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Open Access
Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2022) Lavale, Shivaji Ajinath; KAU; Deepu, Mathew
Bitter gourd (Momordica charantia), being a rich source of phytonutrients such as carbohydrates, minerals, vitamins, and other medicinal compounds, has a great importance in healthy dietary habits. Breeders always seek to breed bitter gourd varieties for the traits such as early maturity and high yield. However, limited investigations have been made to identify the genetic loci governing yield related traits. Marker assisted selection (MAS) assures the presence of favourable alleles and fast recovery of recurrent parent genome in the cultivar under improvement. The success of MAS mainly depends on the availability of a marker-dense genetic linkage map locating quantitative trait loci (QTL) for the target traits. The present study “Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)” was carried out during October, 2018 to December, 2021 with the objective to map the quantitative trait loci and to develop chromosome-wise maps for the yield traits in bitter gourd. To develop the mapping population, high yielding bitter gourd cultivar Priyanka (Momordica charantia var. charantia) and a wild bitter gourd accession IC634896 (M. charantia var. muricata), were used as parents. A set of 450 microsatellites were screened for polymorphism using genomic DNA of parents and 47 were found polymorphic. Bitter gourd genome (GenBank acc. no. GCA_013281855.1) was scanned and new hypervariable microsatellites were identified using Genome wide Microsatellite Analysing Tool (GMATo) and named as KAUBG_n where n is a serial number. From the 75 microsatellites identified, 69 were validated through successful PCR amplification and 38 among them were polymorphic between the parents. This led to the development of a set of 85 markers polymorphic between the parents. Crosses were made between the parental lines and hybrids from the cross Priyanka × IC634896 yielded more number of fruits and total fruit produce compared to the reciprocal hybrid. An F2:3 population was developed through single seed descent method from the cross Priyanka × IC634896. A panel of 200 F2:3 plants were evaluated for twenty seven traits, including fruit-, flower-, seed-, vine-, and leaf-related traits, contributing directly or indirectly to the total yield. Wide variation was observed among the F2:3 plants for the traits studied. A group of ninety plants was selected from 200 F2:3 plants such that they represent the variation of the base population. Genomic DNA of these plants were genotyped using 85 polymorphic markers. Genotypic data from the screening of 85 markers in the mapping population were used to generate a linkage map spanning 1287.99 cM distance across eleven linkage groups (LGs) corresponding to eleven chromosomes, using IciMapping software. LG 7 (28 markers) consisted of maximum number of markers followed by LG 2 and LG 9, each having 11 markers. LG 1 had 10 markers whereas LG 3, 4 and 8 had seven markers each. LG 5, 6, 10 and 11 had only one marker each. LG 7 covered maximum map distance of 384.19 cM where LG 8 covered least map distance of 68.58 cM. The genetic map and phenotypic data were used to generate the QTL maps, using Inclusive Composite Interval Mapping (ICIM) method to locate twenty seven traits on Momordica genome. Sixty QTL, including 37 major QTL with LOD values ranging from 3.1 to 15.2, explaining 1.8 to 35.9 per cent of the phenotypic variation were identified for 24 traits, on seven chromosomes. Twenty three QTL were identified for fruit-traits with LOD values ranging from 3.1 to 7.6, explaining 5.5 to 35.9 per cent of phenotypic variation. Thirteen QTL were identified for flower-related traits with LOD value ranging from 3.1 to 15.2, explaining 7.0 to 26.0 per cent of phenotypic variation. Seven QTL each were identified for seed and leaf-related traits with LOD values ranging from 3.2 to 10.8 and 3.5 to 6.5, explaining 5.6 to 26.3 and 3.2 to 15.8 per cent of phenotypic variation, respectively. Ten QTL were identified for vine-related traits with 3.2 to 8.7 LOD values and explaining 1.8 to 17.6 per cent of phenotypic variation. Single marker analysis was performed to identify markers co-segregating with the yield contributing traits. There were 129 hits for the marker-trait association with LOD values more than 3.0, explaining 11.62 to 29.34 per cent of the phenotypic variation. Using the least and best performing F2:3 plants, markers S13, KAUBG_5 and KAUBG_11 were validated for co-segregation with fruit breadth, first pistillate flower node, and number of pistillate flowers and fruits per plant, respectively. This study gives insights into the relative locations of microsatellites and major effect QTL for yield traits in Momordica genome. QTL with shorter marker interval (qFrtL-8-1, qDPF-3-1, qDSF-3-1, qDSF-7-1, qFrtShp-8-1) can be directly used in MAS for improving yield characters. Linkage observed between microsatellites identified in this study with yield traits signifies their importance in further fine mapping as well as marker assisted selection. The linkage map constructed in this study, being the first with microsatellites from Momordica genome, paves the path for comparative and consensus map generation with other marker types. Further, fine mapping using markers within the identified QTL hotspots can lead to possible identification and cloning of genes underlying the yield traits.
