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Open Access
DEVELOPMENT AND VALIDATION OF EST DERTVED SSR MARKERS WITH FUNCTIONAL RELEVENCE TO BIOTIC AND ABIOTIC STRESS RESPONSES IN GROUNDNUT 1980
(JAU,JUNAGADH, 2015-03) Bosamia Tejas Chimanlal; Dr. Radhalcrishnan T.
Availability of polymorphic markers in a crop species is a prerequisite for genetic improvement through marker-assisted selection, construction of high density genetic linkage map and mapping of Quantitative Trait Loci (QTL). Simple sequence repeat (SSR) markers are widely employed in molecular breeding programmes and thus are needed to be developed and validated. Conventional methods for developing SSR markers are laborious and expensive. The alternative and cost effective way is to explore the existing public databases harboring abundant number of genomic and genie (expressed sequence tag, EST) sequences for the development of new SSR markers. The SSR markers developed from ESTs leverages functional aspect of transcripts and thus associated with its function. Insufficient number of polymorphic molecular markers is an obstacle for current molecular breeding research in groundnut (Arachis hypogaea L.), an important legume grain as well as oilseed cash crop of the semi-arid tropics. It becomes exceedingly essential to develop more number of polymorphic SSR markers for potential use in groundnut improvement. Therefore, the present study was carried out to develop EST-SSRs from publically available groundnut EST resources. In the present study, a total of 178490 EST sequences of Arachis hypogaea ^ere downloaded from NCBI public databases and pre-processed for assembling. In order to find novel non-redundant EST-SSR markers, 7170 publically available SSR primer sequences were subjected to sequence similarity search with the downloaded EST sequences. A total of 23696 (13.28%) very similar sequences were excluded and the remaining non-redundant sequences (154794) were further pre-processed for removai of low corapiexity sequences, poiy A and poly T tail and vector sequences. A total of 138628 high quality sequences were assembled using TGICL software, which yielded 16424 unigenes comprising of 13428 (81.76%) contigs and 2995 (18.24%) singletons with an average length of 857 bp and N50 value of 942 bases. The assembly by TGICL software reduced the redundancy by 82.21% A total of 2784 sequences were indentifled from unigenes which harbored 3373 SSR motifs The number of sequences harboring more than one SSR was 487 (17.49%) and 289 (10.38%) sequences comprised of compound SSRs. The avemge frequency of SSR was found to be one in 4.17 kb which was relatively higher frequency than previous reports in groundnut.
Open Access
DEVELOPMENT AND VALIDATION OF EST DERTVED SSR MARKERS WITH FUNCTIONAL RELEVENCE TO BIOTIC AND ABIOTIC STRESS RESPONSES IN GROUNDNUT 1982
(JAU, JUNAGADH, 2015-03) Bosamia Tejas Chimanlal; Dr. Radhalcrishnan T.
Availability of polymorphic markers in a crop species is a prerequisite for genetic improvement through marker-assisted selection, construction of high density genetic linkage map and mapping of Quantitative Trait Loci (QTL). Simple sequence repeat (SSR) markers are widely employed in molecular breeding programmes and thus are needed to be developed and validated. Conventional methods for developing SSR markers are laborious and expensive. The alternative and cost effective way is to explore the existing public databases harboring abundant number of genomic and genie (expressed sequence tag, EST) sequences for the development of new SSR markers. The SSR markers developed from ESTs leverages functional aspect of transcripts and thus associated with its function. Insufficient number of polymorphic molecular markers is an obstacle for current molecular breeding research in groundnut (Arachis hypogaea L.), an important legume grain as well as oilseed cash crop of the semi-arid tropics. It becomes exceedingly essential to develop more number of polymorphic