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Open Access
Development of biodegradable films from enzymatically modified cassava starch
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Edwin, K Wilson; KAU; Sajeev, M S
Cassava forms an important food crop in the tropical countries and are rich in starch (20-40%) having desirable physico-chemical and functional properties. Starch and starch derivatives form an important constituent in biodegradable film preparation due to its renewability, abundance availability, low cost, film forming properties, high oxygen barriers, odorless, tasteless, colourless, nontoxic, low solubility, biodegradability etc. But films from the native cassava starch often possess poor physico-mechanical and hydrophobic properties. Hence modified starches by chemical, physical and enzymatic methods offer better scope for the production of biodegradable films which has got wide applications in the food packaging industry. The objectives of the present study was to find out the film forming properties of enzymatically modified cassava starch viz., liquefaction by -amylase and debranching by pullulanase enzymes added with glycerol as plasticiser. Rheological properties were measured in terms of the dynamic mechanical spectra of the film forming solutions viz., storage modulus, loss modulus, phase angle and complex viscosity. Filmogenic solutions based on -amylase was prepared with starch 5%, glycerol 20%, amylase concentration: 100, 200 and 300 μl from the stock solution (0.l ml amylase in 100 ml distilled water), temperature: 80, 85 and 90°C and time: 20, 30 and 40 min for gelatinization. For developing the pullulanase modified starch based films, the starch (5%) was incubated with pullulanase at 2, 3 and 4 units concentrations at 45, 50, 55 0C for 8, 16 and 24 h and the filmogenic solutions added with 20% glycerol were gelatinised at 90 0C for 20 min. Both the experiments were designed using response surface methodology using Box- Behnken design. The physico-mechanical, functional hygroscopic, biodegradation and storage studies of the films were carried out. The dextrose equivalent of the filmogenic solutions varied between 1.6-8.4 with the amylase and 2.2-16.0 with the pullulanase treated starch. The higher values of storage modulus, complex viscosity and low phase angle of the solution containing 153pullulanase treated starch compared to -amylase treated solutions showed that the gel formed during gelatinization of the solution is having more solid nature in their visco-elastic character. The films containing -amylase treated cassava starch showed better whiteness properties. Thickness, moisture content, tensile force, elongation at break and swelling capacity of the films containing pullulanase treated starch was higher than that of the films with -amylase treated starch. The higher solubility of the -amylase based starch films helps easy degradation of the films in the soil whereas offers poor packing ability. Pullulanase film‘s packing ability is better owing to low permeability and solubility. Though the films with both the modified starch is easily biodegradable, pullulalanase took 4 weeks for completely degrade into the soil as evidenced from the soil burial test. The microbial analysis studies showed that the soil in which -amylase treated film buried for degradation had highest bacterial, fungal and actinomycetes population than that of the soils with pululanase treated films. Considering various physical, mechanical and functional properties, the pullulanase modified starch offers better scope for the production of biodegradable packing materials.
Open Access
Development of molecular markers for anthracnose resistance in vegetable cowpea [vigna unguiculata (L.) walp]
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2015) Dolagobinda, Pradhan; KAU; Deepu, Mathew
Cowpea is one of the most important food legume crops in the semi-arid tropics, covering Asia, Africa, Southern Europe and Central and South America. Nigeria is the centre of origin for cowpea and in India, it is exclusively known as a kharif season pulse but the demand of cowpea as vegetable is increasing. In India, cowpea is grown in about 0.5 million ha but productivity is as low as 600-750 kg grains ha-1. All the pole type vegetable cultivars are highly susceptible to anthracnose caused by Colletotrichum lindemuthianum, which is the most destructive disease, not only for cowpea but also in other legumes. Since no effective measures are capable to manage this disease, the losses can be up to 74-100 per cent and hence breeding the resistant cultivars is suggested as the only promising strategy. Molecular marker assisted selection offers precision in the breeding process but so far no markers are identified in this crop for the anthracnose resistance gene. With this background, the study on “Development of molecular markers for anthracnose resistance in vegetable cowpea [Vignaunguiculata (L.) Walp.]” was carried out at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, during August 2013 to June 2015. The