Open Access
RNA mediated resistance to Yellow vein mosaic virus in okra
(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2021) Kelkar Vipul Ganesh; KAU; Deepu Mathew
Okra (Abelmoschus esculentus L. Moench, Malvaceae) is one of the leading vegetable crops in hot and humid tropics. Unfortunately, this climate is conducive for many of the pests and diseases. Okra is susceptible to viruses such as Yellow vein mosaic virus (YVMV) and Enation leaf curl virus (ELCV), belonging to the genus Begomovirus (family Geminiviridae). Because of the favourable conditions prevailing in the coastal region, the losses in Kerala state are 60-100%, depending upon the stage of plant growth and the severity of infection. RNAi is one of the promising molecular biology approach against the viral diseases. Keeping the above facts in view, the present study “RNA mediated resistance to Yellow vein mosaic virus in okra” was taken up at the Centre for Plant Biotechnology and Molecular Biology, CoA, Thrissur from September 2017 to May 2021. The high yielding and YVMV susceptible popular okra cv. Salkeerthi was selected for the development of resistance using RNAi mechanism. Total DNA was isolated from the YVMV infected plant and part of the βC1 gene (187 bp) of the virus was amplified using primers VβC1F and VβC1R. Sequence information of PCR product has revealed that the gene is 90-95% identical with the Indian isolates. The βC1 gene sequence was analysed using IDT software and 10 siRNAs were found at three different position (19-44, 34-59, 99-124 bp). Through Restriction Mapper, it was confirmed that the sequence selected for the preparation of sense and antisense strand, do not possess recognition sites for SmaI, HindIII and MauBI restriction enzymes which are present in the pRNAiLIC vector. The output of VSupPred revealed that the fragment does not contain any Viral Suppressor Regions (VSRs), with a high prediction score (0.625). The hairpin RNAi construct harbouring the region of βC1 gene of β satellite of Begomovirus of okra was generated using pRNAi-LIC (CD3-1285) vector. The SmaI digested plasmid produced three fragments, vector backbone (9842 bp), Pdk intron (1641 bp) and ccdB gene (614 bp) and the digested plasmid was treated with dTTP. Product-1 was PCR amplified (215 bp) with VLIC1 and VLIC2 primers, using the DNA from YVMV infected plant as template. Product-2 was PCR amplified (243 bp) with VLIC3 and VLIC4 primers using product-1 as template. Product-1 and product-2 were eluted from the gel and treated with dATP. The dATP treated PCR products and dTTP treated SmaI digested plasmid were mixed together and ligated by incubation at 65ºC for 5 min. followed by 22ºC for 15 min. Ligated product was successfully transformed in competent cells of E. coli (DH5α) and incubated on LB medium containing Kanamycin and Chloramphenicol. Colony PCR was performed, the transformation efficiency was found to be 80%. Plasmid was isolated from the positive DH5α colony and sequenced using the primers VLIC5 and VLIC6. The sequence data had shown that both sense and antisense strands are at right position and direction. Plasmid containing ihpRNA-βC1 cassette was successfully transformed into the competent cells of Agrobacterium (GV3101) and incubated on LB medium containing Kanamycin, Chloramphenicol and Rifampicin. Colony PCR was performed, the transformation efficiency was found to be 100%. Plasmid was isolated from the positive GV3101 colony and sequenced using the primers VLIC5 and VLIC6. Sequence data has further confirmed that both sense and antisense strands are at right position and direction. The ihpRNA-βC1 cassette was successfully transformed into okra cv. Salkeerthi using in planta method of Agrobacterium mediated transformation. The transformation efficiency observed was 11.42% and the transformation was confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5. cDNA was prepared from the total RNA isolated from transformed and control plants. siRNA synthesis was confirmed using the primers VLIC1 and VLIC5 (400bp) and Ubiquitin gene was confirmed using the primer UBQ7 (187 bp). Silencing potential of the RNA interference of βC1 gene and the development of resistance was evaluated by keeping the 15-day old transformed and control plants along with YVMV infected plants inside containment facility, with whiteflies released into insect cage for infection. All the control plants and one transgenic plant have shown the YVMV symptoms after 10 days. Three transgenic plants were healthy with no symptoms. The present investigation was successful in the development of YVMV resistant okra plants carrying ihpRNA-βC1 using pRNAi-LIC (CD3-1285) plasmid vector. The further evaluation is needed in the coming generations for the identification of stable transgenic lines.