SSR markers for potential use in groundnut improvement. Therefore, the present study was carried out to develop EST-SSRs from publically available groundnut EST resources. In the present study, a total of 178490 EST sequences of Arachis hypogaea ^ere downloaded from NCBI public databases and pre-processed for assembling. In order to find novel non-redundant EST-SSR markers, 7170 publically available SSR primer sequences were subjected to sequence similarity search with the downloaded EST sequences. A total of 23696 (13.28%) very similar sequences were excluded and the remaining non-redundant sequences (154794) were further pre-processed for removai of low corapiexity sequences, poiy A and poly T tail and vector sequences. A total of 138628 high quality sequences were assembled using TGICL software, which yielded 16424 unigenes comprising of 13428 (81.76%) contigs and 2995 (18.24%) singletons with an average length of 857 bp and N50 value of 942 bases. The assembly by TGICL software reduced the redundancy by 82.21% A total of 2784 sequences were indentifled from unigenes which harbored 3373 SSR motifs The number of sequences harboring more than one SSR was 487 (17.49%) and 289 (10.38%) sequences comprised of compound SSRs. The avemge frequency of SSR was found to be one in 4.17 kb which was relatively higher frequency than previous reports in groundnut. Regarding functional annotation, 2784 unigenes harboring SSR motifs were subjected to BIast2G0, an online tool for functional annotation. Among the 2784 un,genes, 2027 (72.81%) unigenes were annotated and assinned f . ontology (GO) temrs (4124) were ascribed to unigenes and categorizeriUrr components (391), molecular functions (1120) and hiol • , "^^I'tilar ntaximum homology of groundnut unigenes through aiyclne n,ax (48.2%) followed by Cto orterimm, (16.6o/„). and the sequences of rep^ ml^C rl'XT'''' motifs were categorized in to class I flonap tu ^ (^^-47%) SSR motifs in to short length range class II a' ^ <«»"%) SSR were the most predominant with 33.86% foUowel 77 "-ofifs Tetra-, penta- and hexa-nucleotide repeats meas L (27-51%). r ~AAT/ATT (4.80%) and 1^1" ^^<='ATG (fss^ assembly. The searrvi, ^ repeats wert^ i ~ -* '■ i. .N. ..z,Pnmers were designed hv BatchPrimer3 tool and 2456 • 2784 SSR com • • Pnmers were designed A ^'"^"8 sequences by 5>ica. A set of . 366 pr,mer pairs with functional relevance to biotic and abiotic stresses were selected, synthesized and screened for polymorphism in eleven genotypes which represents the parents of six mapping populations. A set of 339 (92.62%) primer pairs yielded scorable amplicons and 39 (10.66%) primer pairs showed polymorphism among eleven parental genotypes. The 339 primers detected 2 to 12 alleles with an average of 3.77 alleles per marker where as among the 39 polymorphic primers yielded 2 to 12 alleles with an average of 5.10 alleles per marker. The Polymorphic Information Content (PIC) value for these 39 polymorphic markers ranged form 0.028 to 0.375 with an average of 0.325 per marker. The polymorphism screening indicated that compound SSRs were the most predominantly polymorphic (26.09%) followed by di-nucleotide (13.34%) and tri-nucleotide (11.72%) motifs. Among the SSR motif repeats, di-nucleotide repeat SSR markers showed higher PIC value (average 0.357 per marker) followed by penta (average 0.335 per marker) and compound (average 0.332 per marker). The newly designed novel SSR markers have added to the repository of groundnut genomic resources utilizing almost all the available ESTs in public databases as on available till date. The EST-SSR markers showing polymorphism in the parental genotypes could be further screened for polymorphism on a panel of mapping population segregating for biotic or abiotic stress for finding markers linked with that trait. These EST-SSRs could also be effectively utilized for saturation of genetic map or transcript map, QTL mapping and marker-assisted selection for cultivated groundnut.
