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Mapping of Ascochyta blight resistance gene(s)/QTLs from exotic Cicer arietinum L. germplasm in cultivated kabuli chickpea
(Punjab Agricultural University, Ludhiana, 2019) Lovepreet Kaur; Ajinder Kaur
Chickpea is an important pulse crop in the world after dry beans. Ascochyta blight caused by Ascochyta rabiei is a major fungal disease of chickpea and can cause upto 100% yield losses under favourable conditions. It is both air-borne and seed-borne disease that spreads rapidly to all the aerial parts including leaves, petioles, flowers, pods, branches and stems leading to rapid collapse and death of plants. The present investigation was undertaken for mapping of Ascochyta blight resistance gene using an interspecific population. The chickpea cultivar, L 552, besides having several agronomically important characters is susceptible to Ascochyta blight and the exotic accession, FLIP 05-43, is resistant to Ascochyta blight. An F2 interspecific population was developed from cross between L 552 (female parent) and FLIP 05-43 (donar parent). Inheritance studies in F2 and F2:3 populations revealed that inheritance of Ascochyta blight resistance was controlled by monogenic recessive gene, that was designated as arr6. A total of 300 SSR markers were used for polymorphism survey of the parents. A considerably low polymorphism of 15.30 % was found between the parents, and 46 polymorphic markers were used for genotyping of F2 population. For generating linkage map, these 46 SSRs were subjected to linkage analysis using MAPDISTO (1.7.6.5) software at LOD of 3, only 31 markers were mapped generating a linkage map of 377.14 cM with eight linkage groups. Using this linkage map, arr6 gene was mapped onto linkage group 4 at a distance of 8.6 cM distal to CGMM072 marker and from NCPGR247 marker, it was located at a distance 16.1 cM. There is a need to identify more SSR markers in the region lying between the markers CGMM072 and NCPGR247 in order to minimize the distance from the arr6 gene and its transfer to the elite cultivar (L 552) for providing durable resistance against Ascochyta blight. Thus, this study provides further prospective for fine mapping and map based cloning of the arr6 gene.
Open Access
Genetic mapping of brown plant hopper resistance from Oryza rufipogon (Griff.)
(Punjab Agricultural University, Ludhiana, 2019) Pavneet Kaur; Kumari Neelam
Brown planthopper (BPH) Nilaparvata lugens (Stal) is one of the most serious insect-pest of rice accounting for the loss up to 52% of annual production. Under high infestation, complete drying and plant death occurs causing “hopper burn”. The primary strategy for controlling BPH is the application of chemical insecticides, which is already proven detrimental not only to the environment, but also to human health. On this account host-plant resistance serves as an important strategy to reduce the damage caused by BPH. Out of 1003 wild species germplasm accessions screened against BPH, O. rufipogon acc. CR100441 has shown consistent resistance reaction (score 1-3) during 3 years of screening under greenhouse conditions. The F1 plant was obtained by making a cross of O. rufipogon acc.CR100441 and PR122. The BC2F2 plants were generated by further back crossing with PR122 and selfing then after. The BC2F2 plants were screened by the standard seedbox technique at seedling stage against BPH biotype 4. The BC2F2 plants were scored phenotypically on a 0-9 scale according to the Standard Evaluation System (IRRI, 1996) as 1-3 resistance, 5 moderate resistance, and 7-9 susceptible plant. From 276 BC2F2 plants, 66 plants were found to be resistant (1-3 score) and 210 plants (5, 7 and 9 score) were susceptible which fitted in 3:1 ratio (susceptible: resistant) indicating the recessive nature of the BPH resistance gene. Genotyping using polymorphic microsatellite markers between PR122 and O.rufipogon acc.CR100441 spanning all the 12 chromosomes of rice was done. The QTL governing BPH resistance was identified on the short arm of chromosome 4 between marker interval RM551 and RM6659. The RM16335 was peak marker, with LOD score of 10.8 and 35.23% contributing towards the phenotypic variation. This information can be used for further fine mapping and cloning of the gene. The pre-breeding BC2F4 lines with BPH resistance can be further utilize in breeding programmes aimed in development of resistant cultivars.