Open Access
In vitro synthesis of gingerol and analysis of expressed sequence tags for gingerol production in ginger(Zingiber officinale Rosc.)
(Centre for plant biotechnology and molecular biology,College of Horticulture, Vellanikkara, 2020) Manjusha, Rani; KAU; Shylaja, M R
Black pepper (Piper nigrum L.), often described as the ‘King of spices’ is the most important spice crop, grown for its berries in the world. Indian pepper is preferred across the globe due to its intrinsic qualities. Foot rot is a devastating disease of black pepper. In the changing climate, drought can be a major threat in black pepper production. Hence, the present study was taken up at College of Horticulture, Vellanikkara and ICAR-IISR, Kozhikode to characterise and to identify superior accessions of black pepper for yield, quality and tolerance to biotic and abiotic stresses. Fifty accessions of black pepper in the bearing stage maintained in the National Active Germplasm Site of ICAR-IISR, Kozhikode formed the base material for the study. The accessions were characterised for fifty qualitative and fifty quantitative characters following the descriptor developed by IPGRI (1995). Wide variability was observed among the accessions for ten qualitative characters. Quantitative characters of shoot, leaf, spike and fruit also showed wide variability. Field tolerance to foot rot disease and pollu beetle infestation was observed among the accessions. Twenty accessions were selected from the base collection based on superiority of yield (> 450g green berries/vine) , field tolerance to foot rot disease infection (biotic susceptibility score 1) and pollu beetle infestation (biotic susceptibility score 1-3). They were further evaluated for biochemical principles of quality, tolerance to foot rot disease under artificial inoculation and tolerance to drought by physiological and biochemical analyses. Piperine, essential oil and oleoresin ranged from 3.61 - 6.96 per cent, 3.00 - 5.87 per cent and 7.10 - 11.18 per cent, respectively, across the accessions. The accessions with high value of piperine, essential oil and oleoresin were identified as 7293, 7211 and 7289 respectively. The two accessions identified viz. 7293 and 7252 contained more piperine than the highest of Panniyur 2 (6.6 per cent) reported among the released varieties . Artificial inoculation of selected accessions using Phytophthora capsici culture for screening for foot rot disease resistance based on over all disease severity index of both stem and leaf lesions showed that accession 7259 was moderately resistant. The selected accessions did not exhibit significant variation for various physiological and biochemical parameters at field capacity. However higher value of photosynthesis, chlorophyll content, chlorophyll stability index, relative water content and membrane stability index and low leaf temperature were observed for accessions viz. 7215, 7240, P 5 and 7241 after five days and ten days of moisture stress induction following field capacity compared to other accessions. Higher values of proline, SOD, catalase and peroxidase were also observed for these accessions. The visual scoring showed that accessions with higher values for most of physiological and biochemical parameters of drought tolerance viz. 7215, 7240, P5, and 7241 had lesser number of fallen leaves and more number of leaves retained at permanent wilting point (PWP). The accessions 7215 and 7240 took twenty days to reach PWP compared to eleven accessions which took only 16 days to reach PWP. Foliar nutrition with sulphate of potash, IISR - Power mix and Pink Pigmented Facultative Methylotrophs (PPFM) had positive effect on drought tolerance for the accessions (7215, 7240, P5 and 7241) having natural tolerance. The identified accessions with high yield , quality and tolerance to biotic or abiotic stress can be used for further breeding programme.
