (Helianthus annuus L.)
(Punjab Agricultural University, Ludhiana, 2016) Kirandeep Kaur; Dhillon, S.K.
The present investigation was undertaken in the Department of Plant Breeding & Genetics, Punjab Agricultural University, Ludhiana during the year 2015 to study heterosis and combining ability in relation to genetic diversity in sunflower (Helianthus annuus L.). There was significant variation between the parental lines based on diversity analysis. The genotypes were grouped into five clusters on the basis of D2 analysis. Euclidean distance values indicated maximum diversity between cluster III and cluster IV (inter-cluster distance = 103982). The dissimilarity coefficient on the basis of SSR marker analysis ranged from 0.20 to 0.78, signifying divergence among the genotypes. The genotypes having utmost genetic dissimilarity of 78% were P198R and P206R, while P180R and P174R had minimum genetic dissimilarity of 20%. On the basis of combining ability analysis the female parents viz., 67A, 47A and 11A were observed to be good general combiners, however none of the female parent was good combiner for all the traits. Similarly, the male parents viz., P173R, P175R and P184R were good general combiners for most of the traits. There was predominant role of male × female interaction components in the inheritance of most of the traits. The hybrids CMS 11A × P174R, CMS 47A × P190R and CMS 11A × P175R were identified as best specific combiners for seed yield and CMS 11A × P178R, CMS 11A × P196R and CMS 67A × P192R for oil content. Maximum standard heterosis over check PSH1962 was recorded as 26.8 per cent (CMS 67A × P184R) for seed yield and 8.45 per cent (CMS 47A × P197R) for oil content. Simple correlations were worked out to find out the relationship of heterosis with morphological diversity and genetic diversity. The correlation between heterosis and D2 distances revealed that there was negative association of heterosis with morphological diversity for days to 50% flowering and oil content, whereas for days to maturity and test weight there was significant positive correlation. However, the genetic diversity (based on SSR polymorphism) was positively associated with oil content and linoleic acid but negatively associated with days to maturity, test weight, volume weight, stearic acid and oleic acid.
Open Access
Response of poplar clones to different soil types and growth regulator at nursery stage
(Punjab Agricultural University, Ludhiana, 2014) Mitu Bala; Avtar Singh
In present investigation the response of two types of soils (Sandy loam and Sandy clay loam soil) and foliar application of growth regulator i.e. Triacontanol at three levels (0ppm, 3ppm, 5ppm) was investigated on nine poplar clones (PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7, L47/88, L-48/89) at nursery stage, conducted at Research farm area of department of Forestry and Natural Resources, Punjab Agricultural University, Ludhiana. Various attributes like growth characteristics, physiological parameters and nutrient content in leaves were recorded at different interval of growth. The results of the study revealed that Triacontanol showed non significant and soil types showed significant results for morphological characters Viz. Plant height, collar diameter, no. of branches/plant, no. of leaves/plant, leaf area. Sandy clay loam soil showed good response than sandy loam soil for all morphological characters. For physiological characters like chlorophyll content, soil types showed non significant results and growth regulator showed significant results. Triacontanol at 5ppm concentration showed higher chlorophyll content during July and October 2013 by 30.88 and 36.21 SPAD units, respectively. For nutrient content analysis like nitrogen, phosphorus and potassium in leaves of poplar clones, growth regulator and different soil types showed significant results. After critical analysis it was concluded that PL-7, PL-4, PL-2 and PL-1 were better clones among all other clones studied for morphological characters. PL-1, PL-4 and PL-7 showed higher growth characters in sandy loam soil and PL-1, PL-5 and PL-7 clones in sandy clay loam soil.
