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Open Access
Fine mapping of QTL (qSLB8.1 and qSLB9.1) for southern corn leaf blight resistance in maize (Zea mays L.)
(Punjab Agricultural University, Ludhiana, 2020) Simranjit Kaur; Sharma, Priti
Southern corn leaf blight (SLB) caused by Cochliobolus heterostrophus, is a fungal disease of maize. It is prevalent in warm region and can cause yield losses upto 40 percent. Therefore the present investigation was undertaken using 192 RILs developed from cross between LM5 (resistant parent) and CM140 (susceptible parent). RILs along with parental lines were evaluated for SLB disease reaction by inoculating plants at V7-9 stage and data was recorded three times in an interval of 15 days. Disease reaction data showed normal distribution which indicates the quantitative nature of inheritance. Days to anthesis (DTA), days to silking (DTS) and anthesis-silking interval (ASI) were also evaluated and low correlation was observed between flowering traits with disease data. A total of 100 SSR markers of chromosome 8 and 50 SSR markers of chromosome 9 were developed using MISA. Among 221 markers, 71 showed polymorphism between the parental lines. A linkage map was constructed with genomic coverage of 56.3 cM for chromosome 8 (within marker interval umc1075 and bnlg1067) with 20 markers and 9.6 cM for chromosome 9 (within marker interval bnlg1626 and bnlg1091) with 10 markers. QTL analysis was done using QTL cartographer and previously identified QTL qSLB8.1 has been narrowed down with PAU_63-PAU_116 (qSLB8.1) as new flanking markers from distance of 7 cM/110 Mb to 3 cM/42.4 Mb; and in qSLB8.2 distance has been narrowed down to 3.9 cM from 5.6 cM by QTL flanked by markers PAU_95-umc1872 (qSLB8.4). Further, more markers can be designed and identified flanking markers can be used for marker assisted selection.
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High resolution genetic mapping of xa43(t) gene for bacterial blight resistance in rice (oryza sativa l.)
(Punjab Agricultural University, Ludhiana, 2020) Bhatia, Sukhpreet; Vikal, Yogesh
Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the epidemic diseases in rice causes significant yield losses worldwide, especially in Southeast Asia. Due to the lack of effective chemical control, most effective strategy to combat the BB disease is the finding of new resistance sources and its deployment in the elite rice cultivars. Since the durability of resistance is always at stake due to the rapid change in the pathogenicity of the Xoo pathogen, there is a need to identify and characterize novel BB resistance genes. Tagging and fine mapping is pre-requisite for transfer and pyramiding of BB resistance genes through marker-assisted selection (MAS). Therefore, the present investigation was undertaken to develop high resolution genetic map of novel bacterial blight resistance gene, xa43(t) identified from Oryza rufipogon acc. CR100098A. The backcross introgressed lines (BILs, BC1F7) were developed from cross between PR114 and Oryza rufipogon acc. CR100098A. The genetic studies using BC1F2 population and BC1F3 progenies showed single recessive locus conditioning resistance to the Xoo pathotype #7 (PbXo-7). The BILs were segregated in the ratio of 197 resistant : 195 susceptible which is consistent with the expected allelic frequency of 1:1 ratio for single gene inheritance. The xa43(t) gene was tagged with two SSR markers viz. RM27154 and RM2136 on long arm of chromosome 11 using bulked segregant analysis. To fine map the gene, 85 SSR and 86 KASP markers of chromosome 11 were surveyed for parental polymorphism. A total of 22 SSR and five KASP markers were found to be polymorphic between parents. The polymorphic markers were analysed on 392 BILs and genetic analysis placed the xa43(t) BB resistance gene between two flanking SSR markers, PAU11_39 and PAU11_44 within an approximately 447 kb region. Bioinformatic analysis revealed a total of 73 genes within that region and seventeen of them were putative candidate genes involved in biotic stress resistance. The novelty of xa43(t) gene was confirmed by testing the linked markers on other cultivars as well as by surveying the linked markers of other BB resistance genes present on chromosome 11 on parents and bulks.