objective of the study was to identify an ISSR or RAPD molecular marker linked with anthracnose disease resistance in vegetable cowpea, using bulked segregant analysis to enable marker assisted selection. The dual purpose semi-trailing cultivar Kanakamony, which is reported as immune to anthracnose and the high yielding trailing vegetable type cultivar Sharika, which is susceptible, were used for the development of the mapping population for BSA to identify the marker. The parental populations were grown in separate fields in the month of October 2014 as per the package and practices recommended from KAU. Controlled crosses were made to develop F1 seeds during November and December months. Thirty F1 plants were grown during January - March 2015 and the F2 seeds were developed through selfing. The F2 segregating progenies were raised during May-June, in the pots and maintained in greenhouse. Evaluation of parents with F1 of Sharika × Kanakamony and reciprocal cross for morphological characters revealed that pole type and black seed colour characters of Sharika were dominant over semi-trialing and red colour seed of Kanakamony. To isolate the fungal spores for artificial inoculation on the F2 plants, infected plant parts were collected and initially cultured in 3 different media viz. PDA, NGA and CDA. NGA and PDA medium were selected for artificial inoculation based on the characteristics of fungal spores. Artificial inoculation was initially done on 15 days old seedlings and was repeated after 15 days with an approximate concentration of 106 spores/ml and the highly resistant and susceptible F2 plants were identified for BSA. Good quality DNA was isolated from the parents and 47 RAPD and 43 ISSR primers were screened for their capability to generate polymorphic bands. Subsequently, based on the initial screening, 12 RAPD and 10 ISSR primers were selected for bulk segregant analysis. The bulked segregant analysis was done using the DNA from the resistant parent, susceptible parent, resistant F2 bulk and susceptible F2 bulk. BSA with the RAPD primer OPA 02 revealed a polymorphic band at 850 bp which was present in the susceptible parent and susceptible bulk and absent in the resistant parent and bulk. BSA with ISSR primers UBC 810 and UBC 811 produced polymorphic bands at 1.4 kb and 1.5 kb, respectively, which were present in resistant parent and resistant bulks. The co-segregation analysis has to be performed, the marker has to be characterized and validated for the use in breeding programmes.
Open Access
Identification and characterization of viruses infecting lesser yam (Dioscorea esculenta (Lour.) Burkill)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Sudheer, K S; KAU; Jeeva, M L
The study entitled “Identification and characterization of viruses infecting lesser yam (Dioscorea esculenta ( Lour. ) Burkill )” was carried out at the Division of Crop Protection, ICAR-Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram during 2014-2015 with an aim to identify and characterize the viruses of lesser yam at molecular level and to design virus specific primers for detection. Lesser yam is a vegetatively propagated crop, grown for its high calorific value tubers. Yam viruses causing significant reduction in tuber yield and quality are poorly characterized which is restrict the international exchange of germplasm. Lesser yam leaf and tuber sample with different virus symptoms were collected from the germplasm repository and experimental fields of ICAR-CTCRI. The serological and nucleic acid based methods were employed for the detection of Yam Mild Mosaic Virus (YMMV), Yam Macluravirus and Yam Badnavirus which were reported in other yams from India. The leaf and tuber samples of lesser yam were indexed for YMMV, Yam Macluravirus by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT-PCR) whereas Yam Badnavirus was by triple antibody sandwich enzyme linked immunosorbent assay (TAS-ELISA) and polymerase chain reaction (PCR). Yam Mild Mosaic Virus is the most prevalent virus detected in 74.28% of the samples followed by Yam Badnavirus (42.85%) and Yam Macluravirus (40%). Mixed infections of YMMV-Macluravirus (34.28%), YMMV-Badnavirus (31.42%), Macluravirus-Badnavirus (14.2%) and combination of these three viruses (14.2%) were also observed. Two pairs of novel species specific primers were developed to amplify the partial coat protein gene of YMMV and Yam Macluravirus. After identification, one sample each for YMMV, Yam Macluravirus and Yam Badnavirus were cloned and sequenced. The sequence data was analyzed through BLAST and111 sequence similarity was studied. YMMV has maximum similarity of 86% to Yam Mild Mosaic Virus isolate CN20, complete genome, whereas Yam Macluravirus, 95% to YMCTCRI-01 polyprotein gene and Yam Badnavirus, 99% to Dioscorea bacilliform virus 1 gene for polyprotein, isolate FJ65c De. The result obtained from this study will be useful for indexing the planting materials which helps in production of healthy planting material and germplasm there by helping the farming communities to get potential yield.
