Genetic Diversity Analysis of Iranian Improved Rice Cultivars through RAPD Markers
Ghaffar KIANI
Department of Agronomy and Plant Breeding, Sari Agricultural Sciences and Natural Resources University, P.O. Box: 578, Sari, Iran; [email protected]
Abstract
The aim of this study was to evaluate the genetic diversity of Iranian improved rice varieties. Sixteen rice varieties of particular interest to breeding programs were evaluated by means of random amplified polymorphic DNA (RAPD) technique. The number of amplification products generated by each primer varied from 4 (OPB-04) to 11 (OPD-11) with an average of 8.2 bands per primer. Out of 49 bands, 33 (67.35%) were found to be polymorphic for one or more cultivars ranging from 4 to 9 fragments per primer. The size of amplified fragments ranged between 350 to 1800 bp. Pair-wise Nei and Li’s (1979) similarity estimated the range of 0.59 to 0.98 between rice cultivars. Results illustrate the potential of RAPD markers to distinguish improved cultivars at DNA level. The information will facilitate selection of genotypes to serve as parents for effective rice breeding programs in Iran.
Keywords: rice, genetic variation, RAPD, cluster analysis, improved cultivars
Introduction
The development of DNA marker technology has pro- vided an efficient tool to facilitate plant genetic resource conservation and management. Compared to morpho- logical analysis, molecular markers can reveal differences among accessions at DNA level. They represent an op- portunity to provide information on the variation that ex- ists in a particular species within a local region as well as among different countries. They serve as a valuable guide for effective collection and use of genetic resources too.
Molecular markers provide information that helps in de- ciding the distinctiveness of species and their ranking ac- cording to the number of close relatives and phylogenetic position (Rahman et al., 2007).
Several types of molecular markers are available for evaluating the extent of genetic variation in rice. These include RFLP (Botstein et al., 1980), RAPD (Williams et al., 1990), AFLP (Vos et al., 1995) and SSR (Tautz, 1989). Of these RAPD markers is increasingly being em- ployed in genetic research owing to its speedy process and simplicity (Williams et al., 1990). This technique always allows the examination of genomic variation without prior knowledge of DNA sequences (Hadrys et al., 1992) and is especially useful for unzipping the variations in species with low genetic variability when other techniques such as isozyme analysis fail to reveal differences among the indi- viduals. Moreover, varietal distinctiveness and relativeness can unambiguously be estimated by RAPD fingerprinting in commercially important crops (Thomas et al., 2006).
RAPD markers are considered to be unbiased and neutral markers for genetic mapping applications (Michelmore
et al., 1991), in population genetics (Haig et al., 1994), taxonomy (Chapco et al., 1992) as well as for genetic di- agnostics. RAPD has been used for classification and as- sessing diversity and relatness of rice genotypes by several groups (Ko et al., 1994; Mackil, 1995; Raghunathachari et al., 2000; Porreca et al., 2001; Rahman et al., 2007; Rab- bani et al., 2008).
This study aimed to use RAPD markers to evaluate the genetic variation within a collection of improved rice va- rieties and to reveal genetic relationships among them for future use in selection, hybridization, biodiversity assess- ment and conservation of diverse gene pools.
Materials and methods Plant materials
Sixteen cultivars namely ‘Neda’, ‘Hashemi’, ‘Shiroudi’,
‘Tabesh’, ‘Pouya’, ‘Fajr’, ‘Khazar’, ‘Shafagh’, ‘Nemat’, ‘Dasht’,
‘Champa’, ‘Amol-3’, ‘Ghaem-1’, ‘Ghaem-2’, ‘Ghaem-3’ and
‘Sepidrood’ were used in this study. Seeds were obtained from Rice Research Institute and University of Agricul- ture Sciences and Natural Resources, Sari, Mazandaran province, Iran. These genotypes were grown in cropping season of 2010 at research farm of the mentioned Univer- sity.
DNA extraction, PCR reaction and electrophoresis DNA was extracted from leaves of rice genotypes as de- scribed by Dellaporta et al. (1983). PCR were performed using 15 ten-mer RAPD markers (Cina Gene, Tehran, Iran). A 25 µl mixture was prepared for the PCR reaction which containing 50 ng template DNA, 2.5 µl of 10X buf- Received 17 April 2011; accepted 14 August 2011
Where Na = the total number of fragments detected in individual ‘a’; Nb = the total number of fragments shown by individual ‘b’ and Nab = the number of fragments shared by individuals ‘a’ and ‘b’. The resulting similarity coefficients were used to evaluate the relationships among cultivars with a cluster analysis using an unweighted pair group method with arithmetic averages (UPGMA). The analysis was plotted in the form of a dendrogram. All com- putations were carried out using the NTSYS-pc, Version 2.2 package (Rohlf, 1992).
