Oryza Vol. 47. No.3, 2010 (173-178)

Genetic diversity of saline based on RAPD markers

M. Venkatesan, Y. Anbuselvam, P. Karthikeyan and R. Elangaimannan Department of Agricultural Botany, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India

ABSTRACT RAPD markers were used to evaluate genetic relationships in 20 cultivars of saline tolerant rice including few tolerant varieties. Twenty primers generated 207 highly reproducible and discernible loci, among which 160 were polymorphic. Percentage of polymorphism varied from 50 to 100 with an average of 74.8. Two primers, OPBA-03 and OPBC-10 showed 100 percent polymorphism among the accessions studied. A relatively high genetic diversity was detected among all the samples with the similarity coefficient value ranging from 0.45 to 1.00. The uweighed pair group method with arithmetic averages (UPGMA) dendrogram clustered 20 accessions in four clusters at 0.75 coefficient level. Principal coordinate analysis (PCA) further indicated that the genetic diversity of saline rice cultivars was presented by a clustered distribution pattern. Thus, the results from the UPGMA which grouped the accessions into 4 clusters lies more or less on par to the results of PCA. The cluster II and IV consisted of 6 and 10 accessions, respectively and cluster I and III consisted of 1 and 3 accessions, respectively. The similarity co-efficient was maximum between IR 61920-3B-22-2-1 and IR 42 (1.00) indicating less divergence among them. Lower similarity coefficient indices were observed between IR 61920-3B-22-2-1 and HP 3319-2WH-6-3-1-1-3 (0.26), indicating more divergence. Neither the UPGMA dendrogram analysis nor the PCA exhibited strict relationship with geographic distribution for the characters studied.

Key words: saline rice genetic diversity, RAPD markers.

More than 7 m. ha of the cultivable land is affected by prospective parent for production. Molecular salinity problem (IRRI, 1998). This ultimately reduces genetic polymorphism is also used to analyse the the yield as the salt concentration increases. High variability which has been the most efficient approach yielding rice varieties of irrigated ecosystem has not in plant genetics. RAPD analysis is widely used to study been productive under saline conditions. Hence, it is the genetic diversity of various cultivars and lines. imperative to identify high yielding and stable genotypes RAPD assay is rapid and easy to perform and also with increased salt tolerance. This requires new genetic requires only limited amount of DNA. In the present sources of salt tolerance and more efficient techniques investigation the assessment of genetic diversity in saline for identifying salt tolerant germplasm. As a valuable tolerant lines of rice has been studied with a new for genetic resource, and wild relatives comprise their utilization in breeding programme. an extraordinarily important gene pool for breeding. Knowledge on the extent and distribution of genetic MATERIALS AND METHODS variation within a species is fundamental for Twenty accessions collected from different sources conservation of genetic resources and breeding (Ayana (Table 1) were used for RAPD marker analysis in the et al. 2000). DNA markers made possible a fast survey present study. These accessions were grown in nursery of a species, genetic diversity and genetic structure. beds of 3x1m in the Farm, Annamalai Randomly amplified polymorphic DNA (RAPD) University, Tamil Nadu. A total of five leaves were technique is a simple and powerful DNA marker tool. collected from 25 days old seedling in each accession.The seedlings were raised under natural saline The genetic diversity studies using D2 statistics condition. The salinity level of the experimental field of Mahalanobis (1936) is being used for selection of was EC 4.0dsm-1 and pH 7.8.

