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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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Agrobacterium mediated genetic transformation of pigeon pea (Cajanus cajan L. Millsp.) for resistance to spotted pod borer Maruca vitrata
(Punjab Agricultural University, Ludhiana, 2020) Manjinder Singh; Ajinder Kaur
The present investigation dealing with introduction of cry1Ab gene for resistance to Maruca vitrata into pigeonpea (Cajanus cajan L. Millsp.) through Agrobacterium-mediated in planta transformation was carried out using three pigeonpea genotypes i.e. AL 15, AL 201 and PAU 881. Two methods of in planta transformation were used for generating transgenic plants, including inoculation of pricked embryo axes and floral dip transformation. Out of seven experiments on in planta transformation method involving inoculation of pricked embryo axes, only two (OD600 of Agrobacterium broth = 0.6 - 0.7 containing 100 mM acetosyringone, 1 h dipping of 2-day old pricked seedlings) gave positive results. A total of 5,022 half seeds were treated that gave rise to 4059 plants, out of which 15 (representing AL 15 and AL 201 genotypes) were PCR-positive with an overall transformation frequency of 0.36 %. Out of 15 primary transformants, only 11 plants showed transcript accumulation by semi-quantitative RT-PCR. The primary transformants were also analyzed for chimerism by ELISA-based protein accumulation in selected branches. Out of a total of 182 branches of 10 RT – PCR positive plants analyzed, 57, 43 and 49 branches had Cry1Ab protein content equal to or more than 1.08 µg/g [Positive calibrator, (PC) 5.0 ppb], 0.58 µg/g (PC, 2.5 ppb) and 0.08 µg/g (PC, 0.5 ppb), respectively. Besides, there were 33 such branches which did not show accumulation of Cry protein indicating the presence of chimerism in primary transformants. The putative transgenics were advanced to T1 generation, out of 904 plants analyzed, 97 were PCR-positive, thus exhibiting transformation efficiency of 11.70 % (with genotype AL 201) and 10.24 % (with genotype AL 15). Twenty three T1 plants were randomly taken for determination of Cry1Ab protein content, out of which 12 showed high protein content of more than 0.72 µg/g. These plants showed positive results in PCR and RT-PCR, and further used for determining the efficacy of cry1Ab gene against second instar larva of Maruca vitrata using insect bioassay. Both flowers and pods of these 12 T1 pigeonpea plants were used for bioassay experiments. All 12 T1 plants showed restricted increase in larval weight as compared to nontransgenic (control) plant. No insect mortality was observed, but larvae fed on two transgenic plants i.e. 201-344 and 15-537 showed no adult emergence as compared to other transgenic and control plants where adult emergence was normally observed. Both of these two plants (201-344 and 15-537) had high Cry protein content (0.88 µg/g) as compared to other transgenic plants, which established clearcut positive correlation between amount of protein accumulated and inhibition of M. vitrata larval growth. Further, T1 plants were advanced to T2 generation that were maintained in transgenic glasshouse. In case of floral dip transformation, a total of 139 flower buds were treated and 454 seeds were obtained. Ten out of 454 putative T1 plants were observed to be PCR-positive with a transformation frequency of 2.2 %.
Open Access
Mapping and transfer of GENES/QTLs for nematode (Meloidogyne graminicola) resistance from Oryza glaberrima into Oryza sativa L.
(Punjab Agricultural University, Ludhiana, 2020) Gurwinder Kaur; Vikal, Yogesh
Rice root knot nematode (Meloidogyne graminicola) is one of the emerging constraints for rice production, causes about 50% yield losses in field conditions and 98% yield losses in pot experiments. The identification and exploitation of resistant rice genotypes is one of the economic and sustainable option to minimize the yield losses caused by M. graminicola infestations. The present investigation was undertaken to identify the QTLs associated with rice root knot nematode resistance as well as the transfer of resistance to elite rice cultivars. A total of 42 accessions of O. glaberrima along with O. sativa cultivar PR121 were screened in triplicates during kharif 2016 and kharif 2017 in nematode infested sick plot at initial nematode population density of one juvenile per gram of soil. Reproduction factor of rice root knot nematode among O. glaberrima accessions was found to be <1 while in O. sativa cultivar PR121, reproduction factor was >1. Out of 42 accessions, three accessions (IRGC102196, IRGC102538 and IRGC102557) were found to be highly resistant, thirty-three accessions were resistant, and six accessions were moderately resistant. Data on different morphological traits (plant height, root length, fresh shoot weight, fresh root weight, dry shoot weight and dry root weight) revealed that no statistically significant differences were found among O. glaberrima accessions whereas PR121 exhibited significant reduction in all growth parameters in nematode infested sick plot as compared to controlled conditions. The BC1F1 population derived from the cross of O. glaberrima acc. IRGC102206 × PR121 was used for mapping of rice root knot nematode resistance. The BC1F1 plants were screened against M. graminicola in triplicates and genotyped using 84 polymorphic SSR markers. Both phenotypic data and genotypic data was analyzed and a total of 13 QTLs associated with gall number, gall index and different morphological traits were mapped on chromosomes 1, 3, 4, 5, 6 and 8. Out of 13 QTLs, three QTLs associated with gall number were identified; two QTLs designated as qGN4.1and qGN4.2 were mapped on chromosome 4 and one QTL, qGN6.1, was mapped on chromosome 6. QTL associated with gall index (qGI6.1) was found to be co-localized with qGN6.1. Similarly, qGN6.1 QTL was co-localized with plant height and root length. Due to less coverage by SSR markers, we simultaneously performed BSA-QTLseq (Bulked segregant analysis coupled with QTL-seq approach) analysis and identified QTLs for nematode resistance on chromosomes 1, 2, 3, 4, 5, 6, 11 and 12. The genomic interval of all QTLs was narrowed down to 1–2 Mb using QTL-seq analysis. The development of SNP based molecular markers from identified QTL regions will further help to saturate the linkage map and to identify closely linked markers to rice root knot nematode resistance. The identified markers will further fasten the improvement of genotypes for rice root knot nematode resistance through marker assisted breeding approach. Based on introgressed genomic regions from O. glaberrima carrying QTLs for nematode resistance the BC1F1 resistant plants were selected and backcrossed to generate BC2F1 and subsequently BC3F1 progenies for transfer of nematode resistance in the background of PR121. The data generated from this study can serve as valuable genomic resources for rice breeding programmes.