Open Access
Molecular characterization of cassava mosaic disease (CMD) resistant varieties and wild relatives of cassava (Manihot esculenta Crantz) using SSR and SNP markers
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Dhanya, O G; KAU; Mohan, C
In the present study an attempt was made to estimate the extent of genetic diversity between 25 CMD resistant, 16 susceptible and seven wild relatives of cassava using 14 SSR and two SNP primers. Out of the 16 primers, nine primers was analysed using PAGE and remaining seven using capillary electrophoresis on genetic analyzer. The primers produced a total of 53 alleles across the 48 cassava accessions. NS198 was found to be highly polymorphic with 6 alleles followed by NS169, SSR36 and SSR39 (5allelles). The two SNP marker analysis on genetic analyzer namely SNPAPX3 and SNP ERF revealed that out of the two peaks generated, one of the peak at a range of 650-690 and 500-530bp respectively was common to all the 48 accessions and the other peak was variable between samples. The dendrogram constructed with 16 primers using UPGMA had four major clusters which clearly distinguished the resistant, susceptible and wild collections of cassava. The close observation made on one of the sub cluster within major resistant cluster revealed the resistant cultivars TME3 and TME4 were closely related with a similarity coefficient of 0.98. Clustering analysis was well supported by PCA, made representation of distinct location of CMD resistant, susceptible and wild relatives of cassava on 2D and 3D dimensions. Comparison of PIC value for all the 16 primers found out the PIC value for individual SSR markers is higher than the individual SNP PIC value at a range of 0.19 - 0.24. SSR PIC values ranged from 0.347 in SSR32 with observed heterozygosity (He) 0.447 to 0.72 in NS198 with He 0.76. Based on the PIC ranges NS198, NS169, SSRY39, RME-1, SSR106 and NS158 were selected as highly polymorphic markers.
Open Access
Genetic diversity analysis and identification of molecular markers associated with leaf blight resistance in taro (Colocasia esculenta (L.) Schott)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Aswathy Anand, A; KAU; Asha Devi, A
The project titled “Genetic diversity analysis and identification of molecular markers associated with leaf blight resistance in taro (Colocasia esculenta (L.) Schott)” was carried out at the Division of Crop improvement, ICAR-CTCRI, Sreekaryam, Thiruvananthapuram, during 2014-15. Thirty Kerala accessions were employed to study the genetic diversity and 12 genotypes comprising 6 susceptible and 6 resistant which were screened in the fields of ICAR-CTCRI was taken for the study. DNA was isolated from all the 30 varieties by a method standardized by Doyle and Doyle, (1987) and later by Sharma et al., (2008) which was slightly modified by Vinutha, K. B. (2014). A purity level of 1.7-2.0 was achieved by most of the genotypes. PCR conditions already standardized in the molecular biology lab of Crop Improvement lab of ICAR-CTCRI were used to amplify the SSR markers. After preliminary screening, 5 primers were eliminated from the study because of its poor amplification profile. 10 primers out of the 15 screened were utilized for PCR. The PCR products were made to run in 6% acrylamide gel (denaturing urea gel) and the bands were studied after staining with silver nitrate. PIC and cluster analysis was done to analyze its diversity. By calculating polymorphic information content, the highest heterozygosity was obtained for the primer uq 97-256 with a value of 0.871 and the lowest was obtained for the primer uq 84-207 with a value of 0.694. Number of alleles per locus ranged from 1.9-4.56 with the maximum alleles shown by uq 97-256 and the minimum showed by uq 84-207 and uq 201-302. The first cluster comprised of the accessions from C-406 to TCR 450. The second cluster consists of C-218 to C-384 and the third large cluster groups most of the genotypes ranging from TCR 424 to TCR-296 and ADS2014-20 has formed an 126 outlier. The third cluster is further subdivided into four sub clusters and the second sub cluster consists of major number of genoytpes and has an outlier C-273. In the next part of the work, 12 sets of DNA was isolated comprising 6 susceptible and 6 resistant varieties each, to identify molecular markers associated with TLB. 13 ISSR primers were tried to identify the same. The product was amplified at 56⁰C. The PCR products were resolved in 2% agarose Out of the 13 primers, 5 of them gave an extra band for resistant varieties. UBC 825 and (AG)₉AC gave an extra band in all the resistant varieties in 685 bp and 808 bp regions, respectively. The primers UBC 827, UBC 817 and UBC 808 gave bands in 5 resistant varieties in the region, 598, 675 and 667 bp, respectively. The dendrogram analysis using UPMGA clustered resistant and susceptible varieties into two different clusters which gave a more promising result. But the SSR primers employed for the same study using the primers Ce1 A08, Ce1 F04, Ce1 CO3, Ce1 F12, Ce1 H12, uq 97- 256, uq 201- 302, uq 73-164, uq 115-71 and uq 88-94 failed to show any specific bands in the expected product size. However, more number of accessions showing different levels of tolerance and susceptibility to TLB as well as more number of markers could lead to the identification of promising markers associated with TLB resistance.
