Abstracts and Short Communications 901

PS-IV-34

Assessment of genetic diversity in aerobic rice genotypes usingmicrosatellite markers

Puja Kumari* and V. K. Sharma

Department of Agricultural Biotechnology and Molecular Biology, Rajendra Agricultural University, Bihar, Pusa(Samastipur) 848 125; e-mail: pjkm1987@gmail.com

Abstract

A study was undertaken to evaluate the nature of microsatellite sites based simple sequence length polymorphism in 18aerobic rice entries in order to characterize them on the basis of simple sequence length polymorphism and to determine thenature and extent of genetic differentiation and diversity among them using twenty six microsatellite primer pairs, coveringall the chromosomes in the genome of aerobic rice. A total of 110 shared and 115 unique allelic variants were generated inthe form of amplified products. Altogether 225 allelic variants were detected at 33 microsatellite loci amongst the entries andthe number of alleles per locus ranged from 5 to 13, with an average of 6.81 alleles per locus. The polymorphism informationcontent value, a measure of gene diversity, varied from 0.607 to 0.913 with an average value of 0.795 per microsatellite primer.A dendrogram generated from allelic diversity data using similarity coefficient based sequential agglomerative hierarchicalnon-overlapping clustering module indicated that the aerobic rice entries AER-05 and AER-06 were relatively more closelyrelated with the highest similarity coefficient amongst eighteen aerobic rice entries under evaluation in the present study,which were broadly divided into three clusters. The use of 26 microsatellite markers in the analysis exhibited a remarkablyhigher level of genetic polymorphism and revealed unique or entry specific allele which could be useful as DNA fingerprintsin the identification and preservation of these aerobic rice entries as well as invaluable genetic resources for improvingeconomically important traits in rice, such as tolerance to drought stress.

Key words: Aerobic rice, genetic diversity, polymorphism, microsatellite

Rice is one of the most important food crops in the world with regard to human nutrition and caloric intake,providing more than one fifth of the calories consumed worldwide by the human species. Production of ricerequires a great deal of water. But, when the rice is grown in non-puddled aerobic soils or grown on rain-fed,naturally well-drained fields without surface water accumulation, it saves water considerably and protects soilhealth as well. Molecular marker assisted selection and determination of genetic differentiation and geneticdiversity facilitate to a great extent in the breeding programs directed towards the improvement of availablevarieties or development of elite varieties in short time period and more easy way. The SSR markers are knownto detect a significantly higher degree of polymorphism in rice which becomes ideal for studies on geneticdivergence. A carefully chosen set of microsatellite markers is considered to provide genome-wide coverage,facilitating an unbiased assessment of genetic difference and genetic divergence, besides enabling an unambiguousdescription of the entries and molecular profiling of genotypes of rice. Therefore, in the present investigation, anattempt was made to characterize and estimate the extent of genetic divergence among these aerobic ricegenotypes using microsatellite markers for their further utilization in future hybridization programme.

A total of 18 aerobic rice entries were used as experimental material for isolation of genomic DNA from theiryoung leaves using modified CTAB method [1]. The amplification of the extracted DNA was carried out using apanel of twenty six primer pairs specific to the unique flanking sequences of the microsatellites distributedamong all the twelve chromosomes present in the genome of rice. The polymerase chain reaction was performedin the Eppendorf Master Cycler gradient in a total volume of 15µl containing 1.5µl template DNA, 10X buffer,25mM MgCl2, 2mM dNTPs, 10µM of each primers and 1.25U/µl Taq DNA polymerase. The thermal profile cyclingconditions of denaturation at 940C for 1 min; annealing at 530-580C for 1 min and extension at 720C for 2 min for35 cycles was preceded by an initial denaturation at 940C for 5 min. In the end, a final extension at 720C for 7 min

902 National Symposium on Crop Improvement for Inclusive Sustainable Development, Nov. 7-9, 2014, Ludhiana

was given. The amplicons were resolved on 2% agarose gel containing ethidium bromide (0.5µg/ml) in 0.5X TBEbuffer at 100 volts. Frequencies of incidence of amplified allelic variants were calculated and used for determinationof polymorphism information content of each primer pair. The efficacy of the individual primer pairs in differentiationof the genotypes was assessed by computing polymorphism percent, discrimination coefficient and non-discrimination coefficient. The similarity matrix was subjected to construct a dendrogram to produce anagglomerative hierarchical classification [2].