Open Access
Development of resistance against banana bract mosaic virus in musa spp. var. grand naine using small interfering RNA (siRNA)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Jadhav Pritam, Ramesh; KAU; Soni, K B
The study entitled “Development of resistance against Banana bract mosaic virus in Musa spp. var. Grand Naine using small interfering RNA (siRNA)” was carried out during 2015-2019 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to develop resistance against Banana bract mosaic virus in banana var. ‘Grand Naine’ using siRNA mediated technology. Embryogenic calli of banana var. Grand Naine were used for agrobacterium mediated transformation of ihpRNA cassette as the embryogenic cells have single-cell origin and do not produce chimeric plants. The rapid and efficient protocol standardised for somatic embryogenesis in Nendran developed in the Department of Plant Biotechnology, College of Agriculture, Vellayani was followed for inducing somatic embryogenesis in banana var. Grand Naine. Immature male flowers from position 0 to 11 were used as explants. Pale white embryogenic callus was initiated within 45 days on Murashige and Skoog (MS) medium supplemented with 6-Benzyladenine (BA) (8 mgL-1) and Thidiazuron (TDZ) (0.6 mgL-1) under dark conditions. The percentage of embryogenic calli obtained was 13.88. For regeneration of somatic embryo, the embryogenic calli were transferred to MS medium supplemented with BA (2 mgL-1) and incubated under light (16 hr), which resulted in 100 per cent germination and plantlet regeneration. The plantlets were transferred to coir pith compost and were hardened for one month in mist chamber. Plants were then transferred to polybags with soil and cow dung (1:1) mixture and kept in shade net house for secondary hardening. An intron hairpin RNA (ihpRNA) vector was constructed to produce small interfering RNA (siRNA) against the coat protein gene of BBrMV. The construct was designed using the partial coat protein gene sequence of BBrMV isolated from the infected banana var. Grand Naine. The coat protein gene was amplified from the cDNA prepared from the total RNA isolated from the infected plants by RT-PCR. For this coat protein gene specific primers were designed using the whole genome sequence of BBrMV retrieved from NCBI GenBank. The partially amplified coat protein gene fragment of 745bp was sequenced and analysed using BLASTn tool for similarity with the sequences of BBrMV deposited in NCBI genome database. Sequence was closely related to BBrMV infecting cardamom with 97.83 percent similarity. The sequence was subjected to miRNA target prediction for selection of the target site to be used in preparation of ihpRNA construct. The sequence that frequently occurred in probable dicer substrate sites was selected. It was then checked for the presence of restriction sites of AscI, PacI, KpnI and SpeI as these sites were the cloning sites of the primary vector, pSTARLING. Based on this information the primers with anchored restriction sites were designed to amplify a fragment of 326bp towards the 5’ end. The sense fragment of coat protein gene (326bp) was amplified with the primers anchored with AscI and PacI sites and the antisense fragment (326bp) was amplified with the primers having KpnI and SpeI sites. The respective restriction sites were anchored at 5’ end of forward primer and 3’ end of reverse primer. The use of two restriction sites helped in proper orientation of sense and antisense strand in ihpRNA construct. The amplified sense and antisense fragments were eluted from agarose gel and cloned in pJET1.2 cloning vector. The cloned fragments were released with sticky ends from pJET1.2 using the corresponding restriction enzymes and integrated in pSTARLING vector flanking the cre intron to favor the formation of the hairpin structure. Presence of the inserts was confirmed by restriction digestion and PCR. The ihpRNA casette in pSTARLING consisted of