Open Access
Oxidative stress and protein profiling in cassava (Manihot esculenta Crantz) under abiotic stresses
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Sreekuttan, K S; KAU; Ravi, V
The present study an “Oxidative stress and protein profiling in cassava (Manihot esculenta CRANTZ) under abiotic stresses” was conducted during the period 2014- 2015 in the Division of Crop Production, CTCRI (Central Tuber Crop Research Institute) Thiruvananthapuram. The objective of the study was to elict information on the antioxidative enzyme activity, protein profiling, growth parameter and yield parameter of four varieties / genotypes of cassava under irrigated, high temperatures stress (40 o C ±2 o C) and water deficit stress (WDS) conditions and identify marker physiological parameter which can be used for identifying / developing cassava varieties for tolerance to abiotic stress conditions. The study indicated that the plant height of four different cassava varieties were not significantly affected by heat and WDS. The leaf production of four different cassava varieties were significantly affected by both heat and water stress, and the same was maximum in the case of water stress. The leaf retention of four varieties / genotypes were differentially affected by both heat and water stress. There was negligible difference in yield parameters between stress and control condition. However, there was significant difference was yield observed for parameters among the four varieties. The effect of abiotic stress on various physiological parameters like relative water content, chlorophyll and carotenoid content showed significant difference in control plants. Among the given WDS and HTS treatments, the chlorophyll and carotenoid content decreased in all the four varieties. Chlorophyll and carotenoid content were significantly reduced in plants exposed to HTS. Relative water content of leaves varied under both WDS and HTS conditions. A higher RWC was observed in plants exposed to HTS condition compared to WDS condition. Total soluble protein content of leaf and tuber was found to be lower in plants exposed to stress conditions. Total soluble protein in tuber forming roots had increased under HTS conditions. Proline status of plants under stress conditions followed an increasing trend compared to stress conditions. The effect of abiotic stress on various enzymatic antioxidants such asGlutathione Reductase (GR), Superoxide Dusmutase (SOD), Catalase (CAT) and Ascorbate Peroxidase (APX) were studied. Among the four different cassava varieties. Kunguma rose showed maximum GR activity showed an increasing trend in WDS compared to HTS and stress free conditions. Catalase activity of four varieties of cassava plant varied among different stress condition (WDS and HTS) compared to control. The catalase activity was significantly reduced due to stress conditions and. SOD activity increased under stress conditions. The ascorbate peroxidase (APX) activities had significant variation in WDS and HTS compared to control. The AA content in cassava leaves was negligible or below detectable amount under both control and stress conditions. Under HTS condition GR activity increased in H-165 and Sree Athulya alone whereas all the four varieties showed increase GR activity under WDS. Kunguma rose showed maximum SOD activity in HTS whereas Sree Athulya had highest SOD activity in WDS. APX activity was high in all the varieties under HTS. From the present study it is inferred that GR can be used as a biochemical marker for developing WDS tolerant varieties as it showed consistent changes under both WDS and HTS. APX activity can be used as a marker for identifying HTS tolerant varieties. Detailed molecular biochemical and physiological study will be necessary using different varieties for getting appropriate activities as the marker to screen abiotic stress tolerant varieties.