By using a panel of 26 microsatellite markers, 225 allelic variants were detected at 33 SSR loci with anaverage of 6.82 alleles per locus. Among the primer pairs used in present study, the nineteen primer pairsnamely, RM 5359, RM 250, RM 60, RM 114, RM 280, RM 335, RM 538, RM 153, RM 461, RM 510, RM 427, RM219, RM 524, RM 591, RM 5373, RM 332, RM 224, RM 7003 and RM 558 produced single but polymorphicamplicons in combination with the eighteen aerobic rice entries evaluated. These 19 primer pairs amplifed thetargeted regions in the genome of rice, which belonged to simple sequence repeats located on chromosome 1, 2,3, 3, 4, 4, 5, 5, 6, 6, 7, 9, 9, 10, 10, 11, 11, 12 and 12, respectively. The number of allelic variants per locusranged from five in the case of RM 524 to thirteen in the case of RM 538. Altogether 225 allelic variants weredetected at 33 SSR loci with an average of 6.82 allele per locus. However, the proportion of alleles per locusrecorded in the present study is comparatively lower than that documented earlier [3]. A total of 110 shared and115 unique allelic variants were generated in the form of amplified products by using the twenty six primer pairs.The number of shared alleles per locus ranged from three out of five alleles in RM 524, six alleles in RM 289 andRM 407, seven alleles in RM 3873, RM 461 and RM 510, nine alleles in RM 219 and RM 335, eleven alleles in RM591and thirteen alleles in RM 538 to seven out of twelve alleles in the case of RM 337. The number of uniquealleles per locus ranged from one out of six alleles in the case of primer pairs RM 114 and RM 5373 to ten out ofthirteen and sixteen alleles in the cases of primer pairs RM 538 and RM 234, respectively. The primer pairs RM538, RM 591, RM 263, RM 335, RM 234, RM 5359, RM 3530 , RM 3873, RM461, RM 510, RM 219, RM 332, RM280, RM 7003 and RM 407 generated considerably greater polymorphism percentage in descending order ofmagnitude. Considerably greater magnitude of PIC value was obtained in the cases of primer pairs RM 591, RM5359, RM 538, RM 263, RM 224, RM 153, RM 337, RM3530, RM 114, RM 60, RM 332, RM 335 and RM 219 indescending order of magnitude. Among these, the primer pairs RM538, RM 5359, RM 263, RM 337, RM 591, RM224, RM 153, RM 3530, RM 332, RM 335and RM 219 generated considerably greater number of allelic variantsand appeared to be more informative primers. Occurrence of null allele for a particular repeat locus was noticedreflecting failure of locus specific generation of amplified products. The repeat locus associated with primer pairsRM 3873, RM 280, RM 114 and RM 591 showed null allele in some of the entries evaluated. The number ofentries showing null allele varied from one in combination with RM 280, two in combination with RM 3873 andthree in combination with RM 114 and RM 591. Occurrence of null alleles has also been reported by earlierresearchers. Therefore, the result of the present study corroborates the previous reports [46]. The estimates ofdiscrimination coefficient ranged from 0.647 in the case of primer pair RM 407 to 0.960 in the case of primer pairRM 591. Considerably greater ability to discriminate pair-wise combinations of entries was exhibited by theprimer pairs RM 591, RM 5359, RM 538, RM 263, RM 335, RM 153, RM 337, RM 224, RM 3530, RM 114, RM 60and RM 332 in descending order of magnitude. The estimates of non-discrimination coefficient ranged from 0.039in the case of primer pair RM 591 to 0.719 in the case of primer pair RM 263. Comparatively greater inability todiscriminate pair-wise combinations of entries was exhibited by the primer pairs RM 263, RM 524, RM 537, RM510, RM 427, RM 280 and RM 3873. The repeat loci with di, tri nucleotide and complex repeat motifs tended todetect greater number of alleles than the repeat loci with tetra-nucleotide repeat motifs. The results did notindicate any relationship between the repeat number involved in the simple sequence repeat and the number ofidentified alleles. Appearance of more than one band in the same entry was noticed reflecting the existence ofthe duplicated region in the genome of some of the entries. Taking into consideration the number of allelesgenerated by different primer pairs in concomitance with the level of polymorphism detected in the presentinvestigation, the primer pairs primer RM538, RM 5359, RM 263, RM 337, RM 591, RM 224, RM 153, RM 3530,RM 332, RM 335and RM 219 appeared to be more informative primers. The estimates of similarity coefficientclearly indicated the existence of a considerably greater extent of genetic variation at the molecular level. The