Open Access
Phenotypic evaluation and molecular profiling of Brassica juncea (L.) lines carrying introgressions from Erucastrum abyssinicum
(Punjab Agricultural University, Ludhiana, 2014) Shruti; Atri, Chhaya
Introgression lines (ILs), created in Brassica juncea, following hybridization with Erucastrum abyssinicum were studied to catalogue their genetic diversity and crop performance under stressed ecologies. Molecular data generated following amplification with 72 SSR markers (35 (A genome) and 37 (B genome)) suggested the occurrence of high genetic diversity. ILs like JA-85, JA-128, JA-77, JA-30, JA-72, JA-96, JA-196, JA-95, JA-75 fell in Cluster 1 along with E. abyssinicum and were amongst those lines that carried a very large chunk of genomic information from the wild donor species. Graphical genotyping revealed that percent introgression varied from 12.5% to 40% with maximum introgression in JA-30, followed by other ILs like JA-85 (39.9%), JA- 53 (38.3%), JA-128 (37.4%), JA-75 (37.4%), JA-77 (36%), JA-96 (35.9%), JA-95 (35.1%), JA-72 (34.9%), JA-196 (34.1%), JA-245 (33.7%). Least introgression from the donor parent was recorded in JA-24 (12.5%). On the whole, maximum introgression was seen in chromosome 3 of ‘B’ genome which had 43.2 percent introgression from the donor parent. Morphological evaluation helped to identify useful ILs for earliness and components associated with crop productivity. Some of the ILs like JA-344 (number of primary branches), JA-92 (number of siliqua on main shoot), JA- 42 (siliqua length), JA-159 and JA-235 (seeds per pod), JA-192 (seed yield) performed equally well in both the environments. Grain yield was positively associated with flowering, plant height, leaf area index etc. which indicated that higher yield can be achieved by bringing changes in these parameters. GG Biplot analysis also confirmed a high level of variation for all the traits studied. Morphological data for test characters was also used to generate Euclidean distance graphs based on Mahalanobis D2 analysis and the ILs were clustered in 10 groups. The growing unpredictability of climate requires breeding of cultivars adapted to broad environments. Three ILs namely JA-59, JA-65, JA-240 were able to tolerate high temperature while 16 ILs viz. JA-22, JA-31, JA-35, JA-38, JA-45, JA-65, JA-127, JA-147, JA-149, JA-174, JA-193, JA-233, JA-331, JA-343, JA-352 and JA-36 could tolerate low temperature stress during flowering. These ILs can further be used in future breeding programmes suited for the development of varieties for stressed ecologies.
Open Access
Genotype x environment interaction and biplot analysis of multi-environment trials data from low erucic acid indian mustard (brassica juncea l.) Genotypes.
(Punjab Agricultural University, Ludhiana, 2016) Singla, Raman Kant; Kandhola, S S
The present study was conducted on sixteen mustard genotypes evaluated across three locations of Punjab with the objective to assess stability of performance using Eberhart and Russell model and GGE biplot analysis in visualization of interrelationships of genotypes, environments and their interactions for seed yield per plant (g), oil content (%), seed size (1000 seed weight), number of siliquae on main shoot, seeds per siliqua and other yield contributing characters. In terms of utility of GGE biplot, the association between environments was stratified under positive and negative correlations. Two environments E1( Bathinda): E2 (Faridkot) were positively correlated for most of the traits, hence same information about the genotypes could be obtained from fewer test environments, depicting the potential to reduce testing cost by dropping one of them. However, for two traits viz. oil content (%) and seed size, all the three environments were positively correlated. The ideal test environments (most discriminative and also most representative) were E1 (Bathinda) and E2 (Faridkot) for seed yield per plant, oil content, seed size, no. of siliquae on main shoot and main shoot length, therefore, be considered the best for evaluation of genotypes for these traits. ELM 123 (G4) was identified as an ideal genotype with high mean performance for seed yield per plant, oil content, seeds per siliqua and early flowering, thereby suggesting that this variety can be commercially exploited for the Punjab state. This quality mustard genotype ELM 123 (RLC 2) has also been identified at national level for Zone II that includes Punjab in the year 2011. Genotype JC 37-6-1-2 (G5) has high mean and is responsive to favourable environment for seed yield per plant, no. of siliquae on main shoot and early maturity, so it can be used as a donor parent for improvement of these traits. Similarly genotype JC 75-8-2 (G6) has high mean and is responsive to favourable environment for oil content, no. of siliquae on main shoot and early maturity. One of the unique features of GGE biplot technique was which-won-where pattern of genotype x environment interactions. GGE biplot is more logical than AMMI in terms of explanation of PC1 score, which represents genotypic effects rather than additive main effects. So, it is better to exploit GGE biplot technique for estimation of G x E interactions, which is also depicted from the variances captured by different models in the present study. The GGE Biplot has captured maximum variance for each trait showing its efficacy for stability for all traits.