Open Access
In Vitro propagation and identification of molecular markers linked to dwarfness in white yam (Dioscorea rotundata Poir.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Parvathy Harikumar; KAU; Sheela M N
White yam (Dioscorea rotundata Poir) is a leading source calories for over 300 million people in Africa, Asia, parts South America as well as the caribbiean and the South Pacific Island. It is an important tuber cropgrown in Kerala owing to its very high potential and taste. Use of stakes for trailing white yam led to high cost of cultivation limiting the spread of the crop. Recent studies revealed that the most critical problems facing the farmerscultivating yams includes, non availability of adequate staking materials and its high cost that accounts for 30 percent of the cost of cultivation. Hence the present study was carried out with an objective to identify molecular markers linked to genes affecting plant architecture (plant height/dwarfness) in white yam (Dioscorea rotundata Poir.).The study also aims at assessing variability and genetic diversity among white yam genotypes using molecular markers like RAPD, ISSR and SSR and also to standardize in vitro protocol for the rapid propagation of dwarf/bushy white yam. In present study, DNA was isolated from 30 white yam (Dioscorea rotundata Poir.) genotypes having dwarf, semidwarf and tall plant type. Based on preliminary screening, 15 ISSR, 10 SSR and 8 RAPD primers with high polymorphism were selected for the molecular characterization of white yam genotypes. The total number of bands per ISSR primer ranged from 1 ((ACC)6Y) to 10 (UBC 825). UBC 825 that recorded the highest number of polymorphic bands (10) followed by UBC 827 with UBC 864 with nine bands. In the present study, the ISSR primers produced an average of six polymorphic alleles with a mean Hobs and PIC values of 0.738 and 0.707 respectively. Among the ten SSR markers studied, YM15 recorded the maximum number of polymorphic alleles (5) followed by Dab2D06 and YM26 with four alleles. The percentage of polymorphism ranged from 50 (Dab2C05, Dab2E07). The polymorphism information content ranged from 0.3398 (Dab2C05) to 0.7388 (Dab2D06).YM15 also recorded high PIC value of 0.7377. On average the SSR markers recorded Hobs value of 0.5715 and polymorphism information content of 0.4866. Among the eight RAPD primers studied in white yam, OPW -16 was found be the best that produced ten polymorphic bands followed by OPG-02 and OPG-13 with six polymorphic bands. All the primers recorded high PIC value of >0.6. The comparison of the different type of markers (ISSR, SSR, RAPD) studied, showed that the number of polymorphic bands are higher for ISSR and RAPD, the average polymorphism information content was lesser for SSR as compared to RAPD and ISSR primers evaluated. The results implied that the all the different type of markers used were efficient in discriminating the genotypes studied. Cluster analysis done based on Euclidean distance of 30 accessions for ISSR, SSR and RAPD. Dendrogram showed the partition of most of the dwarf and tall genotypes in to two different clusters. Semi dwarf comes under the cluster of dwarf genotypes. The correlation between different clusters was found to be high (>0.5). The cluster data from dendrogram was in agreement with data from cluster analysis after PCA suggesting that most of the tall and dwarf genotypes grouped separately. A preliminary study was also carried out to standardize the micropropagation protocol for the regeneration of plants from nodal explants taken from field. Sodium dichloroisocyanurate solution of 2 per cent for 15 minutes treatment found optimum for surface sterilization and got 76.6% recovery without any microbial contamination, when nodal cuttings collected from field were used as explants. MS medium with 2mg kinetin enhanced rooting compared to other media. Major problem faced during micropropagation is the delay of sprouting and contamination due to systemic microbes. However initial establishment of the explants is very slow and took 82-115 days. Hence further research is needed for hastening the in vitro establishment in white yam. The results indicated the association of three SSR markers (Dab2C05, Dpr3F04, Dab2E07) with dwarfness in white yam. One of the SSR markers Dab2C05 showed unique band of size 1.5Kbp for dwarf genotype. The ISSR primer UBC 836 was also identified as better marker that showed specific band for dwarf plant type. In addition to identifying molecular markers linked to dwarfness, the present study helped to identify a high yielding (8 kg/plant), highly divergent white yam genotype, DR17 that could be used for the genetic improvement of white yam in future.