Results
DNA amplification and polymorphism detection The genetic diversity and the relationships among rice genotypes were evaluated by RAPD markers using 15 primers. Out of this, 6 primers that gave rise to amplified products were selected for evaluating genetic relationship of rice cultivars (Tab. 1). Fig. 1 shows the amplification profiles generated with the primers OPB-04, OPA-12, fer, 0.3 µl of 10 mM dNTPs, 1 µl of 50 mM MgCl2, 1 µl of
each of the primers, and 1 unit of Taq polymerase. DNA Molecular Weight Marker (100 bp ladder; Roche) was used to estimate PCR fragment size. The PCR reaction was per- formed at 94°C for 5 min; then for 40 cycles of 94°C for 1 min; 35°C for 1 min; 72°C for 2 min followed by 72°C for 10 min. PCR products were fractioned by 1.5% agarose gel electrophoresis; then stained with ethidium bromide fluoresce under ultraviolet light and photographed.
Data analysis
RAPD bands were scored visually. Their presence was scored with 1 and absence with 0, separately for each cul- tivar and each primer. If a product was present in a geno- type, it was designated as ‘1’ and if absent it was designated as ‘0’. Estimates of genetic similarity (F) were calculated between all pairs of the cultivars according to Nei and Li (1979) based on following formula:
Similarity (F) = 2Nab/(Na + Nb)
Fig. 1. RAPD profiles of sixteen different cultivars of rice using primer OPB04 (A), OPA-12 (B), OPD-11 (C), OPH-20 (D), OPH-12 (E) and OPA (F). M: Molecular weight marker (100 bp DNA ladder), Lines 1: ‘Neda’, 2: ‘Hashemi’, 3: ‘Shiroudi’, 4: ‘Tabesh’, 5: ‘Pouya’, 6: ‘Fajr’, 7: ‘Khazar’, 8: ‘Shaf- agh’, 9: ‘Nemat’, 10: ‘Dasht’, 11: ‘Champa’, 12: ‘Amol-3’, 13: ‘Ghaem-1’, 14: ‘Ghaem-2’, 15: ‘Ghaem-3’, 16: ‘Sepidrood’
OPD-11 and OPH-20 across 16 improved cultivars. A considerable level of variability was observed among dif- ferent cultivars.
A total of 49 reproducible and scorable amplification products were generated across 16 cultivars (Tab. 1) and these were in the size range of 350 to 1800 bp. The number of amplification products generated by each primer varied from 4 (OPB-04) to 11 (OPD-11) with an average of 8.2 bands per primer. Out of 49 bands, 33 (67.35%) were found to be polymorphic for one or more cultivars rang- ing from 4 to 9 fragments per primer. Primers OPB-04 and OPA-12 gave the highest percentage of polymorphic bands, while the minimum polymorphism was observed using OPH-20 primer.
Similarity matrix
A similarity matrix based on the proportion of shared RAPD fragments was used to establish the level of relat-
edness between improved cultivars. The pairwise Jaccard’s coefficients for the genetic similarities among the 16 cul- tivars are presented in Tab. 2. Relatively high similarity index was observed in ‘Ghaem-1’ vs ‘Ghaem-2’, ‘Champa’
vs ‘Amol-3’, ‘Fajr’ vs ‘Nemat’, ‘Shafagh’ vs ‘Sepidrood’ cul- tivar pairs comparing to the other cultivar pairs. The low- est similarity index were observed in ‘Hashemi’ vs ‘Pouya’
(0.59) followed by ‘Hashemi’ vs either of cultivars ‘Neda’,
‘Champa’, ‘Amol-3’ and ‘Ghaem-1’ (Tab. 2).
Cluster analysis
Genetic similarities obtained from RAPD data were used to create a cluster diagram. Cluster analysis based on Nei and Li’s (1979) similarity coefficients using UPGMA grouped 16 cultivars into 5 main clusters (Fig. 2) at the similarity coefficient of 0.82. Cluster I consisted of three sub-clusters and the majority of cultivars were placed in it.
