ГЕНЕТИКА, 2012, том 48, № 1, с. 62-71
ГЕНЕТИКА РАСТЕНИЙ
УДК 575.1:582.542.1
GENETIC DIVERSITY IN BASMATI RICE (Oryza sativa L.) GERMPLASM AS REVEALED BY MICROSATELLITE (SSR) MARKERS © 2012 г. M. Ashfaq12, A. S. Khan2
department of Plant Science McGill University, Canada e-mail: ashfaq_qs@yahoo.com, muhammad.ashfaq@mail.mcgill.ca 2Department of Plant Breeding and Genetics, University of Agriculture Faisalabad, Pakistan e-mail: drabdussalamuaf@yahoo.com Received January 13, 2011
Genetic diversity among rice genotypes, including 15 indica basmati advance lines and 5 basmati improved varieties were investigated by 28 SSR markers including one indel marker. The SSRs covered all the 12 chromosomes that distributed across the rice genomes. The mean number of alleles per locus was 3.60, showing average number of polymorphism information content was 0.48. A total of 101 alleles were also identified from the microsatellite marker loci. A number of SSR markers were also identified that could be utilized to differentiate between rice genotypes. Pair wise Nei,s genetic distance between rice genotypes ranged from 0.07 to 0.95. The dendrogram based on cluster analysis by using SSR polymorphism that grouped the 20 genotypes of rice in to five clusters based on their genetic similarity. The result could be useful for the identification and selection of the diverse genotypes for the future cross breeding program and development of new rice varieties.
Rice (Oryza sativa L.) is very important cereal food crop of the world. Two cultivated species Oryza sativa L. and Oryza glabberrima L. are very well known in the world for their importance and these are belonging to AA genome [1]. Rice (Oryza sativa L.; 2n = 24; estimated genome size = 430 Mb) is a major cultivated species in the world. Moreover, rice is also an ideal model plant for the study of grass genetics and genome organization due to its diploid genetics, relatively small genome size [2, 3] significant level of genetic polymorphism [4—6] large amount of well conserved genetically diverse material. About 10000 years ago rice first originated in china. Oryza sativa has three subspecies i.e. indica, japonica and javanica growing in tropical temperate and intermediate climate conditions.
There is great genetic potential and diversity in the basmati rice for the improvement and development of new rice varieties. Characterization and quantification of genetic diversity has long been a major goal in evolutionary biology. There is a lot of diverse germplasm collection in different national and international rice research institute i.e. National Rice Research Institute Kala Shah Kaku, IRRI, WARDA, IITA and USDA. It is estimated that more than 400,000 rice germplasm accessions are conserved in gene banks around the world, majority of the germplasms are stored in the in six to seven gene banks located in Asian countries i.e. China, Japan, Korea, India, Pakistan, Thailand and Philippines [7].
Pakistan is among world's countries having an abundance of basmati landraces. A number of traditional varieties and improved cultivars have been released for cultivation in different regions of Pakistan
since early nineteen century. Aromatic cultivars and advanced breeding lines have a narrow genetic base as they are all related to selected basmati lines. Further, a very limited number of basmati breeding lines were used repeatedly in breeding programs to advance grain quality. In Pakistan, out of seven basmati varieties currently under cultivation, five have 'Basmati-370' as one of the parents [8]. There is a strong need to not only conserve landrace genotypes but also broaden the gene-pool of aromatic rice for future utilization in breeding of high yielding, superior quality and better-adapted varieties in the country.
The main basmati rice growing area is old Punjab likely to include present western Punjab (Sialkot, Sheikhupura and Gujranwala very famous for giving high quality and aroma to basmati) in Pakistan. Basmati rice generally identified by three main factors: appearance, aroma and taste. Basmati rice grows better in warm humid climatic condition with better grains characteristics.
Diversity based on phenological and morphological characters usually varies with environments and evaluation of these traits requires growing the plants to full maturity prior to identification. The world rice production has doubled during the last 25 years, largely due to improved technology such as high yielding varieties, better crop management, variability and diversity of plant breeding programmes and new bio-technological tools [9]. Phenotypic traits are also a very important tool for the evaluation of phenotypic diversity to screen out the diverse germplasm lines.