Open Access
BIOCHEMICAL AND PHYSIOLOGICAL ANALYSIS AND PROTEIN PROFILING IN WHEAT {Triticum aestivum L.) GENOTYPES UNDER HEAT STRESS 1939
(JAU, JUNAGADH, 2014-10) Hitesh Ranchhodbhai Raman!; Dr. M. K. Mandavia
The present experiment on "Biochemical and Physiological Analysis and Protein Profiling in Wheat {Triticum aestivum L.) Genotypes under Heat Stress" was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh. The experiment-1 was carried out using fourteen wheat genotypes and four heat treatments using Factorial CRD design, where seeds were grown in germination bag filled with soil for 10 days. The seedlings were subjected to control and heat treatments at 35°C, 40°C and 45°C for four hour and samples were analysed for relative water content, membrane stability and injury, lipid peroxidation, hydrogen peroxide content and chlorophyll stability index. Heat tolerant genotype GW-190 genotype showed highest membrane stability and relative water content and lowest membrane injury, lipid peroxidation and hydrogen peroxide compared to other genotype so it was selected as best heat tolerant genotype. Heat susceptible genotype J-2010-11 showed lowest membrane stability (MS) and relative water content and highest membrane injury, lipid peroxidation and hydrogen peroxide content so it was selected as highly susceptible genotype. Above physiological and biochemical parameters may be used for screening the susceptible and tolerant wheat genotypes against heat stress. H2O2 and MS are more effective indicators for screening heat tolerant genotypes under stress condition. From results of the experiment-1, one heat tolerant (GW-190) and heat susceptible (J-2010-11) wheat genotypes were selected and the plants were subjected to two groups; control and heat treatments where 40°C and 45°C heat treatments given for 2 h and 4 h of duration and physiological, biochemical, antioxidant enzyme activities, Isoenzymes and Protein profiling by 2D electrophoresis analysis were performed. Relative water content and membrane stability were found to be higher in heat tolerant genotype GW-190 compared to heat susceptible genotype J-2010-11 at tillering and grain filling stages. As heat stress and duration of heat stress increased the relative water content and membrane stability of heat tolerant and heat susceptible genotypes were decreased at both the stages of wheat development. Compared to tillering stage, relative water content and membrane stability were found lower in grain filling stage because of increased temperature. Protein, Proline and glycine betaine, Glutathione reductase, Peroxidase and Superoxide dismutase acitivities were found to be higher in heat tolerant genotype GW-190 compared to heat susceptible genotype J-2010-11 at tillering and grain filling stages. As heat stress and duration of heat stress increased, the biochemical constitutes and antioxidant enzymes activities of heat tolerant and heat susceptible genotypes also increased at both the stages of wheat development. As well all the biochemical constitutes and all three antioxidant enzymes activities were found to be higher in tillering stage compared to grain filling stage. At tillering stage, in case of Peroxidase, band No. 5 (Rm= 0.289) and band No. 6 (Rm=0.495) were present only in heat tolerant genotype while it was absent in heat susceptible genotype. At grain filling stage, band No. 3 (Rm=0.160) was present only in heat tolerant genotype. In case of Superoxide dismutase at tillering stage all the bands were present in heat tolerant as well in heat susceptible genotypes. At graing filling stage, band No. 2 (Rm—0.072) was present only in heat tolerant genotype. So isoenzymes may be useful for screening the heat tolerant and heat susceptible genotypes. At tillering stage, more total protein spots (1207) were recorded compared to that of (972) spots at grain filling stage. Compared to control, in heat stress condition expression of spots were increased. This was not true for grain filling stage. At tillering stage, highest numbers of protein spots (207) were found at 45°C for 4h duration in heat tolerant genotype GW-190 while it was true (148) spots at 40°C for 2h duration in heat tolerant genotype GW-190 at grain filling stage. The protein spots showed the differential expression pattern in treated heat tolerant genotype might be responsible for the stronger heat tolerance. Scanning electron microscopy of wheat leaves showed that analysis of variance indicated significant differences for stomatal length existed among heat tolerant and susceptible genotype as well significant differences were found for stomatal width among heat tolerant and susceptible genotype. Total 12 Operon series RAPD primers were amplified to generate the 105 fragments. The percent polymorphism obtained for RAPD primers were ranged from 71.4% to 100% with an average value of 92.33% per primer. Subcluster A1 (b) of cluster-1 consisted of only one heat tolerant genotype J-2010-06 with more than 66% of similarity. Subcluster A2 of cluster-I consisted of only one heat susceptible genotype J-2010-13 similarity of more than 85%. Cluster-II consisted of two genotypes J-2010-05 and GW-190 sh " similarity of more than 85% that belongs to heat tolerant groups. These primers be used to screen the genotypes against heat stress.
