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RAPD and Isozyme markers for genetic diversity and their correlation with heterosis in Rice (Oryza sativa L)

S. Manonmani1, S Senthilvel2, A.K. Fazlullah Khan3 and M. Maheswaran4

1Assistant Professor, Plant Breeding and Genetics, Department of Rice, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore - 641 003. Email swamimano@ahoo.co.in
2
Project Scientist, Advanced Genome Laboratory, ICRISAT, India Email senthilvel@cgiar.org
3
Professor (Retired), Plant Breeding and Genetics, Centre for Plant Breeding and Genetics, TNAU, Coimbatore-641 003. India.
4
Associate Professor, Plant Breeding and Genetics, Centre for Plant Molecular Biology, TNAU, Coimbatore-641 003. Email mahes@tnau.ac.in

Abstract

Success of rice breeding depends on the ability to exploit the vast amount of genes and gene combinations in the rice gene pools that produce higher heterosis. Magnitude of heterosis depends on the choice of appropriate parental lines. Fourteen indica rice plants were assessed for diversity among them through isozyme and RAPD markers. The four isozyme systems viz., esterase, peroxidase, super oxide dismutase and polyphenal oxidase grouped the genotypes into eight clusters based on polymorphic marker loci. Diversity analysis using Randam Amphified Polymorphic DNA (RAPD) markers grouped the genotypes into a maximum of nine clusters. The genotypes viz., CR 1009, TNAU 841434 and AS 95035 were identified as more divergent by both the methods though these three genotypes share a common ancestral parent, Peta. Forty crosses were made using (four lines and 10 testers) the above fourteen parents and F1s were evaluated in two seasons along with their parents for expression of heterosis in plant height, number of productive tillers, days to 50% flowering, panicle length, number of filled grains per panicle, 100 grain weight and grain yield. Genetic distance based on isozyme analysis were correlated with mean performance and heterosis showed that there was no significant correlation between genetic distance and heterosis for any of the biometric characters in both the seasons. But positive correlation was observed between RAPD marker distance and statistical parameters viz., F1 per se performance, relative heterosis, heterobeltiosis and standard heterosis for number of productive tillers per plant and panicle length. The maximum heterotic value was observed in CR 1009 / HA 891037 in which both the parents were from different clusters. Thus RAPD's provide an effective tool for exploiting heterosis in rice.

Media Summary

Fourteen indica rice parents were assessed for genetic diversity through isozyme and RAPD markers. There was positive correlation between genetic distance measured through RAPD markers and heterosis of forty hybrids studied.

Key words

RAPD - isozyme - genetic diversity - correlation - heterosis.

Introduction

Rice germplasm collection in India is considered as a rich source of diversity due to the existence of high degree of phenotypic variability (De et al., 1988). Hybrids showing strong heterosis were usually developed from parental lines diverse in genetic similarity, ecotype, geographic origin etc., (Lin and Yuan, 1980; Yuan and Cheng, 1986 and Yuan 1985). Genetic diversity in Rice has been studied by various means such as biometrical analysis, biochemical analysis (isozyme) and molecular markers (RAPD). Isozymes are enzymes that share a common substrate but differ in electrophoretic mobility (Market and Moller, 1959). Isozymes have been utilized for the classification of varieties within Oryza sativa. The recently developed Random Amplified Polymorphic DNA (RAPD) (Williams et.al., 1999) based on polymerase chain reaction has been readily adopted in gene mapping and finger printing studies. RAPD technology was feasible for the identification of the phylogenetic relationship among rice varieties (Yu and Nguyen, 1994). Zhang et al., (1994, 1995) studied the relationship between marker genotype heterozygosity and heterosis in yield from a diallel cross involving eight indica type parental lines commonly used in hybrid rice breeding programmes in China. DNA polymorphism has been utilized to study genetic diversity and its relationship to heterosis in rice (Xie, 1993). While in the present study, isozyme and RAPD markers were employed to study the genetic diversity among fourteen elite parental lines. The genetic distance was correlated with heterotic performance of hybrids in rice.

Methods

Studies on isozyme and RAPD polymorphism

The isozyme variation among the 14 genotypes was studied using native polyacrylamide gel electrophoresis for the esterase, peroxidase, polyphenal exidase and superoxide dismutase as per the method described by Glaszman et.al. (1988). The DNA isolation for RAPD analysis was fallowed by Dellaporta et al., (1983). The PCR reaction condition reported by Williams et al., (1990) was performed by using PTC-100 TM programmable Thermal controller. (MJ Research Inc). A total of 14 primers were used.

DATA Analysis

The data generated from the detection of polymorphic fraqments were analysed. Specific amplification products were scored as present (1) or absent (0) for each of the fourteen varieties with fourteen primers. Genetic similarity between all fourteen parents were estimated by simple matching co-efficient (Sokal and Michener , 1958).

Field Experiments and statistical analysis.