Open Access
Identification and characterization of high temperature stress responsive genes in maize (Zea mays L.)
(Punjab Agricultural University, Ludhiana, 2020) Ashok Babadev Jagtap; Vikal, Yogesh
Heat stress due to climate change is an emerging issue for maize breeders as it largely affects the yield. The present study focuses to elucidate molecular mechanisms and underlying genes, or QTLs associated with heat stress tolerance in maize. The transcriptional studies of maize leaves, pollens and ovules using heat stress susceptible and tolerant inbred lines, namely LM11 and CML25, respectively revealed a total of 2,164 (1127 up-regulated and 1037 down-regulated) differentially expressed genes (DEGs) between LM11 (HS) and CML25 (HT) samples, with 1151, 451 and 562 DEGs were identified in comparisons of corresponding leaf, pollen and ovule samples, respectively. Functional annotations of DEGs showed that many of them were related to transcription factors (TFs) viz. AP2, MYB, WRKY, PsbP, bZIP and NAM, heat shock proteins (HSP20, HSP70 and HSP101/ClpB), as well as genes related to photosynthesis (PsaD and PsaN), antioxidation (APX and CAT) and polyamines (Spd and Spm). KEGG pathways analyses explicated that metabolic overview pathway and secondary metabolites biosynthesis pathway, with involvement of 264 and 146 genes, respectively were highly enriched in response to heat stress. A bioinformatics pipeline was used to call and type SNPs from RNA-seq reads and applied it to transcriptomic data of LM11 and CML25. A total of 554,423, 410,698 and 596,868 polymorphic SNPs were identified respectively among leaf, pollen and ovule of the LM11 and CML25. A total of 100 genome-wide SNP based KASP assay markers were developed and validated as well as subsequently genotyped on 90 F2 individuals derived from the cross of LM11 × CML25. The success of SNP conversion rate was 71%. In addition, F2 population and their parental inbreds were genotyped using 94 polymorphic SSR markers. The 175 F2:3 families during late March (Spring 2017) were evaluated for heat stress under field and glass house conditions. Heat stress significantly affected all the morpho-physiological and yield contributing traits. Grain yield was positively associated with ear weight, number of kernels per ear, pollen viability, pollen shedding durations and chlorophyll content. Furthermore, secondary traits like membrane thermostability, days to anthesis and silking, anthesis-silking interval, canopy temperature, leaf firing and tassel blast showed significant negative impact on grain yield in both field and glass house conditions under heat stress. Both genotyping and mean phenotypic data of each component trait was analyzed for single marker analysis (SMA) and composite interval mapping (CIM) using WinQTL Cartographer. A linkage map of 1857.1 cM in total length was constructed by applying both SSR and SNP markers. A total of 11 QTLs were detected for 7 traits on chromosomes 1, 3, 4, 6, 7 and 9 with phenotypic variance ranged from 8.67 to 29.62 per cent. Four of these QTLs, qKPE6.1, qPV6.1, qCC9.2 and qLF4.1, accounted for above 15 per cent of phenotypic variation, and might be considered as major QTLs for heat tolerance. The data generated in present investigation laid the foundation for future work to uncover genes and mechanisms critical for the development of heat-resilient maize using genetic and biotechnological approaches.