Open Access
Genetic transformation of guava (Psidium guajava L.) with RNAi construct for fruit fly [Bactrocera dorsalis (Hendel)] resistance
(Punjab Agricultural University, Ludhiana, 2020) Gursimran Kaur; Mohanpuria, Prashant
Guava (Psidium guajava L.) is one of the most nutritious fruit due to its abundance in antioxidants, tannins, vitamins A, C and minerals. But its production is severely affected by fruit fly [Bactrocera dorsalis (Hendel)] infestation. There is no fruit fly resistant guava germplasm reported so far globally. RNAi approach against fruit fly provides an attractive approach to overcome this devastating problem. Here we report generation of marker-free RNAi guava lines of cv. Allahabad Safeda targeting ecdysone receptor (ECR) gene of fruit fly through in planta genetic transformation. For floral dip method, completely developed closed bud and bud with calyx split were used and effects of different dipping time in infiltration medium were observed. Dipping time for more than 10 seconds led to necrosis of floral buds and pre-mature flower drop. For floral drop method, different amount of dropping inoculum on bud with calyx split and open flower were used. Dropping 4-6 drops of Agrobacterium inoculum led to necrosis and flower drop, while dropping 1-2 drops of inoculum found optimum led to development of subsequent guava fruits. RNAi cassette integration was detected in transformed guava lines through PCR using gene specific and vector specific primers. No transformants were obtained with floral dip method in guava, but floral drop transformation showed a higher transformation efficiency of 4.95% with bud with calyx split stage of guava used as explant. The present optimized, efficient floral drop transformation method will be very useful for genetic manipulation of guava.
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Development of transient assay for analyzing the efficacy of antifungal genes against different fungal pathogens of rice
(Punjab Agricultural University, Ludhiana, 2020) Anroop Kaur; Sandhu, Jagdeep Singh
Two gene cassettes containing β-1,3 glucanase and chitinase genes under the control of CaMV 35S promoter and nos terminator mobilized in Agrobacterium strain LBA4404 were used to agroinfect rice calli for testing their antifungal activity against rice pathogens. The callus cultures were induced by culturing mature rice seeds (Oryza sativa L. cv. PR114) on MS media supplemented with 1mg/l 2,4-D and 0.15mg/l BAP. The Agrobacterium broth at O.D-0.6 was used for agroinfection of rice callus in the presence of 100 μM of acetosyringone. The agroinfected calli were transferred for first selection cycle of 14 days on MS media supplemented with 2,4-D (1mg/l) + BAP (0.5mg/l) + cefotaxime (250mg/l) and kanamycin (50 mg/l). The calli were transferred for second and third selection cycle for 10 days and 7 days on MS media supplemented with 2,4-D (1mg/l) + BAP (0.5mg/l) and kanamycin (50 mg/l). The presence of β-1,3 glucanase and chitinase transgene was verified on all surviving calli through PCR using transgene-specific primers. The amplification of 1764 bp and 1275 bp sequence confirmed the presence of β-1,3 glucanase and chitinase gene, respectively with the transformation efficiency of 26.78% and 26.73%. The calli showing amplification of β-1,3 glucanase and chitinase gene were analyzed for the antagonism against Rhizactonia solani, Magnoporthe oryzae and Fusarium moniliforme through dual culture technique. The transformed calli did not inhibit mycelium growth on R. solani and F. moniliforme probably due to the ineffectiveness of β-1,3 glucanase and chitinase transgenes against the pathogens. Whereas, inhibition of M. oryzae growth was observed in vitro. So, β-1,3-glucanase and chitinase gene integration serve as an effective control measure for the control of rice blast disease caused by M. oryzae.
Open Access
Cloning of doublesex gene, dsx for its rnai-mediated silencing in fruit fly [Bactrocera dorsalis (Hendel)]
(Punjab Agricultural University, Ludhiana, 2020) Vasundara Devi S; Mohanpuria, Prashant
Bactrocera dorsalis (Hendel) is a highly invasive pest of fruits and vegetables in the oriental region. The management of fruit fly is highly challenging due to its high fecundity, wider climatic adaptability and unexposed developmental stages. Bactrocera dorsalis is a major pest in several northern and northeastern parts of India. Effective management of fruit fly requires collective input of cultural, chemical and post-harvest techniques. One of the widely used methods for control of B. dorsalis is the Sterile Insect Technique (SIT). We have investigated the potential of RNAi mediated silencing of doublesex gene for its use in SIT, by feeding dsx-dsRNA expressing bacteria to fruit flies. By designing male and female specific dsx-dsRNA sex specific knockdown of the target gene was achieved. Distortion in sex ratio was observed by feeding dsRNA expressing bacteria to maggots. Feeding bacteria expressing dsRNA to adults delayed the sexual maturity in both males (3 days) and females (4 days). The number of eggs laid were found to be reduced due to dsx-dsRNA treatment, a maximum reduction of 20 percent was observed in dsxf silencing and 26 percent reduction in dsxm silencing. The egg hatching efficiency of treated females and males were found to be reduced up to 52 percent and 51 percent respectively. These results demonstrate the ability of this method to replace the radiation induced sterilization technique for SIT due to its cost-efficiency and simplicity. It can also be used for sex sorting or sex selection for SIT.