In this cluster the coefficient of similarity ranged from 0.73 (‘Tabesh’ vs ‘Sepidrood’) to 0.98 (‘Ghaem-1’ vs ‘Ghaem-2’) indicating relatively less divergence among the cultivars of this cluster due to originating from closely related ances- tors. For example, both ‘Ghaem-1’ and ‘Ghaem-2’ devel- oped from the cross between ‘Sepidrood’ and ‘Sange-Jo’.
So, it is necessary to avoid crossing between cultivars of this cluster.
The remaining cultivars ‘Khazar’, ‘Ghaem-3’, ‘Pouya’
and ‘Hashemi’ individually formed next clusters which were more isolated from the other cultivars. Low similar- ity indices were observed between ‘Pouya’ and ‘Hashemi’
(0.59) and among ‘Hashemi’ with either of cultivars ‘Neda’,
‘Champa’, ‘Amol-3’ with 0.61 similarity index which indi- cated more divergence. Crossing between the genotypes with low similarity coefficient will manifest high hetero- sis. As regards to higher quality status of ‘Hashemi’ among cultivars, it is essential that to pay more attention to it in hybridization programs.
Tab. 1. Primers used for RAPDs in 16 Iranian improved rice cultivars
Name Sequence (5’-3’) Amplified Polymorphic Polymorphism (%) Fragment
OPA-04 AATCGGGCTG 8 4 50 500-1200
OPA-12 TCGGCGATAG 7 7 100 400-1250
OPB-04 GGACTGGAGT 4 4 100 550-1800
OPD-11 AGCGCCATTG 11 9 81.82 350-1250
OPH-12 ACGCGCATGT 9 5 55.56 400-1250
OPH-20 GGGAGACATC 10 4 40 500-1300
Total 49 33
Tab. 2. Similarity matrix for Jaccard’s coefficient for 16 rice cultivars based on bands obtained from RAPD markers
No. Cultivars 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 ‘Neda’ 1
2 ‘Hashemi’ 0.61 1
3 ‘Shiroudi’ 0.92 0.61 1
4 ‘Tabesh’ 0.78 0.63 0.82 1
5 ‘Pouya’ 0.73 0.59 0.73 0.80 1
6 ‘Fajr’ 0.84 0.69 0.88 0.77 0.73 1
7 ‘Khazar’ 0.71 0.69 0.67 0.65 0.61 0.75 1 8 ‘Shafagh’ 0.78 0.71 0.86 0.80 0.67 0.86 0.65 1 9 ‘Nemat’ 0.82 0.71 0.86 0.84 0.75 0.94 0.77 0.84 1 10 ‘Dasht’ 0.82 0.67 0.82 0.80 0.75 0.82 0.69 0.88 0.84 1 11 ‘Champa’ 0.84 0.61 0.88 0.86 0.77 0.84 0.67 0.86 0.86 0.86 1 12 ‘Amol-3’ 0.84 0.61 0.88 0.90 0.77 0.84 0.67 0.86 0.90 0.90 0.96 1 13 ‘Ghaem-1’ 0.86 0.67 0.86 0.80 0.71 0.90 0.77 0.80 0.92 0.84 0.82 0.86 1 14 ‘Ghaem-2’ 0.84 0.65 0.88 0.82 0.69 0.88 0.75 0.82 0.90 0.82 0.84 0.88 0.98 1 15 ‘Ghaem-3’ 0.78 0.75 0.73 0.71 0.75 0.82 0.77 0.71 0.84 0.71 0.69 0.73 0.88 0.86 1 16 ‘Sepidrood’ 0.84 0.69 0.84 0.73 0.65 0.84 0.67 0.94 0.82 0.86 0.80 0.80 0.82 0.80 0.73 1
The present work revealed genetic variation and re- latedness among the sixteen rice germplasm. The em- ployment of RAPD markers in genetic diversity analysis helped in grouping the genotypes. Cluster analysis based on the RAPD data revealed that genotypes ‘Pouya’ and
‘Hashemi’ are clustered in a separate cluster as they have far genetic background from each other and from the rest of the investigated genotypes. In addition, the pres- ent study showed that cultivars in first cluster possess high degree of genetic similarity due to originating from closely related ancestors. .
Acknowledgements
This project (02-1389-02) conducted by financial sup- port of Agricultural Sciences and Natural Resources Uni- versity, Sari, Iran. Author thank to Dr. M. Sattari and M.
Noruzi from Rice Research Institute of Iran for their valu- able comments.
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