Possibility of using ISSR Marker


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Possibility of using ISSR Marker for the development of drought tolerant genotypes in chickpea


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Possibility of using ISSR Marker for the development of drought tolerant genotypes in chickpea Geetika Mehta * 1 , Geeta Devi 2 and Sonali Sangwan 3 1,2 Department of Plant breeding & Genetics, CCSHAU, Hisar, Haryana 3 Department of MBBB, CCSHAU, Hisar, Haryana SUMMARY Chickpea ( Cicer arietinum Linnaeus), a member of family Fabaceae, is an ancient self pollinated leguminous crop. It is ranked 3rd after common bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.) and is mainly concentrated in semi-arid environments. Drought is the most important constraint to yield in chickpea, accounting for about 50% yield reduction globally. Therefore, it is essential to develop varieties which can make use of available water resources and produce maximum yield. Most of the physiological traits associated with drought tolerance are quantitative in nature. An important research strategy that has been widely used over the past two decades to deal with such complexity is to use molecular markers to identify quantitative trait loci (Qtls) and their applications in marker-assisted selection (MAS) programmes. The present study on chickpea was conducted in the dryland area of CCS Haryana Agricultural University, Hisar for two consecutive years to screen 21 genotypes for rainfed conditions. In addition to morphological and physiological traits recorded on these genotypes, their ISSR DNA profile was also analyzed. Lots of genetic diversity was recorded at DNA level among all the genotypes. NTSYS pc2.0 was used to cluster them. One of the ISSR markers UBS841 was found to be associated with deeper root length in chickpea. Morphological, physiological and molecular observations in combination can be used to identify high yielding plants in chickpea for rainfed conditions (drought). The ISSR marker thus identified can also be successfully employed in MAS programmes for drought tolerance. Theme: Transgenics for biotic and abiotic stress tolerance Sr. No. Varieties / Genotype Source Pedigree 1 H11-03 CCSHAU HISAR HC1×HC2 2 H11-11 CCSHAU HISAR HC1×HC2 3 H11-1516 CCSHAU HISAR HC1×PCG84 4 H11-21 CCSHAU HISAR HC1×H00-216 5 H11-22 CCSHAU HISAR HC1×WR315 6 H11-31 CCSHAU HISAR HC5×Amethyst 7 H11-36 CCSHAU HISAR HC5×Amethyst 8 H11-37 CCSHAU HISAR HC5×Amethyst 9 H11-38 CCSHAU HISAR HC5×Amethyst 10 H11-41 CCSHAU HISAR (HC5×PDG84-16) × PDG84-16 11 H11-45 CCSHAU HISAR (HC5×ICCV96029) ×ICCV96029 12 H11-47 CCSHAU HISAR H208×ICC4958 13 H11-54 CCSHAU HISAR (HC1×H00-256)×ICC4958 14 H11-58 CCSHAU HISAR HC3×IPC94-132 15 C235 CCSHAU HISAR (State 1976) IP58×C1234 16 HC-5 CCSHAU HISAR (State 2005) H89-78×H89-84 17 HC-1 CCSHAU HISAR (National 1990) F61×L550 18 DCP92-3 IIPR Selection from germplasm 19 GNG1581 ARS, Sriganganagar GPF2×H82-2 20 H08-18 CCSHAU HISAR H99-264×H00-256 21 HC-3 CCSHAU HISAR (State 2000) L550×E100YM BACKGROUND MATERIALS AND METHODS RESULTS AND DISCUSSION Agarose gel electrophoresis after isolating DNA of 21 chickpea genotypes to test the quality and quantity of DNA Sr. No. Primer name Primer sequence Annealing temp.