Open Access
BIOCHEMICAL AND MOLECULAR ANALYSIS OF CMS, MAINTANER AND RESTORER LINES IN PEARLMILLET (Pennisetum glaucum (L.) R.Br.) 1725
(JAU,JUNAGADH, 2013-02) Jogia Zeel Vinodray; Dr. R. B. Madariya
Pearlmillet (Pennisatum. glauccum [L.] R.Br.), commonly known as pearl, cat tail, spiked millet is a member of the poaeeae family and has a relatively small diploid genome (2n=2x=14) with a DNA content of lC=2.36pg (Martel et al, 1997). It is the staple food and fodder crop of millions of people living on the most marginal agricultural lands of sub-Saharan Africa and the Indian subcontinent. The present investigation on "Biochemical and molecular analysis of CMS, maintaner and restorer lines in pearlmillet [Pennisetum glaucum (L.) R.Br.)" was planned with three main objectives, (1) To study molecular markers (ISSR and SSR) in different pearl millet CMS, maintaner and restorer lines. (2) To study the protein profiling of different pearl millet CMS, maintainer and restorer lines. (3) To study isoenzymatic patterns in different pearl millet CMS maintainer and restorer lines. Ten ISSR primers produced 31 allelcs with 95.5% polymorphism with an average of 2.8 alleles per primer. 100% polymorphism was m'>'T
Open Access
“MOLECULAR AND ISOENZYMIC CHARACTERIZATION OF INDIAN BEAN (Lablab purpureus L.) GENOTYPES”
(jau,junagadh, 2018-08) Mr. Dholakia Hemang P.; Dr. D. R. Mehta
The present experiment was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives to analyze molecular diversity of 20 Indian bean genotypes using various molecular markers (RAPD, ISSR, and SSR) and to examine the isoenzymes pattern of Indian bean genotypes by polyacrylemide gel electrophoresis. The genomic DNA was isolated from ten to twelve days old seedlings of 20 Indian bean genotypes and their purity was in the range of 1.7 to 2.08. Thirteen RAPD primers generated total of 78 polymorphic bands/alleles in showing 100% polymorphism. The average band per primer was 6.00. The polymorphic information content (PIC) ranged from 0.58 (OPA-09 and OPA-17) to 0.89 (OPA-20). Similarly RAPD primer index (RPI) ranged from1.00 to 10.56 with an average of 4.52 per primer. The highest RPI value was obtained by OPB-04 and the lowest was obtained by OPA-09. Jaccard’s coefficient of similarity of 20 Indian bean genotypes ranged from 10.0 % (between GP- 155 and GP-159) to 62.2% (between GP-157 and GP-161). The phylogenetic tree constructed by UPGMA method generated two main clusters which was again subgrouped in their respective sub-clusters. Ten ISSR primers produced 61 polymorphic bands/alleles showing 100 % polymorphism with an average of 6.10 bands per primer. The polymorphic information content ranged between 0.49 (ISSR-888) and 0.88 (Oligo-03). Likewise, ISSR primer index (IPI) ranged from 1.96 to 8.8 with an average of 4.54 per primer. The maximum IPI value was obtained by Oligo-03 and the minimum was obtained by UBC-888. Jaccard’s coefficient of similarity between 20 Indian bean genotypes ranged from 3.80 % (between GP-158 and GP-165) to 57.8 % (between GP-153 and GP-154). The cluster analysis of ISSR revealed two main clusters, which was further divided into various sub-clusters. Total 20 Indian bean genotypes were subjected to SSR analysis using 10 SSR primers. The polymorphic information content ranged between zero and 0.75. The highest PIC value of 0.75 was noticed in GATS911 AF483842, while lowest PIC value of zero was noticed in BM-142 and PVgaat001 with an average of 0.14 per primer. Likewise, SSR primer index (SPI) ranged from zero to 4.80 with an average of 1.71 per primer. The maximum SPI value was obtained by AGB-8 and the minimum was obtained by BM-142 and PVgaat001. Jaccard’s coefficient of similarity between 20 Indian bean genotypes ranged from 57.1 % (between GP-140 and GJIB-11) to 96.4 % (between GJIB-2 and GJIB-11). The cluster analysis of ISSR revealed two main clusters, which was further divided into various sub-clusters. The pooled study of molecular markers through RAPD, ISSR and SSR was done to confirm the differences and similarity among 20 Indian bean genotypes. Jaccard’s similarity coefficient and UPGMA method showed the highest (66.3%) similarity between GP-157 and GP-161 and the lowest (21.8%) similarity between GP-140 and GJIB-2. The cluster analysis on pooled basis revealed two main clusters, which was further divided into various sub-clusters. Among the studied techniques, RAPD and ISSR primers gave 100 % polymorphism. However, more number of polymorphic fragments, more PIC and higher percentage polymorphism per primer were amplified by RAPD and ISSR as compared to SSR markers. Both RAPD and ISSR markers gave distinct clustering patterns. Isoenzymes were used for the characterization of 20 Indian bean genotypes. Peroxidase generated two polymorphic bands at 15 DAG with Rm value of 0.841 and 0.856. Four polymorphic bands of esterase isoenzyme were detected at 15 DAG with Rm value of 0.146, 0.205, 0.291, and 0.593. Two polymorphic bands were generated though Polyphenol oxidase isoenzyme at 15 DAG with Rm value of 0.261 and 0.535.