Abstracts and Short Communications 903

genetic relationships among the entries wereevaluated by matrix based on Dice’s similaritycoefficients [7]. The magnitude of similaritycoefficient between AER-05 and AER-06(0.542) was found to be the maximum amongstpair-wise combinations of entries. Theclustering analysis was conducted based onthe similarity matrix by UPGMA method anddendrogram has been constructed (Fig. 1).Considering broad classification of entries, asindicated by dendrogram, basically the entrieswere divided into three groups and mainlygrouped into seven main groups with differentsubgroups viz., IA, IB, IIA, IIB, IVA, IVB, VAand VB and sub-sub groups viz, IAa, IAb, IIAa,IIAb, IIBa and IIBb (Table 1 and Fig. 1).

Among the aerobic rice entries underevaluation in the present study, AER-05 andAER-06 exhibited the maximum extent ofsimilarity. The microsatellite site specific SSR

Fig. 1. Dendrogram of 18 aerobic rice varieties based on average similarity coefficients for 26 microsatelliteprimer pairs dependent allelic diversity

Table 1. Composition of clusters based on similarity coefficient innumerical taxonomic approach of cluster analysis.

Clusters identified at different Entries included in eachphenon levels cluster

25 50 75

I (6) IA(4) IAa(3) AER-04, AER-05, AER-06IAb(1) NDR-1140IB(2) IB(2) AER-02, AER-03

II(4) IIA(2) IIAa (1) VandanaIIAb (1) IR-36

IIB(2) IIBa(1) KMPIIBb (1) NDR-1143

III(2) III(2) IIIA(1) SahbhagiIIIB(1) Rasi

IV(2) IVA(1) IVA(1) IR-64IVB(1) IVB(1) Rajendra Bhagwati

V(2) VA(1) VA(1) MGD1104VB(1) VB(1) MGD1206

VI(1) VI(1) VI(1) Rewa

primer based analysis revealed unique or entry specific allele which could be useful as DNA fingerprints in theidentification and preservation of aerobic rice entries. The use of twenty six SSR markers in the evaluation ofaerobic rice entries exhibited a remarkably higher level of genetic polymorphism, which allowed unique genotypingof eighteen aerobic rice entries included in the analysis.

The microsatellite markers manifested a high level of polymorphism was observed as revealed by the useof twenty six primer pairs specific to the unique flanking sequences of the repeats providing a molecular database

904 National Symposium on Crop Improvement for Inclusive Sustainable Development, Nov. 7-9, 2014, Ludhiana

of the eighteen aerobic rice entries evaluated, in the present investigation. The marker based identification anddifferentiation of aerobic rice could be helpful to preserve the integrity of these high quality aerobic rice genotypesto benefit farmers, breeders and consumers.

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