With regard to the pre-breeding and breeding, knowledge of the genetic diversity of elite advance
Table 1. A list of rice genotypes used in the present study
Sr. No Lines/Varieties Indica Upland Parentage History Collection site
1 CB-13* N/A Advance line RRI-KSK
2 CB-14* N/A » RRI-KSK
3 CB-15* N/A » RRI-KSK
4 CB-16* N/A » RRI-KSK
5 CB-17* N/A » RRI-KSK
6 CB-19* N/A » RRI-KSK
7 CB-21* N/A » RRI-KSK
8 CB-27* N/A » RRI-KSK
9 CB-32* N/A » RRI-KSK
10 CB-33* N/A » RRI-KSK
11 CB-36* N/A » RRI-KSK
12 CB-38* N/A » RRI-KSK
13 CB-39* N/A » RRI-KSK
14 CB-40* N/A » RRI-KSK
15 CB-42* N/A » RRI-KSK
16 Basmati-385* Basmati 3704 x TN1 Commercial Variety Cultivated Field
17 Super Basmati* Basmati 370 x 10486 » »
18 Basmati-198* Basmati 3703 x TN1 » »
19 Basmati-2000* Basmati 385 x 4048-3 » »
20 Bamati-Pak* CM7-6 x Basmati 370 » »
lines, improved varieties and cultivars from different breeding programmes provide a very reliable basis for expanding the gene pool of cultivated rice. Efficient use of wild relatives as a source of desirable genes for cultivated species requires a detailed understanding of their genetic structure and diversity [10].
Molecular markers also play a very important role in the mapping population and determination the genetic diversity studies. For molecular characterization, rice has the advantage of having more than 3,000 SSRs (Simple Sequence Repeats) freely available, allowing the identification, selection and use of markers with specific and robust amplification, high genome coverage and a precise genome position. SSRs have been used in the identification of novel marker alleles linked to genes involved in the expression of important traits, which can be extensively explored during cultivar development in breeding programs [11—13].
Simple sequence repeats (SSRs) or microsatellites are considered the markers of choice for most of ge-netic/genomic application, because of the significant level of allelic diversity that may be revealed [14]. SSR markers have many advantages over the other markers due to their co dominant nature, high degree of polymorphic genetic information content, PCR based, high reproducibility, more distributed in genomes and associated with non repetitive DNA. For rice, hundreds of SSR derived from structural genome, includ-
ing expressed and non expressed DNA sequences, are available to be used in genetic studies and germplasm characterization.
In present study, allelic diversity assessed by SSR markers was used to estimate the genetic diversity of the rice germplasm lines. The objectives of the study were to: 1) screen rice germplasm lines by using SSR markers, 2) study the genetic diversity among the rice germplasm lines at molecular levels.
MATERIALS AND METHODS
Plant materials
Seeds of twenty genotypes of rice were collected from rice research institute Kala Shah Kaku Pakistan and farmers cultivated field. The genotypes consisted on 15 advance lines and 5 commercial varieties i.e. CB-13, CB-14, CB-15, CB-16, CB-17, CB-19, CB-21, CB-27, CB-32, CB-33, CB-36, CB-38, CB-39, CB-40, CB-42, Basmati-385, Basmati-Super, Bas-mati-198, Basmati-2000, and Basmati-Pak. The list of genotypes, basmati commercial varieties and advance lines their growing areas and collection sites also shown in the Table 1 and Fig. 1. Seeds of each genotype were sown separately in to the paddy fields for raising the rice nursery.
Fig. 1. Basmati commercial varieties rice growing areas, collection site (located in zone II) and rice advance lines collected from RRI-KSK (Rice Research Institute Kala Shah Kaku Lahore Pakistan).
Genomic DNA extraction
The fresh leaves of each genotype were collected from the paddy fields at seedling stage. The total DNA of each genotype was extracted from fresh leaves by the cetyl tri-methyl ammonium bromide (CTAB) method [15]. The DNA pellet of each sample was diluted with TER solution (Tris base EDTA RNase). The purity and concentration of extracted DNA of each rice genotypes were determined spectrophotometrically at 260 and 280 nm by using the Nano Drop (ND 1000 Spectrophotometer). The DNA of all the genotypes was good quality. All the samples were diluted to a concentration of 40 ng/ul with ddH2O (Double Distilled Water) for PCR analysis.
Selection of SSR primers
Twenty eight SSR primers pairs were selected for the genetic diversity analysis on the basis of published rice microsatellite framework map. These primers cover almost all the 12 chromosomes of rice. The original source of primer sequence and the chromosomal positions of these markers can be found from rice genome database (http://www.gramene.org). A total of 28 microsatellite primer pairs were obtained from Eu-rofins mwg/operon Canada.
SSR analysis
For SSR analysis 28 selected primer pairs were used for the PCR amplification. PCR conditioned were maintained as described by Panaud et al. [16]. Each PCR reaction were done in total volume of 20 ^l containing 0.2 ^M of each forward and reverse primer, 200 ^M of deoxy ribonucleotides, 50 mM KCL, 10 mM Tris HCL (pH 8.3), 1.5 mM MgCl2, 40 ng DNA of each genotype and 0.5 unit of Taq DNA polymerase. Each PCR reaction was set at (depending upon the annealing t
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