Open Access
“MOLECULAR AND ISOENZYMIC CHARACTERIZATION OF INDIAN BEAN (Lablab purpureus L.) GENOTYPES”
(JAU,JUNAGADH, 2018-08) Mr. Dholakia Hemang P.; Dr. D. R. Mehta
The present experiment was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives to analyze molecular diversity of 20 Indian bean genotypes using various molecular markers (RAPD, ISSR, and SSR) and to examine the isoenzymes pattern of Indian bean genotypes by polyacrylemide gel electrophoresis. The genomic DNA was isolated from ten to twelve days old seedlings of 20 Indian bean genotypes and their purity was in the range of 1.7 to 2.08. Thirteen RAPD primers generated total of 78 polymorphic bands/alleles in showing 100% polymorphism. The average band per primer was 6.00. The polymorphic information content (PIC) ranged from 0.58 (OPA-09 and OPA-17) to 0.89 (OPA-20). Similarly RAPD primer index (RPI) ranged from1.00 to 10.56 with an average of 4.52 per primer. The highest RPI value was obtained by OPB-04 and the lowest was obtained by OPA-09. Jaccard’s coefficient of similarity of 20 Indian bean genotypes ranged from 10.0 % (between GP- 155 and GP-159) to 62.2% (between GP-157 and GP-161). The phylogenetic tree constructed by UPGMA method generated two main clusters which was again subgrouped in their respective sub-clusters. Ten ISSR primers produced 61 polymorphic bands/alleles showing 100 % polymorphism with an average of 6.10 bands per primer. The polymorphic information content ranged between 0.49 (ISSR-888) and 0.88 (Oligo-03). Likewise, ISSR primer index (IPI) ranged from 1.96 to 8.8 with an average of 4.54 per primer. The maximum IPI value was obtained by Oligo-03 and the minimum was obtained by UBC-888. Jaccard’s coefficient of similarity between 20 Indian bean genotypes ranged from 3.80 % (between GP-158 and GP-165) to 57.8 % (between GP-153 and GP-154). The cluster analysis of ISSR revealed two main clusters, which was further divided into various sub-clusters. Total 20 Indian bean genotypes were subjected to SSR analysis using 10 SSR primers. The polymorphic information content ranged between zero and 0.75. The highest PIC value of 0.75 was noticed in GATS911 AF483842, while lowest PIC value of zero was noticed in BM-142 and PVgaat001 with an average of 0.14 per primer. Likewise, SSR primer index (SPI) ranged from zero to 4.80 with an average of 1.71 per primer. The maximum SPI value was obtained by AGB-8 and the minimum was obtained by BM-142 and PVgaat001. Jaccard’s coefficient of similarity between 20 Indian bean genotypes ranged from 57.1 % (between GP-140 and GJIB-11) to 96.4 % (between GJIB-2 and GJIB-11). The cluster analysis of ISSR revealed two main clusters, which was further divided into various sub-clusters. The pooled study of molecular markers through RAPD, ISSR and SSR was done to confirm the differences and similarity among 20 Indian bean genotypes. Jaccard’s similarity coefficient and UPGMA method showed the highest (66.3%) similarity between GP-157 and GP-161 and the lowest (21.8%) similarity between GP-140 and GJIB-2. The cluster analysis on pooled basis revealed two main clusters, which was further divided into various sub-clusters. Among the studied techniques, RAPD and ISSR primers gave 100 % polymorphism. However, more number of polymorphic fragments, more PIC and higher percentage polymorphism per primer were amplified by RAPD and ISSR as compared to SSR markers. Both RAPD and ISSR markers gave distinct clustering patterns. Isoenzymes were used for the characterization of 20 Indian bean genotypes. Peroxidase generated two polymorphic bands at 15 DAG with Rm value of 0.841 and 0.856. Four polymorphic bands of esterase isoenzyme were detected at 15 DAG with Rm value of 0.146, 0.205, 0.291, and 0.593. Two polymorphic bands were generated though Polyphenol oxidase isoenzyme at 15 DAG with Rm value of 0.261 and 0.535. Key words: Indian bean, RAPD, ISSR, SSR, Isoenzymes.