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Genetic variability for coleoptile length and seedling vigour in rice species, Oryza sativa L. and O. glaberrima Steud. and molecular characterization of introgression in interspecific progenies
(Punjab Agricultural University, Ludhiana, 2016) Binder Kaur; Sidhu, Navjot
Direct seeded rice (DSR) is a feasible alternative to conventional puddled transplanted rice in the north western plains amidst a looming water crisis and labour scarcity. But it is associated with poor seedling emergence and establishment. Early seedling vigor is the essential trait for direct seeded rice to dominate and smother the weed growth. There is need to identify genotypes specifically adapted to DSR conditions having early vigour. The present investigation was therefore carried out with the objective to determine genetic variability for early vigour and molecular characterization of introgressed segments from O. glaberrima into O. sativa in rice germplasm under dry direct seeded conditions. A set of 100 introgression lines derived from the cross of PR114/O. glaberrima //*2 PR114, 25 accessions of O. glaberrima, 14 cultivars of O. sativa and eleven elite rice breeding lines were evaluated in Kharif 2014 at two sowing depths D1 (2cm) and D2 (5cm). Data were recorded on various morphological traits i.e. seedling emergence, germination percentage, seedling height, SPAD reading, fresh shoot biomass, dry shoot biomass, fresh root biomass, dry root biomass and root length. Genotypes IL2968, IL3030, IR100983 and Pusa Basmati 1509 had best performance for seedling emergence, SPAD, seedling height at D1. The sowing depth significantly influenced the expression of different characters. IL2968, IL2970, IL3030, IL3062, PR 115, PR114, PR123 and Pusa Basmati 1509 had early emergence, good germination percentage, root length and SPAD value at both the depths. Seedling emergence showed significant negative correlation with all observed characters. SPAD reading was positively correlated with root length. Twenty six introgression lines selected on the basis of early vigour as compared to recurrent parent PR114 and best suited variety under DSR, PR115 were evaluated for plant height, days to 50% flowering, days to maturity, spikelet fertility, and grain yield under DSR in field conditions.So the desirable genotypes viz. IL3023, IL3030 and IL3062 showed high spikelet fertility, grain yield with intermediate plant height. A significant positive correlation was observed between spikelet fertility and grain yield. Molecular characterization of introgression of O. glaberrima chromosome segments was assessed. Ninety six markers out of 197 SSR primers screened were polymorphic between PR114 and parental lines of O. glaberrima. Genotyping of the 41 lines was done using 46 markers. Maximum introgression found on chromosome no. 2 and 11.The introgression lines IL3023, IL3030 and IL3062 with recovered background can directly be used in direct seeded rice breeding program for transfer of early vigor traits to the popular varieties.
Open Access
Characterization and molecular analysis of rice, Oryza sativa L. genotypes for high temperature tolerance
(Punjab Agricultural University, Ludhiana, 2016) Yashmeet Kaur; Rupinder Kaur
Rice is very sensitive to high temperature especially at the reproductive and grain filling stage which leads to higher spikelet sterility and ultimately yield losses. The mean, maximum and minimum temperature in Punjab are showing an increasing trend over the last decade. The present investigation was, therefore, carried out with the objective to identify heat tolerant genetic resources for future genetic studies and breeding. A set of 100 Oryza sativa genotypes were screened for heat tolerance in two environments E1 and E2 in the field during kharif 2014. Data were recorded on various morphological characters i.e. days to 50% flowering, panicle exsertion, SPAD reading, flag leaf temperature, panicle temperature, spikelet fertility, 1000 grain weight, grain yield per plant and head rice recovery. Based on spikelet fertility, grain yield per plant, SPAD and 1000 grain weight, genotypes TD 25, Binulawan, Chiem Chanh, Teqing, Ai Lan Ke 1110,Hasan Serai, Jamajigi, CT 6510-24-1-2, UPL RI-7, Karayal, Tsipala Fotsy and Zao Shou 691-11 in environment E1 and genotypes BR 24, Binulawan, Chang Ch'sang Hsu Tao, Chiem Chanh, O-Luen-Cheung, Teqing, Ai Lan Ke 1110, Deng Deng Qi, Ea Houm, IR 77298-14-1-2, Hasan Serai, Jamajigi, CT 6510-24-1-2 and UPL RI-7 in environment E2 were relatively heat tolerant as they had high spikelet fertility and grain yield per plant under high temperature conditions. Some genotypes like IR 64-21, RTS 14, Chang Ch'sang Hsu Tao, Deng Deng Qi, Hong Mi Dong Mao Zhan and Lua Tau Duc in environment E1 and genotypes IR 64-21, Sanhuangzhan No 2, IR 36, Deng Deng Qi, E 5168, Hong Mi Dong Mao Zhan, Lua Tau Duc, Race, Tsipala Fotsy and Way Rarem in E2 were moderately tolerant as they had high spikelet fertility but average or slightly below average grain yield per plant. Grain yield per plant and spikelet fertility showed significant negative correlation with flag leaf temperature as well as panicle temperature. Genetic diversity was assessed between these genotypes using 48 SSR primers. The genotypes were grouped into three distinct clusters I, II and III based on genetic diversity. The desirable genotypes for various heat tolerant traits identified in experiment I were scattered in all clusters. The heat tolerant lines identified in this study could be used as donors in breeding programmes focusing on heat tolerance.
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).

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