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Table 1. studied under natural saline Then, the pellet was dissolved in 500 µ l of TE and stored condition. at -20ºC. The quality and quantity were checked through AccessionName of theOrgin /source 0.8% agarose gel by electrophoresis. DNA No.accessions concentretain for PCR reaction was estimated, by 1HP 3319-2WH-6-3-1-1-3Himachal Pradesh, India comparing the band intensity produced by the known 2IR47547-3B-26-2B-1IRRI, Philippines dilution that gave good amplification. 3IR 6199-313-24-3IRRI, Philippines 4IR 5931-110-1IRRI, Philippines Genomic DNA was used as template for PCR 5IR 6192-3B-15-2-2IRRI, Philippines amplification as described by Williams et al., (1990). A 6IR 28IRRI, Philippines set of 20 arbitrary primers (OPERON technologies. Inc. 7IR 29IRRI, Philippines California, USA) was used to produce distinct marker 8PY 5Pondicherry, India profiles for twenty parental lines (Table 2). Amplification 9IR 63311-B-3R-B-24-3IRRI, Philippines reactions were in the volumes of 20 µ l containing 10IR 61920-3B-22-2-1IRRI, Philippines 10mM Tris HCl (pH8.3), 50mM KCl, 1.5mM MgCl2, 11POKKALIKerala, India 0.001% gelatin, dATP, dCTP, dGTP and dTTPs each at 12IR 36IRRI, Philippines 0.1mM, 0.2mM primes, 25-30 ng of genomic DNA mol 13IR 42IRRI, Philippines 0.5 unit of Taq DNA polymerase (Bangalore Genei Pvt. 14IR 61920-3B-22-2-1IRRI, Philippines Ltd., Bangalore). Amplification was performed with 15IR 71657-5R-B-129IRRI, Philippines thermal controller (MJ Research Inc.) programmed for 16IR 7500-59-1-1-BIRRI, Philippines 40 cycles. After initial denaturation for two minutes at 17ADT 38Tamil Nadu, India 18ADT 45Tamil Nadu, India 94ºC, each cycle consisted of one minute at 94ºC, one 19CR 1009Tamil Nadu, India minute at 36ºC and two minutes at 72ºC. The 40 cycles 20TRY 1Tamil Nadu, India Table 2.List of random primers used in the genetic diversity analysis of saline rice cultivars. DNA was extracted following the protocol described by Williams et al. (from frozen leaves stored SI.Primers nameNucleotide sequence 5'to 3' at -180ºC, 1990) with modification. The leaves cut into No. pieces were completely homogenized in liquid nitrogen. 1OPBA – 015' – TTCCCCACCC – 3' Extraction buffer (100mM Tris HCl (pH 8.0), 20 mM 2OPBA – 025' – TGCTCGGCTC – 3' EDTA, 1.4 mM NaCl, 2 % cTAB per litre) was added 3OPBA – 035' – GTGCGAGAAC – 3' in 50 ml tubes filled with leaf powder to a volume of 15 4OPBA – 045' – TCCTAGGCTC- 3' ml and mixed well. The tubes were incubated at 65ºC 5OPBA – 055' – TGCGTTCCAC- 3' for 30 minutes with repeated shaking. Equal volume of 6OPBA – 065' – GGACGACCGT – 3' chloroform: isoamylalcohol mix (24:1) was added and 7OPBA – 075' – GGGTCGCATC – 3' mixed thoroughly for 15 minutes, followed by 8OPBA – 085' – CCACAGCCGA – 3' centrifugation at 4000 rpm for 30 minutes. Equal volume 9OPBA – 095' – GGAACTCCAC – 3' of isopropanol was added to the supernatant. DNA was 10OPBC – 105' – AACGTCGAGG – 3' hooked out after half an hour and washed in 70% ethanol 11OPBA – 115' – CCACCTTCAG – 3' and suspended in 500 µ l of TE buffer (pH 8.0). The 12OPAX – 065' – ACACTCGGCA – 3' DNA was incubated with 10-15 µ l (10 µ g/ µ l 13OPAK – 105' – CAAGCGTCAC – 3' concentration) of RNAse for 30 minutes. To this, equal volume of chloroform: isoamylalcohol was added and 14OPAY – 185' – AACTTGGCCC – 3' centrifuged at 12,500 rpm for 10 minutes. Twice the 15OPBA – 155' – GAAGACCTGG – 3' volume of absolute ethanol and1/10th volume of 3M 16OPBA –165' – CCACGCATCA – 3' sodium acetate were added to the acqueous layer and 17OPBA – 175' – TGTACCCCTG – 3' kept over night. The contents were centrifuged at 12500 18OPBA – 185' – CTCGGATGTC – 3' rpm for 15 minutes and supernatant was discarded. The 19OPBA – 195' – CCATCCGTTG – 3' pellet was washed with 70 percent ethanol and air-dried. 20OPBA – 205' – GAGCGCTACC – 3'