Open Access
“MOLECULAR CHARACTERIZATION OF GARLIC (Allium sativum L.) GENOTYPES DIFFER IN TOTAL SOLUBLE SOLID CONTENT”
(JAU,JUNAGADH, 2018-07) Ms. UmaretiyaVidisha R.; Dr. G. V. Marviya
The present investigation on “Molecular characterization of garlic (Allium sativum L.) genotypes differ in total soluble solid content” was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives toanalyze different garlic genotypes for proximate and total soluble solid content, molecular diversity based on total soluble solid content using various PCR based molecular markers viz. Random Amplified Polymorphic DNA(RAPD), Inter Simple Sequence Repeats (ISSRs) and Simple Sequence Repeats (SSRs) as well as to find out the phylogenic relationship among different garlic genotypes. Twenty one genotypes were selected for study based on their total soluble solid content. Among twenty one genotypes, 10 had high soluble solid content and 11 were with low soluble solid content and mean values ranged between 46.17°Brix (RGP-270) to 36.43°Brix (RGP-474). The highest value for total sugar content and reducing sugar content were observed in the genotype with RGP-1 with 6.66 and 4.46 mg %, respectively while, the lowest values for total sugar content and reducing sugar content were recorded in the genotype RGP-7 with 2.19 and 1.05 mg %, respectively. Non-reducing sugar content was ranged between 1.14 mg % (RGP-7 and RGP-224) to 4.01 mg % (RGP-270). True protein and crude protein were observed maximum with 5.39 mg % and 10.43% in the genotypes RGP-224 and RGP-585, respectively while, minimum values for these both were observed with 1.11 mg % (RGP-513) and 6.83 % (RGP-3), respectively. Dry matter was varied between 44.14% (RGP-513) to 35.61% (RGP-278). Total moisture content was observed between 64.38% (RGP-278) to 55.86% (RGP-513). Ash content was ranged from 6.92% (RGP-3) to 3.05 % (RGP- 491). Maximum crude fat content was recorded in the genotype RGP-114 with 0.592% while, the minimum (0.112%) was in the genotype RGP-276. Abstract Total 15 RAPD primers generated 84 bands in which all 84 bands were polymorphic having 82 shared and 2 unique bands with an average of 5.6 bands and 100% polymorphism per primer. The RAPD primers augmented fragment size ranged from 128bp in primer OPP-05 to 3781bp in primer OPO-07. The 18 ISSR primers engendered 87 bands in which all 87 bands were polymorphic with 84 shared and 3 unique bands and had 100% polymorphism with an average of 4.83 bands per primer. The amplified fragments were in range of 158bp in UBC-872 to 4757bp in UBC-824. No any RAPD or ISSR primers gave monomorphic band. Total 12 SSR primers generated the 13 fragments in which all bands were polymorphic shared except primer SSR-13, which gave only one monomorphic band with 91.66% polymorphism with an average of 1 band per primer. Size of SSR primer amplified fragments were in the range of 140bp in SSR-13 to 616bp in SSR-14. An average polymorphism information content (PIC) value for RAPD primer, ISSR primer and SSR primer were 0.77, 0.69 and 0.04, respectively. Primer index for RAPD, ISSR and SSR were 4.35, 3.52 and 0.08 respectively. The similarity coefficient of clusters analysis was ranged from 21 to 49 % for RAPD, 44 to 60% for ISSR and 49 to 75% for SSR. Dendrogram construction of all these molecular markers showed that in RAPD and ISSR, RGP-602 genotype of garlic was the most diversified genotype observed having alone separate position in cluster- II. In SSR marker, RGP-560 and RGP-607 was diversified genotype in cluster-II, which was the same genotype observed as most diversified with SSR markers. The pooled analysis study of RAPD, ISSR and SSR generated clustering pattern which was to be similar as RAPD and ISSR clustering pattern. The data generated from the present study may be useful for the identification of the genetic diversity among garlic genotypes which could be further utilized for molecular breeding and marker assisted selection in crop improvement programs. Key words: Garlic, Genetic diversity, Molecular markers, Proximate parameters.