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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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Transformation of antifungal gene β-1, 3-glucanase in rice (Oryza sativa L.) and its expression analysis for sheath blight resistance
(Punjab Agricultural University, Ludhiana-, 2020) Pathania, Shivali; Sandhu, Jagdeep Singh
Trichoderma cell wall degrading β-1, 3-glucanase enzymes hydrolyse β-1, 3-glycosidic linkages of β-glucan rich cell wall of Rhizoctonia solani, a causal agent of rice sheath blight. To this date, no cultivar is known to provide true resistance to the phytopathogen. In this study, β1, 3-glucanase gene, encoding antifungal protein, was introduced into rice through Agrobacterium-mediated genetic transformation. Gene construct was generated in pRI 101-ON binary vector, carrying β-1, 3-glucanase under the control of CaMV35S promoter and NOS terminator and was mobilized into two Agrobacterium tumefaciens strains, LBA4404 and EHA105 that were used to agro-infect Kitaake and PR124 rice calli. A total of 434 and 388 regenerated plants were obtained in Kitaake and PR124, respectively. The presence of transgene was confirmed by PCR, revealing amplicon of size 2,307 bp corresponding to β-1, 3glucanase with transformation efficiency of 0.76 % and 0.46 % in Kitaake and PR124, respectively. The integration and expression of transgene in primary transformants was verified by Real-Time PCR, depicting up to 5.13- and 3.81-fold increase in β-1, 3-glucanase expression in transformed Kitaake and PR124 plants, respectively than non-transgenic control. Moderate resistance to RS-1 isolate of R. solani was found in transgenic plants, indicating potential use of β-1, 3-glucanase from Trichoderma spp. in rice to confer resistance to sheath blight disease.
Open Access
Development of an in vivo haploid induction system in rice through distant hybridization and manipulation of CenH3 gene
(Punjab Agricultural University, Ludhiana, 2020) Karminderbir Kaur; Kumari Neelam
In rice, an in vivo haploid induction system, that can produce both maternal and paternal haploids at promising rates is not available. In the present study, four different experiments were carried out to explore various possibilities of developing an in vivo haploid induction system in rice. Rice X maize and rice X pearl millet crosses were analyzed for pollen tube penetration to study fertilization barriers. A total of 38,468 spikelets were pollinated, but no true caryopsis was observed. Fluorescent microscopy revealed that callose depositions hinder the release of generative nuclei to the ovule. 73 wild species accessions of genus Oryza were re-sequenced for mining alleles of the OsCENH3 gene. Based on the sequence data, 16 haplotypes for OsCENH3 gene were identified. The most consistent variation obtained with respect to Nipponbare reference was SNP A/G at position 69 (codon 23). Phylogenetic analysis of 24 accessions revealed that the O. rufipogon accessions carrying amino acid changes in the N-terminal tail domain region to be closer to O. nivara accessions than they were to OsCENH3 reference. In addition to the wild species, two HFD-region mutants were also identified from a Nipponbare TILLING population. Ros 5783 TILLING mutant showed 50% incidence of seed death upon crossing; indicating it to be a promising haploid inducer candidate. Genome editing was carried out using an M4 specific guide RNA, complemented by three different mutant CENH3 versions, in separate events. The plantlets obtained after transformation with the guide and the complements were genotyped. Cas9 and complementtransgenes were found to be present in four and ten T0 plants respectively. The transformation was successful, but edits could not be detected. Both TILLING mutants and transformed plants are available at PAU for the further crossing to check for haploid induction capabilities.
Open Access
RNAi mediated broad spectrum resistance against begomoviruses of okra (Abelmoschus esculentus L.)
(Punjab Agricultural University, Ludhiana, 2020) Sharma, Nity; Sarao, Navraj Kaur
Okra is an important vegetable crop of Malvaceae family and grown widely in tropical and subtropical areas of the world. Yellow vein, leaf curl and mosaic diseases caused by begomoviruses have emerged as most devastating viral diseases of okra. In the present investigation, RNA interference (RNAi) strategy was employed to control okra-infecting begomoviruses. For this, two hairpin RNAi constructs harbouring overlapping regions of AC1/AC2 and AC1/AC4 genes of DNA-A genome of begomoviruses of okra were generated. Consecutively, agroinfectious clone harbouring dimeric units of whole genome clone of okrainfecting begomoviruses was also generated. Efficacy of three constructs was checked by transient assay and plants having RNAi constructs did not show any symptoms after four weeks of agroinfilteration as compared to plants inoculated with agroinfectious clone. Further, Agrobacterium- mediated tissue culture dependent protocol was utilized to generate transgenic okra plants of variety Punjab 8 expressing AC1/AC2-hp and AC1/AC4-hp RNA. Hypocotyls were used as explants for transformation and a cytokinin named trans-zeatin riboside of 2mg/l concentration was used for regeneration of plantlets. Transgenic plants were assayed for resistance to okra-infecting begomoviruses using agro-infectious clone and viruliferous whiteflies. Nearly 90% resistance against begomovirus infection was observed in transgenic lines when compared with untransformed plants and the plants transformed with empty vectors. Control plants developed severe viral disease symptoms within 4 weeks. Out of eleven plants, one plant expressing AC1/AC4-hp RNAi constructs displayed appearance of milder symptoms after 6 weeks when attacked with viruliferous whiteflies. The resistant transgenic lines accumulated very low titres of viral DNA. Plants thus formed had normal phenotype with no yield penalty in greenhouse conditions.

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