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were followed by seven minutes of final extension at Table 3.The polymorphism obtained with random primers 72ºC. PCR amplified products were subjected to among all the cultivars of rice elctrophoresis on 1.5% agarose gel in 1XTBE buffer at Sl.PrimerTotalPolymorphicPolymorphism 120V for 4 hrs using Hoefer Super Submarine No.bandsbands(%) electrophoresis unit (Pharmacia Biotech, USA). The 1.OPBA-0110770.0 electronic images of ethidium bromide stained gels were 2.OPBA-029666.6 captured using Kodak digital science DC 120 digital 3.OPBA-031313100.0 camera (Rochester, USA) and gels were documented 4.OPBA-049666.6 using electrophoresis Documentation and Analysis 5.OPBA-0510770.0 System (EDAS 120). 6.OPBA-06111090.9 7.OPBA-079666.6 All the accessions were subjected to RAPD 8.OPBA-086350.0 analysis. The DNA’s were isolated by adopting cTAB 9.OPBA-0910660.0 method and then amplified using PCR. The amplified 10.OPBC-101414100.0 products were then loaded under gel electrophoresis. 11.OPBA-1110660.0 The amplified regions were visualized by means of 12.OPX6-12131291.6 adding suitable dye under trans-illuminator and 13.OPAK-10121191.6 documentated through gel documentation system. The 14.OPAY-18121083.3 data generated from the polymorphic fragments were 15.OPBA-158562.5 scored as present (1) or absent (0) for each of the 16.OPBA-1610770.0 twenty accessions. In order to detect whether genetic 17.OPBA-179666.6 diversity of the rice accessions is evenly distributed, 18.OPBA-18121191.6 principal component analysis was performed based on 19.OPBA-1910770.0 20.OPBA-2010770.0 the data matrix from the DNA amplification products. Total207160- The diversity among the lines was worked out by Mean10.358.074.8 subjecting the RAPD scores to cluster analysis. Sequential Agglomerative Hierarchic Non-overlapping scattered plots, based on the first two principal co- (SAHN) clustering was performed on simple matching ordinates accounting for the total variation, exhibited similarity matrix by Unweighed Pair Group Method with relatively significant differentiation between the Arithmetic Averages (UPGMA). The data analysis was accessions (Fig.1). Group I, group II and group III were done using the software NTSYS pc version 2.02 (Rohlf, mainly clustered rice accessions. The two accessions, 1998). IR 61920-3B-22-2-1 and HP 3319-2WH-6-3-1-1-3 (0.26), showed a distant genetic relationship with other RESULTS AND DISCUSSION accessions, indicating sexual hybridization of rice Two hundred seven bands were detected of which accession either within the same type or between 22.71% bands were monomorphic across all the different types may possibly cause desirable heterotic cultivars studied. The remaining 160 bands (77.29%) effects. Three accessions namely HP 3319-2WH-6-3- were polymorphic with the average number ranging 1-1-3 from Himachal Pradesh, India, IR 61920-3B-22- from 3 to 14 per primer with a mean of 8.0, which 2-1 from IRRI, Philippines and TRY 1 from Tamil Nadu, indicated the high level of polymorphism expressed by India showed different genetic patterns. The Indian arbitrary primers (Table.3). The primers OPBC-10 accession (accession 8) was grouped together with produced maximum number of fragments (14) followed those accession-sharing genes with the IRRI accessions. by OPBA-03 and OPAX-06 each with 13 and 12 The sample matching similarity was calculated fragments, respectively. The primer OPBA-08 recorded using RAPD score and dendrogram was constructed minimum number of fragments (3). The analysis of using unweighed pair group method with arithmetic genetic distance between pair wise individuals revealed averages (UPGMA) employing sequential, a considerable genetic diversity of the rice accessions, agglomerative hierarchic and non-overlapping clustering presenting a clustered distribution pattern. These (SAHN). In UPGMA tree, high genetic diversity was

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revealed in all the rice accessions with the Jaccard 3-1-1-3 (0.263) (Table4). Molecular marker analysis similarity coefficient values varying from 0.45 to 1.00. of the twenty accessions using 20 RAPD primers The accessions were grouped into four clusters at 0.75 produced polymorphism for most of the loci studied. similarity co-efficient level (Fig.2). As per the similarity index, the accessions were grouped into 4 clusters. Crossing between the accessions with Cluster I consisted of one accession i.e. low similarity co-efficient will manifest high . accession 1, indicating different genetic pattern from other accessions. Cluster II had six accessions of IRRI. Our RAPD survey of 20 saline rice accessions Only three accessions (accession 18, 8 and 19) were indicated a high level of genetic diversity with 74.8% grouped in cluster III. Cluster IV included 10 accessions of bands being polymorphic. The high genetic diversity mainly from the IRRI accessions. In addition, all the of saline rice accessions may be attributed to two four clusters in dendrogram did not show any strict reasons. firstly, the geographic isolation and genetic relationship with geographical distribution, morphotypic variation of saline rice accession, though this result did classification, though some accessions exhibited their not show strict geographic relationship among all genetic differentiations among all the accessions. The accessions. Secondly, the sexual hybridization between similarity co-efficient was maximum between accession two accessions also can develop special or IR 61920-3B-22-2-1 and accession IR 42 (1.00). While, breeding lines. Although attempts were made to disclose minimum similarity was observed between accession the relationship between morphophyte and molecular IR 61920-3B-22-2-1 and accession HP 3319-2WH-6- markers, the result was not satisfactory, which may be

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g1 g2 g6 g3 g4 g5 g7 g18 g8 g19 g9 g13 g14 g12 g16 g15 g17 g10 g11 g20 0.45 0.59 0.73 0.86 1.00 Similarity Coefficient Fig. 2. Dendrogram based on genetic similarities among 20 saline accessions of rice.

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