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CRISPR/Cas9-mediated pectate lyase gene editing for enhanced shelf life of tomato (Solanum lycopersicum L.)
(Punjab Agricultural University, 2024) Sumedha; Dr Prashant Mohanpuria
Tomato is one of the most consumed vegetable crops worldwide. Tomato fruits are having an inherently short shelf life which is directly related to fruit softening, caused by cell wall modifying enzymes. Among which pectate lyase (PL) controls fruit softening and thus PL gene was targeted using CRISPR/Cas9 to enhance shelf life of tomato variety Punjab Ratta. In silico study of PL genes from Solanaceous crops including tomato was attempted and three sgRNAs were designed from exon-2 of PL gene. The CRISPR/Cas9 constructs containing sgRNA-1 and 2 were used for transformation of tomato. A new in planta transformation protocol was developed with 10.49 % overall transformation efficiency. Floral buds of 3-8 days of developmental stages were used for dipping in Agro suspension which resulted in setting of 150 fruits. The extracted T0 seeds were selected on kanamycin. A total of 3240 T1 generation plants were regenerated from transgenic tomato fruits (T0), out of which 1620 were found kanamycin resistant which were used for DNA isolation and PCR confirmation of T-DNA integration. The 425 bp PL gene target region from 170 transgenic tomato T1 plants was PCR amplified. Upon sequencing eight T1 tomato transgenic plants were found with PL gene edits containing T insertion, T insertions as well as T substitutions, GC substitution by CG as well as TT insertion downstream to PAM site. PL gene expression and pectin content were decreased in fruits of mutated tomato plants but morphological and biochemical characteristics were more or less similar.
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Identification and characterization of genes for regular and irregular bearing in mango (Mangifera indica L.)
(Punjab Agricultural University, 2022) Harmanpreet Kaur; Sidhu, Gurupkar Singh
The mango, known as the "King of Fruits," is a member of the Anacardiaceae family and is the most important fruit crop grown in India because of its exquisite flavour. Flowering is a crucial phenophase, since it directly impacts the production. Mango flowering, however, is a complicated phenomenon. Typically, it bears a large crop in one year (on year) and produces little to nothing in the following year (off year). The gene expression analysis for flowering genes in regular (Amrapali and Neelum) and irregular (Dashehari) bearing cultivars was done using RNA-Seq. Illumina technology was used to sequence the cDNA libraries made from the leaves, shoot apex, and inflorescence tissues of Dashehari, Amrapali, and Neelum. For Dashehari, Amrapali, and Neelum, paired-end high-quality clean reads of 117 Mb, 74 Mb, and 24 Mb, respectively, were obtained. Dashehari's de novo assembly generated 67,915 transcripts, 25,776 trinity genes, and N50 value of 1,981. The transcripts were annotated using BLAST2GO and PfamScan, and the biological process, molecular function, and cellular component functional categories of GO were used to group the genes. Major pathways include the sucrose and starch metabolism, tryptophan biosynthesis, trehalose biosynthesis, and phenylpropanoid biosynthesis were found by KEGG and Plant Reactome analyses. From the FLOR-ID flowering database, ortholog transcripts were found using reciprocal blast, and 85 genes relevant to flowering were found. In this study, more genes were found to be up-regulated in leaves of Dashehari bearing tree than in non-bearing tree, in inflorescence than in leaf and apex, and in Amrapali than Dashehari among varieties. In particular, genes associated with photoperiod (CO, GI, FTIP1 and FT), vernalization (FRI4 and VIN3), the circadian clock (LHY1, TIC and PRR7), age (TOPLESS and SPL15), and the hormonal pathway (BR1, EIN3, T6P and GA20OX) were identified. Using qRT-PCR, we validated 18 flowering-related genes for regular and irregular bearing in the three genotypes. All the genes demonstrated greater expression values in leaves of Dashehari bearing tree as compared to non-bearing tree and in Amrapali, which is congruent with the expression values revealed from the transcriptome data. These results will help in the discovery of regulatory regions and factors implicated for regularity in mango fruit bearing, and they will establish the foundation for understanding the cellular and molecular mechanisms involved in regular and irregular bearing fruit varieties.