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Genome wide selection for rapid introgression of productivity traits from Oryza rufipogon into O. sativa
(Punjab Agricultural University, Ludhiana, 2019) Malik, Palvi; Kumari Neelam
The genome wide selection for rapid introgression of productivity traits was carried out on a diverse set of 346 accessions of Oryza rufipogon. Population structure investigated using 2271 SNPs in O. rufipogon, indicated a weakly differentiated population which further classified into six sub-populations. Linkage disequilibrium decayed to its half-maximum at 10 kb for Oryza rufipogon and 60 kb for Oryza sativa. Genome wide association analysis was carried out using a set of 44,109 SNPs for seven traits, i.e, plant height, culm thickness, panicle length, number of primary branches, grain length, grain width, hundred grain weight. The study revealed 37 strong SNP associations, out of which 13 SNPs were associated with multiple traits. A total of 14 SNPs localized in 12 previously reported QTL regions of the concerned traits. Genomic selection was carried out on 1751 backcross lines derived from 11 different backcross families. Bayes B and Bayes C were used to build models in training population, comprised of 1223 and 1215 lines for panicle length and plant height, respectively. GS accuracy for above mentioned traits came out to be 0.51 and 0.72, respectively. In conclusion, the study revealed very subtle population structure with low levels of differentiation in Oryza rufipogon population. The analysis yielded 23 novel SNPs, which might serve as putative candidates for further investigation of agronomically important traits. The backcross progenies with good genomic estimated breeding values can be further utilized in breeding programs for improving yield related traits.
Open Access
Fine mapping of QTL on chromosome 9 for drought tolerance in maize
(Punjab Agricultural University, Ludhiana, 2019) Ramandeep Kaur; Sharma, Priti
Drought is considered as one of the major limiting factors in sustainable maize production all over the world as it causes yield reduction by an average of 15% to 20%. Maize is generally grown in Kharif season but spring maize is now coming up in India. However, the water requirement is very high but farmers are reluctant to see the long term effect of maize cultivation during spring season. To meet the growing demand of water during spring season, enhancement of maize yield can be achieved by developing water efficient maize hybrids. The objective of the study was to identify and to transfer QTL associated with drought tolerance into spring maize inbreds through marker assisted backcross breeding (MABB). A total of 135 F8 recombinant inbred lines (RILs) from the cross between CM123 as the susceptible (female) parent and CM140 as the tolerant (male) parent along with parents were evaluated under control and drought stress conditions for two consecutive seasons. The QTL on chromosome 1, 3, 4, 6, 7 and 9 were identified for drought tolerance under both stress and control conditions. The present study focused on fine mapping of QTL for number of kernel per ear (qKPE) present on chromosome 9 (bnlg1401-umc1634) explaining phenotypic variance of 23.14% under stressed environment. This region was narrow down by designing 50 new SSR markers between the bracketed QTL (qKPE). Seventeen SSR markers showed the polymorphism between CM123 and CM140. These markers along-with previous mapped markers were employed on RIL population. The QTL analysis narrowed down the genetic distance to 3.8 cM from 11.5 cM and physical distance to 691 kb from earlier distance of 15 Mb flanked by two new SSR markers viz. PAU_1143 and PAU_1137. The qKPE is also introgressed through MABB into two spring maize inbreds LM23 and LM24 of hybrid PMH10 for water use efficiency. The foreground selection has been carried out in two generations i.e., BC1F1 and BC2F1. Also, Background selection has been done in BC1F1 to check the background recovery of recurrent parent. The plants carrying the QTL with highest recurrent parent background recovery were selected and again backcrossed to respective parent for generation of BC3F1 population. The BC3F1 plants have been raised during Kharif 2019. The development of drought tolerant PMH10 hybrid will lead to overcome frequent irrigations during spring season and helps to conserve ground water depletion.
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Linkage map construction in guava F1 population of Allahabad Safeda x Arka Kiran using molecular markers
(Punjab Agricultural University, Ludhiana, 2018) Jindal, Manish; Mittal, Amandeep
Guava is a perennial fruit tree grown in tropical and sub-tropical regions of the world. As of now, there are about 160 cultivars available in India. Crop improvement work attempted in India has resulted in release of several superior selections or hybrids. However, the maximum area under guava cultivation is occupied by Allahabad Safeda. Being a cross-pollinated tree (25.7 to 41.3 % cross pollination) guava has a heterozygous genome. Molecular mapping can help us to find the relative positions of the markers as well as the markers co-segregating with the trait of interest that could finally be transferred to the cultivated species. In the present study we have attempted Linkage map construction in guava. A cross between white fleshed Allahabad Safeda and colored fleshed Arka Kiran was attempted in Fruit Science, PAU. We genotyped Allahabad Safeda and Arka Kiran using 167 genomic SSR, 22 EST based and 5 apple color specific markers. Forty eight markers showed polymorphism out of 194 total markers. Polymorphic markers applied on a population of 73 F1 individuals showed segregation. Pattern of marker segregation in the population was scored and analysed with software, MAPDISTO version 1.7.7.0.1.1 (XL 2007) and a genetic linkage map was constructed using stringency parameters of LOD score and recombination frequency set to 3.0 and 0.35, respectively. Out of 48 polymorphic markers, thirty markers were mapped on different linkage groups of guava genome and 6 linkage groups were obtained. The genetic linkage map covered a total of 538.68 cM of the guava genome. Fruits were not set on 2 year old F1 trees so color segregation was studied on leaves as a proxy for fruit color trait. Color in young and mature leaves was measured using miniature leaf spectrometer. The data recorded in terms of anthocyanin reflective index 1 (ARI1) was analysed with the help of mapping software. Two markers mPgCIR93 and mPgCIR21 were mapped to linkage group 2 on positions 54.3 cM and 10.3 cM for leaf color traits at young and mature stage.