Open Access
“DIFFERENTIAL GENE EXPRESSION OF AQUAPORIN FOR SALINITY STRESS IN GROUNDNUT”
(JAU,JUNAGADH, 2018-06) JOSHI MEERABEN K.; Dr. G. V. MARVIYA
The present investigation was carried out at the Department of Biotechnology, Junagadh Agricultural University, Junagadh to study the differential gene expression of Aquaporin in salinity stress in groundnut (wild type – Arachis duranensis and GG 20 cultivated variety). Peanut or groundnut (Arachis hypogaea L.) is a species in the legume or "bean" family and native to South America, Mexico and Central America. The groundnut is herbaceous, annual type of plant. Groundnut is an important food and cash crop for resource-poor farmers. It is primarily grown for edible oil (48–50%) as well as for direct consumption by people. Salinity affects at almost all the growth stage of groundnut, i.e. from emergence to maturity. So, salinity in soil during sensitive (critical) stages (flowering, pegging and pod formation) reduces final yield and oil content. The present investigation was carried out with an aim to study the candidate gene expression between control and salinity stressed tissues of wild type Arachis duranensis and GG 20 variety. Variety GG 20 and Arachis duranensis were selected for this study. Arachis duranensis is a wild herb. Wild peanut are genetically diverse and were selected throughout evolution to a range of environments constituting therefore, it is an important source of allelic diversity for abiotic stress tolerance. GG 20 is cultivated in entire Gujarat except North Gujarat. It has high oil content and is high yielding variety. Salt stress was imposed by irrigating both the cultivars with sea water with ECe 4 dS m-1, ECe 6 dS m-1and ECe 8 dS m-1 after 19 days after treatment. Study was conducted with two main objectives. First was to study physio-biochemical changes in control and stressed condition. Second was to determine differential Aquaporin expression during salinity stress through real time PCR. The RNA concentration in the range of 24.9 – 63.0 µg/ml was selected for the study. cDNA synthesis was carried out for differential gene expression of Aquaporin genes. Retrieving of sequences was carried out by searching conserved domain of Aquaporin membrane protein in the genome of Arachis duranensis and primers were designed using online tool batch primer-3 by selecting the generic option. Real time assay was performed for 11 aquaporin genes at 8 ECe salinity level. 19 days salinity stress with constant volume of sea water (8 ECe) showed differential gene expression. In Arachis duranensis all genes were upregulated and in GG 20, 09 were up regulated, only two i.e. Aradu.0NC5M.1and Aradu.YIH2Y.1 were down regulated. Expression studies were conducted with Alcohol dehydrogenase as endogenous control. This information could be used to develop salinity tolerant groundnut. Treatment wise decrease in relative water content was observed from T1 to T4 as the salinity level increased. Reduction of water content was found less in Arachis duranensis as compared to GG 20. Electrolytic leakage (EL) was examined in response to salinity stress. EL was found more in GG 20 (36.45 to 92.04%) as compared to A. duranensis (29.82 to 80.29%) which proved tolerance of wild type against salinity. The highest value (0.871mg.gm-1 fr.wt.) for total chlorophyll content was observed in control (T1) as compared to T4 (0.202 mg.gm-1 fr.wt.). Total chlorophyll content was found less in A. duranensis (0.783 to 0.167 mg.gm-1 fr.wt.) as compared to GG 20 (0.960 to 0.237 mg.gm-1 fr.wt.). Increment in free amino acids was found more in A. duranensis (0.89 to 2.21 mg. g.-1) as compared to GG 20 (0.77 to 1.85 mg. g.-1). Total soluble sugars was found more in A. duranensis (0.41 to 0.90%) as compared to GG 20 (0.37 to 0.81%).