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Mapping Qtl For Heat Tolerance Component Traits In Triticum Durum Desf. X Aegilops Speltoides Tausch. Backcross Introgression Lines And Transfer To Triticum Aestivum L.
(Punjab Agricultural University, 2023) Navaneetha Krishnan J; Chhuneja, Parveen
Wheat, a major cereal crop, is the most consumed staple after rice in India. Frequent episodes of heat waves during the past decade have raised concerns about food security and necessitates the development of heat-tolerant wheat cultivars. A mapping population comprising 315 BC2F10 backcross introgression lines (BILs) was developed by crossing Triticum durum cultivar PDW274 and heat tolerant diploid wild relative Aegilops speltoides accession pau3809 to map QTLs for terminal heat tolerance. The homozygous BILs were evaluated for heat stress tolerance component traits under an optimum environment (OE) and a heat-stressed environment (HE) for the two cropping seasons 2017-18 and 2018-19. Data on spike length, spikelet number per spike, peduncle length, thousand-grain weight, grains per spike, days to heading, days to maturity, grain filling duration, NDVI at heading and plot yield were recorded. Heat tolerance index was calculated for all the traits except plant height and used for QTL mapping. Genotyping-by-sequencing (GBS) of the BILs was carried out, and 3085 polymorphic SNPs were obtained. The QTL mapping for heat tolerance traits detected fifty QTLs on all the chromosomes except 7B. The PVE of the mapped QTLs ranged from 5% to 11.5%, indicating the minor effect contributed by the QTLs on the total phenotypic variability. Several candidate genes reported to play a role in heat stress responses were identified by browsing the 1 Mb physical region flanking the stable QTLs detected under the HE. To introgress the heat tolerance QTLs, three BILs, DS201, DS394 and DS437, which were superior for one or more phenotypic traits, were crossed with the popular bread wheat cultivars PBW723 and PBW725 along with an advanced breeding line CB762 (= PBW550+GpcB1+Yr5). Kompetitive allele-specific PCR (KASP) markers were designed based on the mapped SNPs for targeting the QTLs associated with heat tolerance traits. But these markers failed to differentiate between the donor BILs and the bread wheat parents, making them unfit for marker-assisted selection. Therefore, phenotypic selections were carried out from the BC2F2 generation onwards to select promising lines for important heat tolerance traits. Nineteen BC2F3 lines were selected that performed better than the respective bread wheat parents for one or more of the heat tolerance-associated traits. The selected BC2F3 lines can be advanced further and screened for heat tolerance at multiple locations. A well-developed root system is essential for efficient nutrient and water uptake. The BILs were evaluated for various root architecture traits during the 2019-20 and 2020-21 cropping seasons. The roots were sampled at the maximum tillering stage and data on various root architecture traits namely, total root length, root surface area, root projected area, root volume, average root diameter, number of root tips, number of root forks, number of root crossings and root dry weight were recorded. The QTL mapping for root architecture traits detected twenty-one QTLs for various root architecture traits on chromosomes 1A, 2A, 2B, 3B, 5A and 6B. Scanning of the 1Mb region flanking the mapped SNPs linked to the stable QTLs revealed multiple genes involved in root growth and development. These QTLs can be used in breeding programmes after the development and validation of suitable marker assays.