Open Access
Fine mapping of qtl-qslb.pau-3.04 for southern leaf blight resistance in maize (Zea mays L.)
(Punjab Agricultural University, Ludhiana, 2019) Kirandeep Kaur; Vikal, Yogesh
Southern leaf blight (SLB) caused by Cochliobolus heterostrophus (Drechsler), is a serious disease throughout the world where maize is grown under warm and humid conditions, leading to enormous yield losses. Exploiting genes and quantitative trait loci (QTLs) related to SLB is helpful for improving fungal resistance. In earlier study at Punjab Agricultural University, Ludhiana a set of 325 F2:3 families and F4 progenies derived from the cross of CM139 as the resistant (female) parent and CM140 as the susceptible (male) parent were phenotyped for resistance to SLB under field conditions. A total of 172 polymorphic SSR markers were genotyped on F2 population.Three probable QTL viz. qSLB2.1, qSLB3.1, qSLB3.2 were detected for SLB resistance in bins of 2.05-2.08, 3.04 and 3.06-3.09. The marker interval phi099-umc1729 spanning qSLB3.1 was considered as major putative QTL. In present study, the QTL qSLB3.1 spanning region was saturated with more SSR markers on 298 RIL population of the same cross. The detected QTL region (13.7 cM) was fine mapped to 1.2 cM region flanked with marker MSSR1-MSSR20 explaining phenotypic variance of 15.1 per cent at log-likelihood of 6.9. The 1.2 cM region corresponds to 269.3 Kb and comprises only six candidate genes. The candidate gene based markers were designed, to study the differential expression patterns of candidate genes through quantitative real-time PCR (qRT-PCR). Two of the candidate genes viz. GRMZM2GO88371 and GRMZM5G862219 showed 3.48 and 6.40 fold higher expression in CM140 at 48h and 72h respectively. GRMZM2GO88371 and GRMZM5G862219 have their biological function in lipid metabolism and β oxidation respectively that is involved in the defense pathway and may be one of the potential candidate genes conferring resistance against C. heterostrophus. The SLB QTL linked flanking markers were employed for mobilization of qSLB3.1 QTL into the background of CM140 through marker assisted backcross breeding (MABB). The F1s of cross (CM139 X CM140) were backcrossed to recurrent parent to generate BC1F1 population. A total of 64 plants out of 420 were selected on the basis of foreground selection. Recombinant selection for the carrier chromosome was done to identify single and double recombinants. The plants having maximum recurrent parent recovery for carrier chromosome were selected and backcrossed with CM140 to generate BC2F1 generation. The data generated from this study can serve as valuable genomic resource for maize breeding programmes. It will enable the researcher to multi-thronged and focused approaches for sustainable development of new genotypes by pyramiding it with other desirable genes using MABB.
Open Access
Marker assisted mobilization of cry1Ac gene from transgenic chickpea into cultivated chickpea (Cicer arietinum L.) for pod borer [Helicoverpa armigera (Hübner)] resistance
(Punjab Agricultural University, Ludhiana, 2019) Sharma, Urvashi; Ajinder Kaur
Helicoverpa armigera (Hübner) poses a serious threat to chickpea production world-wide. cry1Ac gene is known to be most effective in controlling infestation of pod borer. The present investigation was undertaken to incorporate cry1Ac gene from T5 transgenic chickpea lines BS 100B-T5 and BS 100E-T5 into cultivated chickpea (Cicer arietinum L.) varieties PBG 7 and L 552, respectively through crossing and recurrent backcrossing. The BC1F1 progenies [F1 (PBG 7 x BS 100B-T5) x PBG 7] and [F1 (L 552 x BS 100E-T5) x L 552] were tested for cry1Ac gene presence using gene specific primers. ELISA on the positive plants revealed Cry1Ac protein content between 10.57 to 11.72 µg/g leaf tissue. Therefore, on the basis of foreground selection and ELISA, positive BC1F1 plants were propagated to obtain BC1F2 populations, which were also subjected to foreground selection. A BC2F2 population of 83 plants was also grown by selfing [F1 (PBG 7 x BS 100E-T5) x PBG 7] x PBG 7. Foreground selection using cry1Ac specific primers on BC2F2 population resulted in ten (12.04 %) cry1Ac positive plants (1, 2, 8, 9, 12, 20, 26, 33, 39 and 44); their background selection using 12 polymorphic SSR markers revealed average recurrent parent genome restoration of 88.9 %. As a result, BC2F3 population comprising 128 plants was obtained from ten cry1Ac positive BC2F2 plants. Each BC2F3 row of positive plants was then tested using cry1Ac specific primers to identify BC2F2 plants homozygous for cry1Ac gene. The analysis revealed that three (30 %) BC2F2 plants 26, 39 and 44 were homozygous for cry1Ac gene, whereas remaining seven plants 1, 2, 8, 9, 12, 20 and 33 segregated in ratios of 6:1, 2.3:1, 1.25:1, 3:1, 1.6:1, 4:1 and 3:1, respectively.