Open Access
Molecular characterisation of sweet potato ( Ipomea batatas (L) Lam ) accessions and wild relatives using SSR markers
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Amritha, M S; KAU; Mohan, C
Sweet potato (Ipomoea batatas (L.) Lam) is one of the major tuber crops in Asian countries including India, yet the interrelationship and genetic variability among the cultivar and its crop wild relatives remain unclear. The present study utilised 12 simple sequence repeats (SSR) marker for analyse the degree of genetic diversity and relationship within and between 20 sweet potato accessions, 23 wild Ipomoea species and two Merremia species. The SSR primers produced 84 polymorphic alleles within the range of 105-393 bp. That showed an average polymorphism of 94.5%. The average PIC value of 0.67 showed that all the primers were polymorphic in nature. Similarity coefficient based dendrogram and PCA scatter plot clearly differentiated all the cultivars and wild species. This dendrogram helped to identify the close relationship of cultivars with I. triloba and I. trifida at a similarity index of 0.75. The dendrogram based on the SSR molecular data shows high degree of genetic variability within and between hexaploid sweet potato and diploid wild species, whereas lower within the cultivars, which shows that there is a high level of genetic diversity exist within the Ipomoea species. The results supported the current taxonomical position of M. dissecta as Merremia species whereas opposed the taxonomical position of M. vitifolia as Merremia as this study revealed more similarity to Ipomoea than Merremia species. The similarity coefficient varied from 0.37 to 0.96 indicating high variability in the genotypes that were included for the present investigation
Open Access
Development of molecular markers linked to yellow vein mosaic resistance in okra (Abelmoschus esculentus (L.) Moench)
(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2015) Vinutha, J S; KAU; Deepu Mathew
Yellow vein mosaic is the most serious disease leading to 50-94 per cent yield loss in okra. None of the chemical measures are successful to save an infected plant and breeding the resistant varieties is the most accepted strategy in the management of this disease. Molecular breeding through marker assisted selection is more advantageous over the conventional breeding since it will help to assure the presence of the gene in the breeding lines and avoid the selection of pseudo-resistant line. As of now, there are no markers for the identification of YVMV resistance gene in okra. Hence the study was undertaken with the objective to identify ISSR and RAPD markers linked with the gene governing resistance to the yellow vein mosaic virus disease in okra through bulked segregant analysis to enable marker assisted selection. In the present investigation, two okra genotypes namely Parbhani Kranthi (YVMV resistant) and Salkeerthi (YVMV susceptible, early bearing, excellent fruit quality) were used. Field screening of both the parental lines was done simultaneously in the same field during February-May 2014. No artificial inoculation methods were followed since the heavy population of white flies observed in the summer months was sufficient to ensure the disease occurrence and crossing was done between the selected plants (Salkeerthi X Parbhani Kranthi). The seeds were harvested from the crossed plants and subsequently used to raise F 1 population. The raising of F 1 plants was carried out in the month of September- December 2014 and the morphological characteristics and disease response of 180 F 1 plants were recorded. All the F 1 plants were free from disease symptoms. The F 1 plants were selfed to produce F 2 seeds. The F 2 population with 200 plants was field screened during January-April 2015. Seven highly susceptible and 7 highly resistant plants were identified, DNA isolated from each, resistant and susceptible DNAs bulked separately and used for Bulked Segregant Analysis (BSA). For theextraction of good quality DNA, the CTAB method (Doyle and Doyle, 1987) may be modified by avoiding the liquid nitrogen while grinding the plant tissue and additionally washing the DNA pellet with wash buffer to remove the mucilage content. Evaluation of quantitative characters on F 1 in comparison with parental lines showed variation for the traits such as plant height, petiole length, days to flowering, days to first harvest, number of fruits per plant and yield per plant. Two molecular marker systems namely, RAPD and ISSR were employed for identification of markers linked with YVMV resistance. A total of 84 RAPD primers and 82 ISSR primers were initially screened for their ability to amplify the DNA fragments. Out of these, 39 RAPD primers and 24 ISSR primers were selected based on the number of bands and nature of amplification. In BSA, two RAPD primers OPB11 and OPL 18 yielded markers linked with resistance to YVMV. OPB11 produced distinct polymorphic bands of 800, 1000 and 1100 bp sizes whereas, OPL 18 produced polymorphic bands of 1000 and 1100bp. Two ISSR primers ISSR 8 and UBC 873 yielded distinct polymorphic bands in relation to YVMV resistance. The ISSR 8 and UBC 873 yielded the markers at 500 and 900 bp, respectively. Another primer ISSR 22 gave a distinct marker at 1100 bp size, linked to susceptibility to YVMV. Co-segregation analysis was performed using individual DNA of resistant parent, susceptible parent, resistant F 2 and susceptible F 2 using RAPD primers OPB 11, OPL 18 and ISSR primers namely ISSR 8, ISSR 22 and UBC 873. OPB 11 produced three distinct markers in all resistant F 2 individuals. ISSR 8 produced distinct marker of 500 bp in all resistant F 2 individuals. ISSR markers were found to be reproducible and they are recommended for use in marker assisted selection for resistance to YVMV in okra.

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