(°C) 1 ISSR-1 5’-ACACACACACACACACG-3’ 47 2 ISSR-2 5’-ACACACACACACACACGG-3’ 50 3 ISSR-3 5’-ACACACACACACACACC-3’ 47 4 ISSR-4 5’-ACACACACACACACACTT-3’ 46 5 ISSR-4 5’-AGAGAGAGAGAGAGAGC -3’ 47 6 ISSR-5 5’-AGAGAGAGAGAGAGAGT-3’ 45 7 ISSR-11 5’-AGAGAGAGAGAGAGAGGT-3’ 48 8 ISSR-15 5’-ACACACACACACACACGA-3’ 48 9 UBC-811 5’-GAGAGAGAGAGAGAGAC-3’ 47 10 UBC-825 5’-ACACACACACACACT-3’ 39 11 UBC-864 5’-ATGATGATGATGATGATG-3’ 41 12 UBC-841 5’-GAGAGAGAGAGAGAGAYC-3’ 48 13 UBC-841 5’-GACACGACACGACACGACAC-3’ 56 14 UBC-818 5’-CACACACACACACACAG-3’ 47 15 UBC-820 5’-GTGTGTGTGTGTGTGTC-3’ 47 16 UBC-857 5’-ACACACACACACACACGG-3’ 50 17 UBC-866 5’-CTCCTCCTCCTCCTCCTC-3’ 55 18 UBC-868 5’-GAAGAAGAAGAAGAAGAA-3’ 41 19 UBC-880 5’-GGAGAGGAGAGGAGAGGAGA-3’ 45 20 UBC-809 5’-AGAGAGAGAGAGAGAGG-3’ 47 21 UBC-808 5’-AGAGAGAGAGAGAGAGC-3’ 47 22 UBC-855 5’-ACACACACACACACACTT-3’ 46 23 RAMP-TAG 5’-TAGAGAGAGAGAGAGAGAG-3’ 49 24 LK7 5’-CCACTCTCTCTCTCTCTCT-3’ 51 25 UBC-823 5’-TCTCTCTCTCTCTCTCC-3’ 47 26 UBC-860 5’-TGTGTGTGTGTGTGTGGA-3’ 48 27 UBC-860 5’-TGTGTGTGTGTGTGTGAA-3’ 46 28 UBC-864 5’-ATGATGATGATGATGATG-3’ 41 29 UBC-803 5’-ATATATATATATATATC-3’ 43 30 UBC-854 5’-TCTCTCTCTCTCTCTCRG-3’ 48 31 UBC-856 5’-ACACACACACACACACYA-3’ 46 32 UBC-872 5’-GATAGATA-3’ 20 33 UBC-873 5’-GACAGACA-3’ 24 34 UBC-873 5’-GACAGACAGACAGACA -3’ 43 35 (ACTG)4 5’- ACTGACTGACTGACTG-3’ 43 36 ISSR-1 5’-GAAGAAGAAGAAGAAGAA-3’ 41 37 ISSR-2 5’-ACACACACACACACACT-3’ 45 38 ISSR-3 5’-TGTGTGTGTGTGTGTGAA-3’ 46 39 ISSR-5 5’-TGTGTGTGTGTGTGTGGA-3’ 48 40 ISSR-6 5’-GAGAGAGAGAGAGAGAC-3’ 47 41 ISSR-7 5’-GAGAGAGAGAGAGAGAT-3’ 45 42 ISSR-8 5’-CTCTCTCTCTCTCTCTG-3’ 47 43 ISSR-14 5’-ACACACACACACACACGG-3’ 50 44 UBC-807 5’-AGAGAGAGAGAGAGAGT-3’ 45 45 UBC-810 5’-GAGAGAGAGAGAGAGAT-3’ 45 46 UBC-817 5’-CACACACACACACACAA-3’ 45 47 UBC-821 5’-GTGTGTGTGTGTGTGTT-3’ 45 48 UBC-824 5’-TCTCTCTCTCTCTCTCG-3’ 48 49 UBC-834 5’-AGAGAGAGAGAGAGAGYT-3’ 45 50 UBC-844 5’-CTCTCTCTCTCTCTCTRC-3’ 48 PCR and gel electrophoresis The polymerase chain reaction (PCR) was conducted in Perkin Elmer Cetus thermocycler . PCR amplification was carried out in the reaction mixture (10 ul ) contained 1 ul ( microlitre )of DNA template, .5 ul MgCl2,1 ul dNTP mix, 1 ul primer , 1 ul 10x Buffer [ (1X TBE = for 1000 ml: Tris = 108 gm, Boric acid = 55 gm, .5 Molar EDTA = 40 ml, Distilled water) ], 6 ul Sterile distilled water and 0.8 units of Taq DNA polymerase . Amplifications were performed for 30 cycles with denaturation at 91 o C for 1 min, annealing at 48 o C for 49 s and extension at 72 o C for 2 min. Initial denaturation was done at 91 o C for 3 min and a final extension step of 5 min at 72 o C was also included .The PCR reactions were stored at 4°C until loading on gel for electrophoresis. ISSR amplified PCR products were resolved on 2.5% agarose gel in 10X TBE buffer (950ml distilled water and 50 ml 1X TBE Buffer) at a constant voltage of 80 V for 3 h. The bands were scored quantitatively for presence (1) or absence (0) in each genotype . DNA seizer or DNA marker 100bp was used to estimate molecular weight. Allele scoring / Gel scoring Bands for ISSR analysis were scored based on the presence (taken as 1) or absence (taken as 0) of bands for each chickpea genotype for each ISSR primer used. Only clear and unambiguous bands were scored. The size (in nucleotide base pairs) of the amplified bands was observed based on its migration and conformation relative to molecular size marker (100 bp ladder). Data analysis Polymorphism was calculated based on the presence or absence of bands. The 0/1 matrix was used to calculate the similarity index genetic distance using ‘ simqual ’ sub-program of software NTSYS-PC (numerical taxonomy and multivariate analysis system program). Dendrogram was constructed by using distance matrix by the unweighted pair-group method with arithmetic average (UPGMA) sub-program of NTSYS-PC

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