Open Access
Heterologous expression and characterization of antifungal chitinase and glucanase genes from Trichoderma viride in Escherichia coli
(Punjab Agricultural University, Ludhiana, 2019) Ramanjeet Kaur; Sandhu, Jagdeep Singh
The endochitinase and β-1,3-glucanase genes isolated from Trichoderma spp. were expressed in Escherichia coli strains Rosetta-gami 2 (DE3) and BL21 (DE3) pLysS using His-tagged pET28a+ and pET6xHN-C vectors. The expression of endochitinase and β-1,3-glucanase recombinant proteins was optimized by varying the incubation conditions, such as incubation temperature, incubation time and inducer concentration. The maximum expression (86.23 ng/ml) of endochitinase was observed with 0.4 mM IPTG at 37 ºC for 4 h in Rosetta-gami 2 (DE3), and maximum expression for β-1,3-glucanase (89.23 ng/ml) was observed in Rosetta-gami 2 (DE3) with 0.6 mM IPTG at 37 ºC for 6 h. The recombinant endochitinase and β-1,3-glucanase proteins were purified from E. coli using Ni-NTA affinity columns. The purified recombinant proteins were verified by SDS-PAGE, Western blotting and ELISA. The verified recombinant proteins were then tested for antifungal activity by confronting with Rhizoctonia solani casual organism of rice sheath blight and Phytophothora parasitica casual organism of citrus foot rot. The inhibition ratio of R. solani growth by endochitinase protein was 30±2.08 mm and by β-1,3-glucanase was 22.6±0.44 mm. This inhibition was higher than clotrimazole fungicide (21.85±0.47 mm). The recombinant endochitinase did not inhibit growth of P. parasitica whereas, β-1,3-glucanase revealed inhibition ratio of 32.6±0.47 mm against the pathogen. The hydrolytic action of the recombinant endochitinase and β-1,3-glucanase proteins on the cell morphology of R. solani was observed through scanning electron microscopy revealing breakage and pores on hyphal cell walls of R. solani whereas, β-1,3-glucanase confronted hyphae were prone to desiccation and have wrinkles and globular structures on hyphal walls eventually leading to hyphal lysis.
Open Access
RNA interference mediated gene silencing in fruit fly [Bactrocera dorsalis (Hendel)]
(Punjab Agricultural University, Ludhiana, 2019) Mohan. G; Mohanpuria, Prashant
The oriental fruit fly, Bactrocera dorsalis (Hendel) is serious pest infesting important fruits and vegetables throughout the world. In Punjab, it infests damage of 70-80 per cent each in peach and Kinnow, 60-80 per cent in pear, 50 per cent in jamun, 30-40 per cent each in loquat, fig, plum and mango, and 100 per cent in guava fruits during rainy season and thus, fruit fly is a major concern in Punjab. Control of fruit fly is highly challenging because of its polyphagous nature and unexposed developmental stages and limitations of existing methods. This has now necessitated the biotechnological intervention for fruit fly management. In present investigation double-stranded RNA (dsRNA)-mediated gene silencing in guava fruit fly was attempted. Specific regions of four genes of fruit fly i.e. ecr (ecdysone receptor), rpl19 (a ribosomal protein L19), noa (fatty acid elongase) and v-ATPase-D important for its growth, development, molting, metamorphosis and energy production were cloned in dsRNA expression vector and transformation was done into HT115 (DE3) Escherichia coli strain for dsRNA production. Silencing effects were observed by feeding artificial diet mixed with bacteria expressing dsRNA corresponding to ecr and rpl19 genes to maggot and adult fruit fly. The results showed severe deformities in pupae and adult fruit fly and mortality indicating the feasibility of dsRNA delivery method and silencing of these potential genes of fruit fly. In future, transgenic fruit crops expressing dsRNA of these potential genes of fruit fly and/or spray based dsRNA formulations could be developed for fruit fly management.

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