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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.
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Identification and isolation of genes responsible for increased shelf-life in guava (Psidium guajava L.)
(Punjab Agricultural University, Ludhiana, 2020) Namita, Namita; Mittal, Amandeep
Guava is a climacteric fruit and ripening continuous even after harvesting leading to various textural changes. Abscisic acids (ABA) triggers the production of ripening hormone ethylene at fruit maturation stage. Most of the post-harvest changes are attributed to biosynthesis and signaling of ethylene further leading to cell wall metabolic changes imparting softness to fruit. Here, we tested 14 guava cultivars for ripening behavior and measured the physio-chemical attributes and biochemical changes at 3 ripening intervals in 2018-19 winter season crop. White fleshed Allahabad Safeda, Hisar Safeda, Punjab Safeda, Thailand guava, Shweta, Sardar guava and CISH-G5 and pink fleshed Punjab Pink, Lalit, Hisar Surkha, Purple local, 17-16, Punjab Kiran and Arka Kiran cultivars were analysed. Average TSS among genotypes varied from 8-11.5(ᵒbrix), Acidity 0.28 - 0.46%, reducing sugar 3.1-8.9%, Vitamin C 97.7 – 121 mg/100g, Firmness 4.66 - 6.73lb in 8 days of shelf life experiment. Highest fruit firmness at 4 days post-harvest was found in Thailand guava (9.8 lb). Our physico-chemical and gene expression analysis for ethylene & ABA biosynthesis, signaling and cell wall degrading enzyme genes reveal that ABA and ethylene biosynthesis is delayed in the highest shelf life cv. Thailand guava. Comparative genome sequence analysis of Thailand and Allahabad Safeda identified the specific nucleotide changes in ethylene biosynthesis gene SAM3 leading to change in glutamic acid to aspartic acid in an important domain. The genes ACO5, EIL3, EIN2, ERF6 and PL have SNPs in the untranslated and/or the promoter regions that might be impacting the docking of transcriptional machinery and concomitant delay in ripening. SNP based molecular markers in Thailand vs Allahabad Safeda might help in marker assisted selection for transferring superior alleles for delayed ripening in popular guava cultivars.
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Evaluation of gamma irradiated mutant population of sugarcane (Saccharum officinarum) for lignin content and its characterization for COMT gene
(Punjab Agricultural University, Ludhiana, 2020) Sharma, Shaweta; Malhotra, Pawan Kumar
Sugarcane is a C4 plant which is grown for sugar and biofuel in many parts of world. The bagasse left after extracting juice from sugarcane stalk is rich in cellulose, hemicellulose and lignin. This makes it an ideal substrate for bioethanol production. Lignin is the single key factor which makes this lignocellulosic biomass recalcitrant for ethanol extraction. Around 200 γ-irradiated mutant plants of sugarcane variety CoJ 85 were used in present study for evaluating variation for lignin content using acetyl bromide method. The mutant plants showed variation in lignin content ranging from 13-28%. The normal plant (control) showed 22.95% lignin content. To know this variation at molecular level the caffeic acid-o- methyl transferase (COMT) gene was amplified from five low lignin mutant plants and control. Two overlapping primers of the reference gene retrieved from sugarcane genome database (https://sugarcane-genome.cirad.fr) were designed using perl primer software for COMT gene for amplification using high fidelity polymerase chain reaction. The eluted PCR products from mutants as well as control were then separately ligated in pGEM-T vector and cloned in E.coli (strain DH5-ɑ). The plasmids were then isolated and Sanger sequencing was done for all these samples. After removing bad sequences from ABI files the gene lengths were predicted in control as well as mutants in comparison to reference gene. The predicted sequence of control was 2566 bp while those of mutants ranged from 2560-2568 bp in length. Sequencing results showed variations such as SNPs and indels in various regions of COMT gene for all the screened mutants and control in comparison to reference gene. All the CDS, TSS, intron and exonic regions were also identified by using FGENESH program in COMT gene isolated from control as well as in field grown γ-irradiated sugarcane mutants.
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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