Open Access
“MOLECULAR CHARACTERIZATION OF BLACKGRAM [Vigna mungo (L.) Hepper] GENOTYPES FOR SALINE WATER TOLERANCE”
(JAU,JUNAGADH, 2018-07) Mukul Kumar Gandhi; Dr. G. V. Marviya
Blackgram [Vigna mungo (L.) Hepper], Family: Fabaceae and sub family: faboideae is the most widely grown type of pulse. To alleviate protein energy malnutrition, a minimum of 50g pulses/capita/day should be available in addition to other sources of protein such as cereals, milk, meat and eggs. The present experiment on “MOLECULAR CHARACTERIZATION OF BLACKGRAM [Vigna mungo (L.) Hepper] GENOTYPES FOR SALINE WATER TOLERANCE” was conducted at Department of Biotechnology, Junagadh Agricultural University, Junagadh with objectives to analyze molecular diversity of different blackgram genotypes by PCR based molecular markers to find out the phylogenetic relationship among different blackgram genotypes tolerance to salinity. On the basis of physiological parameters, the blackgram genotypes were discriminated into tolerant, moderate and sensitive to salinity stress. Out of 22 genotypes, five genotypes viz., SKNU-03-03, SKNU-07-06, SKNU-06-03, SKNU-07-01 and IC-214520 were found to be tolerant to salinity. Thirteen genotypes were found to be moderately tolerant and four genotype viz., GJU-1506, JAWAHAR URD-3, JAWAHAR URD-2 and GJU-1509 are sensitive to salinity. Germination per cent decreased by 67.77 %, root length reduced by 64.11 %, shoot length reduced by 67.91 % while seedling length decreased by 66.44 % in T4 treatment as compared to the T1 (control) treatment among all the blackgram genotypes. Seedling dry weight reduced by 1.40 fold in T4 treatment as compared to the T1 (control) treatment. Looking to the vigor index, seedling vigor index-I (length basis) and seedling vigor index-II (Dry weight basis) decreased by 2.21 and 2.03 folds, respectively in T4 ECe dSm-1. For molecular characterization, 11 RAPD, 10 ISSR and 10 SSR primers were used. Amplification of genomic DNA of 20 blackgram genotypes, using RAPD analysis, yielded 80 polymorphic fragments with an average of 99.99 % polymorphism. Number of amplified fragments with RAPD primers ranged from 3 to 15 bands and varied in size from 102 to 3344 bp. A dendrogram based on UPGMA analysis grouped the 20 blackgram genotypes into two main clusters named cluster-A and cluster-B with 10 % similarity, with Jaccard’s similarity coefficient ranging from 0.047 to 0.857. The 10 ISSR primers produced 44 bands across 20 blackgram genotypes, which were polymorphic. The size of amplified bands varied from 61 to 2267 bp. A dendrogram based on UPGMA analysis of 20 blackgram genotypes generated for ISSR data formed two main clusters, named cluster-A and cluster-B with 35 % similarity and Jaccard’s similarity coefficient ranged from 0.240 to 0.741. The 10 SSR primers produced 14 bands across 20 blackgram genotypes, which were polymorphic. A dendrogram based on UPGMA analysis of 20 blackgram genotypes generated by SSR molecular marker data formed two main clusters, named cluster-A and cluster-B with 40 % similarity and Jaccard’s similarity coefficient ranged from 0.100 to 1.00. The pooled study of all markers revealed a dendrogram consisted of two clusters. Cluster-I consisted of 5 blackgram genotypes, while cluster-II consisted of 17 genotypes in different cluster. From the dendrogram depicted genotype JAWAHAR URD-3 found to be most diverse from the other genotypes. The molecular markers (RAPD, ISSR and SSR) revealed phylogenetic relation of 20 blackgram genotypes and showed the different dendrogram pattern for different genotypes taken for study. RAPD abled to discriminate most diverse genotype SKNU-07-01 into cluster-B separately. The ISSR revealed one genotype JAWAHAR URD-3 in cluster-B found to be most diversified genotype across the 20 blackgram genotypes. SSR depicted one diverse genotypes JAWAHAR URD-3 into one cluster-B which were able to be showed diverse position in cluster.

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