The crossing block was raised during Rabi 1998. Forty hybrids were produced by crossing four lines (CR 1009, ADT 40, CO 43 and Improved white ponni) with ten testers (ADT 43, ASD 20, HA 891037, IS 14, AS 95035, CB 97033, CB (DH) 95298, ACK 198, 1ET 15341 and TNAU 841434). Seeds from 40 cross combinations along with fourteen parents were raised in Randomised Block Design with 3 replications during kharif 1999 and Rabi 1999-2000. Recommended package of practices were followed. Biometrical observations were recorded for the seven characters in parents and F1 generation were used for statistical analysis. The analysis was done using the INDOSTAT statistical package. Estimation of heterosis was fallowed by procedure given by Fonesca and Peterson 1968 and its significance was worked out by the formula given by Snedecor and Cockran 1967.

Results

A total of four enzyme systems viz., esterase, peroxidase, superoxide dismutase and polyphenal oxidase were studied involving all the 14 genotypes. Eight different markers were generated for esterase activity and four markers were generated for peroxidase activity. The clustering of fourteen genotypes showed eight clusters. Genetic distances based on isozyme analysis were correlated with mean performance and heterosis. The results showed that there was no significant correlation between genetic distance and mean performance as well as heterosis for any of the biometrical characters. The genetic distance worked out between the parents based on RAPD analysis was correlated with the per se performance and heterosis of the 40 hybrids (Table 1). The correlation values ranged between -0.217 and 0.505. Genetic distance of parents was found to be correlated with hybrid performance, mid parental heterosis, better parental heterosis and standard heterosis in season 2 as well as in pooled data analysis for only number of productive tillers per plant. Genetic distance was also found to be correlated with both per se performance and standard heterosis for panicle length in first season. The other biometrical traits viz., days to 50 percent flowering, plant height, number of filled grains per panicle, hundred grain weight and grain yield were not significantly associated with genetic divergence with regard to per se performance, relative heterosis, heterobeltiosis and standard heterosis in both the seasons.

Table 1. Correlations between genetic distance derived from RAPD analysis and per se performance and heterosis in the 40 F1 hybrids (x – per se performance, di - Mid parental heterosis, dii - Better parental heterosis and diii - Standard heterosis)

Trait

Season 1

Season 2

Pooled

x

di

dii

diii

x

di

dii

diii

x

di

dii

diii

Days to 50 per cent flowering

0.187

-0.161

-0.135

0.187

0.161

-0.217

-0.147

0.161

0.179

-0.21

-0.142

0.179

Plant height

0.12

0.149

0.208

0.121

0.088

0.244

0.118

0.088

-0.127

0.239

0.193

0.127

Number of productive tillers per plant

0.205

0.182

0.146

0.205

0.375*

0.364*

0.349*

0.375*

0.376*

0.389*

0.389*

0.376*

Panicle length

0.505**

0.156

0.220

0.504**

0.033

0.016

-0.08

0.032

0.263

0.147

0.169

0.263

Number of filled grains per panicle

0.137

0.055

0.061

0.137

0.042

-0.151

-0.132

-0.042

0.031

-0.083

-0.079

0.031

100 grain weight

-0.063

0.067

0.079

-0.064

-0.089

0.078

0.079

-0.088

-0.09

0.092

0.069

-0.090

Grain yield per plant

0.129

0.029

-0.034

0.129

0.212

0.274

0.283

0.212

0.183

0.202

0.211

0.183

* Significant at 5% level ; ** Significant at 1% level

Conclusion

Correlations between marker distance and hybrid performance is dependent on the germplasm used. The results from the current study indicate that RAPD’s offer a reliable and effective means of assessing genetic variation and thus provide an alternative avenue for predicting performance and heterosis in indica rice hybrids.

References

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Dellaporta SL, Wood J and Hicks JB (1983). Plant DNA mini preparation. In: Molecular biology of plants. A laboratory course manual. Plant Molecular Biology Reporter 1, 19-21.

Fonesca S, and Patterson FL (1968). Hybrid vigour in a seven parent diallel cross in common winter wheat. Crop Science 8, 85-88.

Glaszman JC, De Los Reyes BG and Khush GS (1988). Electrophoretic variation of isozymes in plumules of rice (Oryza sativa L) - a key to the identification of 76 alleles at 24 loci. IRRI research paper series no. 134.

Lin SC and Yuan LP. (1980). Hybrid rice breeding in China. International Rice Research Institute. Shanghai Academic and Technical Press, Shenghai, China.

Market CL and Moller F (1959). Multiple Forms of Enzymes, Tissue, Ontogenetic and Species Specific pattern. Proceedings of National Academic Science 45, 753-763.

Snedecor GW and Cochran WG. (1967). Statistical methods. Oxford and IBH publishing Co., New Delhi, p.593.

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Yu LX and Nguyen HT (1994). Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L) Theoretical and Applied Genetics 87, 668-672.

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Zhang QF, Gao YJ, Yang SH and Li JX. (1995). Molecular divergence and hybrid performance in rice. Molecular Breeding 1, 133-142.

Zhang QF, Gao YJ, Yang SH and Li ZB. (1994). A diallel analysis of heterosis in elite hybrid rice based on RFLPs and microsatellites. Theoretical and Applied Genetics 89, 185-192.

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