ubiquitin promoter, ubiquitin intron, sense coat protein strand, cre intron, antisense coat protein strand and termination sequence in the order within the NotI restriction sites. For agrobacterium mediated transformation, the complete cassette was released using NotI and ligated at NotI site within the lacZ gene of the binary vector pART27 containing antibiotic resistance markers nptII and Spec. Integration of cassette within lacZ gene facilitated the selection of the binary vector with the ihpRNA cassette by blue-white screening. The white positive colonies were confirmed with PCR. The binary vector with the insert was transferred to Agrobacterium tumefaciens strain GV 3103 by freeze-thaw method. Transformed colonies were picked and the presence of the vector and the ihpRNA insert was confirmed by PCR and restriction digestion. Embryogenic calli were transformed with A. tumefaciens strain GV 3103 carrying the ihpRNA construct and the transformed embryos were selected under antibiotic pressure (kanamycin 200 mg L-1). Transformed calli were transferred on MS medium containing 2 mg L-1 BA which gave a maximum regeneration of 12 percent. The regenerants were confirmed for the presence of ihpRNA construct using PCR with the primers for insert, npt II and cre intron. The formation of siRNA in the transformed plant was analysed using Northern hybridisation. The small RNAs were isolated from the leaves of transformed plants and separated on 15 percent acrylamide gel containing 7M urea and electroblotted on the positively charged nylon membrane. It was then subjected to hybridization with biotin labelled probes designed against the target site. The small RNAs were detected after DAB staining. The study was successful in developing ihpRNA construct for resistance against BBrMV in Musa spp. var. Grand Naine. This is the first report on development of ihpRNA construct for siRNA mediated resistance against BBrMV. The transgenics developed in this study need to be evaluated for virus resistance by challenging with viruliferous aphids. The technology developed in this study can be applied in other banana varieties also for imparting virus resistance. Compared to other recombinant DNA techniques, RNAi offers specificity and efficacy in gene silencing. Since the small gene fragment used for construct preparation do not code for any protein, this technology does not arise much biosafety concerns. In banana, where traditional breeding for virus resistance is very difficult, this technology is a promising alternative.
Open Access
Isolation, characterisation and evaluation of PINI and BP genes in ralation to inflorescence architecture in black pepper (Piper nigrum L.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Smitha Bhasi; KAU; Swapna Alex
The study entitled “Isolation, characterisation and evaluation of PIN1 and BP genes in relation to inflorescence architecture in black pepper (Piper nigrum L.)” was conducted during 2015-2018 at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram. The objective of the study was to isolate and characterize PIN1 (PINFORMED1) and BP (BREVIPEDICELLUS) genes in black pepper and to evaluate the role of these genes in branching of spikes by studying their differential expression pattern in branching and non-branching varieties of black pepper. Spike samples at their three developmental stages viz. stage-1 (12-15 days after emergence of bud; 1-2 cm length), stage-2 (22-25 days after emergence; 6-8 cm length) and stage-3 (32-35 days after emergence; 9-12 cm length) were collected from bush pepper plants of branching type (Thekken) and non- branching varieties (Panniyur-1, Karimunda and Aimpiriyan) and used for the study. Morphological, histological, molecular (genome, transcriptome and proteome levels) and hormonal analyses were carried out. On morphological analysis of spike characteristics based on IPGRI descriptors, Thekken exhibited six fold increase in number of berries per spike (480) compared to Panniyur-1 (79). Maximum spike length (14 cm) was exhibited by Panniyur-1, and minimum by Karimunda (7.5 cm). Thousand berry weight was maximum for Aimpiriyan (135.6 g) and minimum for Thekken (100 g). Microscopic analysis showed the presence of thick fleshy bracts in Thekken compared to Panniyur-1. Spikelet emergence was noticed at stage-3 in the spikes of Thekken. Histological analysis revealed the presence of a distinct mass of meristematic tissue in stage-1 of Thekken that gradually differentiated during stage-2 and emerged into a spikelet during stage-3. For genome level analysis, DNA isolated by CTAB method was amplified by Polymerase Chain Reaction (PCR) with gene specific primers for PIN1 and BP, designed using Primer-3 software. Two amplicons of size 124 bp and 650 bp were obtained using PIN1 specific primer. Sequence of the 124 bp amplicon on BLASTn analysis revealed 94% identity to PIN1 gene of Canna sp. while the 650 bp amplicon revealed 83% identity to auxin efflux carrier gene of Sorghum bicolor. Amplification of the genomic DNA with primer designed for BP gene produced an amplicon of 145 bp size and its sequence revealed 88% identity to homeobox protein knotted-1 like gene family to which BP belongs. Twenty five per cent variation was noticed between the PIN1 sequences of Thekken and Panniyur-1, whereas only five percent variation could be noticed between BP sequences. For transcriptome level analysis, cDNA reverse transcribed from total RNA isolated using Trizol method was used. Differential expression analysis was carried out using Real Time PCR. The expression pattern of PIN1 was similar in both Thekken and Panniyur-1. However, the expression levels were significantly higher in all stages of Thekken with maximum (14 fold) at stage-1. The expression pattern of BP varied in Thekken compared to Panniyur-1. It showed twenty seven fold over expression in stage-1 and three fold down regulation in stage-2 of Thekken compared to Panniyur-1. Rapid Amplification of cDNA Ends (RACE) of the 124 bp amplicon of PIN1 generated two fragments of size 280 bp and 580 bp. Protein domain analysis of the fragments revealed the presence of zinc finger domains. Proteome analysis using SDS Polyacrylamide gel electrophoresis (PAGE) showed unique protein fragments in all the stages of inflorescence in Thekken (100 KDa fragment in stage-1 and stage-2; 48 KDa and 55 KDa fragments in stage-3). Hormonal analysis of auxin using High Performance Liquid Chromatography (HPLC) revealed that IAA content in Thekken (25 ppb) was one-fourth compared to Panniyur-1 (90 ppb) and one-third compared to Karimunda (60 ppb). Enzyme Linked Immuno Sorbant Assay (ELISA) showed an increase in total cytokinin content in Thekken from stage-1 (2.8 ng/g) to stage-2 (3.5 ng/g), in contrast to non-branching varieties (3.6 ng/g to 3.0 ng/g). The present study is the first report on the isolation and characterisation of coding sequences of PIN1 and BP genes in black pepper. The fragments of PIN1 and BP genes isolated were deposited in the NCBI database. The 580 bp PIN1 sequence revealed numerous zinc finger domains similar to PIN1 gene of Arabidopsis indicating similar function. PIN1, being an auxin efflux carrier, its overexpression noticed in Thekken at the transcriptome level correlated with the lower auxin content in the hormonal analysis. The twenty seven fold expression of BP noticed in Thekken correlated with the increase in cytokinin content and the emergence of a differentiated meristamatic tissue that was prominent in the histological sections of stage-2 of Thekken. The unique proteins of sizes 100 KDa, 48 KDa and 55 KDa in Thekken in proteome analysis suggest the presence of novel proteins in Thekken. The differential expression of PIN1 and BP genes revealed in the present study indicates the significant role of these genes in inducing the spike branching trait in Thekken.

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