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Mapping and transfer of higher grain length and multiple rust resistance from T.dicoccoides to cultivated wheat
(Punjab Agricultural University, Ludhiana, 2022) Manpreet Kaur; Satinder Kaur
Wild emmer wheat, Triticum dicoccoides, the progenitor of modern tetraploid and hexaploid wheats, is an important resource of new variability for disease resistance genes and grain yield traits. T.dicoccoides acc. pau14723 showed resistant to leaf and stripe rust races and was crossed with T.dicoccoides acc. pau4663 (susceptible to leaf and stripe rust) for studying the inheritance and mapping the genes for leaf and stripe rust resistance. Recombinant inbred lines were developed and screened against highly virulent Pt and Pst pathotypes at the seedling and adult plant stages. Inheritance analyses revealed that both the rust infections were controlled by dominant major single gene. For mapping these genes, the markers showing diagnostic polymorphism in the resistant and susceptible bulks were amplified on all RILs. The molecular characterization identified the genes to be present on 1A chromosome of wheat (short arm). MapDisto version 1.7.5. Beta 4 software was used to determining the linkage present between the genes governing resistance and the SSR markers Xbarc148, Xbarc240, Xwmc93, Xwmc818. A total map size of 9.7cM was obtained showing no segregation between Lr and Yr genes and the marker lying closest to the resistance genes was Xbarc148 at 1 cM distance from the resistance genes. The variation in the T. dicoccoides for the yield related traits was studied by crossing the accessions having longer and wider grains with the accessions having shorter grains resulting in three different crosses: T.dico.14723 and T.dico. 4663, T.dico.5219 x T.dico. 4663 and T.dico. TA1027 x T.dico. 5232. The RIL populations developed from these crosses were screened for different traits affecting the yield which include 100 grain wt. (100 Gwt.), spike length (SpL), spike length with awns (SpLWA), no. of spikelets/spike (Splts/Sp), grain length (GL), grain width (GW) and grain area (GA). The interaction between the traits and their contribution to grain yield was studied. The molecular and the phenotypic data was statistically analyzed for the significant contribution of marker alleles. The evaluation of allelic effects of the polymorphic markers for each trait using Kruskal–Wallis test showed that the phenotypic differences in the mean value of GL, GA and GWT were statistically significant for the three different classes of TaGASR7-A and TaGASR7-D. The study suggests the use of variation existing in T.dicoccoides germplasm for wheat breeding programmes.
Open Access
Molecular mapping of yellow mosaic virus resistance in bitter gourd (Momordica charantia L.)
(Punjab Agricultural University, Ludhiana, 2021) Gurpreet Kaur; Navraj Kaur
Bitter gourd (Momordica charantia) is a popular cultivated vegetable in India and other countries. The susceptible parent ‘Punjab-14’ and the resistant parent ‘PAUBG-6’ were crossed to obtain F4 mapping population comprising 101 individuals with the objective of mapping yellow mosaic disease resistance in bitter gourd. In the present study, genotypingby-sequencing (GBS) approach was used to develop the genetic linkage map. The map contained 3,144 single nucleotide polymorphism (SNP) markers, consisted of 15 linkage groups and spanned for 2415.2 cM with an average marker distance of 0.7 cM. By adopting the artificial and field inoculation techniques, F4:5 individuals were phenotyped for disease resistance in Nethouse (2019), Rainy (2019) and Spring season (2020). The QTL analysis using the genetic map and phenotyping data identified three QTLs qYMD.pau_3.1, qYMD.pau_4.1 and qYMD.pau_5.1 on chromosome 3, 4 and 5 respectively with phenotypic variation explained (PVE) from 13.5 to 22.1%. In addition, nine horticultural important traits including days to appearance of first female flower, days to appearance of first male flower, days to fruit maturity, node to appearance of first female flower, seed hardness, seed number, fruit length, fruit diameter and fruit weight were evaluated using quantitative data. As a result, twelve QTLs responsible for these traits were also identified. In another experiment, the relative gene expression of begomovirus genes in susceptible and resistant hosts was studied to understand the mechanism of virus resistance. The expression analysis indicated the high expression of AC4 gene in resistant plant provided the information on the efforts of virus to combat the resistance of plant. The information generated in this study is very useful in future for fine-mapping and for marker-assisted selection for these traits.
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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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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.

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