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Diversity Analysis of Some Selected Rice Genotypes Through Ssr Based Molecular Markers

M. H. Shahriar
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh- 2202

A. H. K. Robin
Assistant Professor
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh- 2202

S. N. Begum
Senior Scientific Officer
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh-2202, Bangladesh

A. Hoque
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh- 2202

Abstract/ Executive Summary:

An experiment was carried out to assess the genetic diversity of advanced rice (Oryza sativa L.) breeding lines using three SSR markers viz., RM147, RM167 and RM215. Thirty T. Aman advanced breeding lines at F₉ generation along with 4 check varieties were assessed. All three primers showed polymorphism. A total of 29 alleles were detected among the rice genotypes with an average of 9.67 alleles per locus. Polymorphism information content (PIC) ranged from 0.47 to 0.88 with an average of 0.71. A dendrogram was constructed based on total microsatellite polymorphism and 34 genotypes were grouped into four major clusters at 0.36 similarity co-efficient differentiating the early maturing genotypes from the late maturing types. The information about the genetic diversity might be utilized in future breeding programs for developing rice varieties with much shorter growth duration. The results also suggested that microsatellite markers which are linked to genes or QTLs responsible for growth duration properties are suitable tools for marker assisted selection (MAS) to select the rice genotypes of shorter growth duration.

Keywords: Rice, SSR marker, Polymorphism, Genetic diversity, Advanced breeding lines

Location: Biotechnology Laboratory of Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh

Start Date: 00-03-2014

End Date: 00-05-2014

Program Area: Variety and Species

Commodity: Rice

Objectives:

Objective of the study were-

To assess the genetic diversity of 30 advanced rice lines along with the check varieties to screen out the short duration lines
To select diversified parents for having desirable genotypes in segregating generations in future

Methodology:

The experiment was carried out at the Biotechnology Laboratory of Bangladesh Institute of Nuclear Agriculture (BINA) during the period of March- May 2014. Plant materials: Seeds of 30 advanced breeding lines of T. Aman rice along with 4 check varieties were collected from the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh. Seeds were germinated in a controlled room by incubating those at 30^oC in petridishes. Collection of leaf samples: Young, vigorously growing fresh leaf samples were collected from 25 days old seedling of each of the thirty four genotypes which were used as the source of genomic DNA. Initially healthy portion of the youngest leaves of the seedlings were cut apart with a pair of sterilized scissors and washed in distilled water and ethanol and dried on a fresh tissue paper to remove spore of microorganisms and any other source of foreign DNA. Collected leaf samples were then put into polythene bags and kept on ice in an ice box. Tag was maintained for each sample. After that, the polythene bags were wrapped by aluminium foil and stored at -80^oC freezer. Genomic DNA isolation: DNA was extracted from the leaves of each genotype using the Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method. SSR Protocol: PCR amplification of SSR markers was undertaken using three primer. Each of PCR reaction tube contained 3.4 μL ddH₂O, 3 μL template DNA, 0.2 μL dNTPs, 1.2 μL MgCl₂, 0.2 μL Taq polymerase, and 0.5 μL each of forward and reverse primers. Amplification was executed using the following conditions: preheated at 94^oC for 3 minutes, 40 cycles of 1 minute denaturation at 94^oC, 1 minute annealing at 55^oC, 2 minutes elongation at 72^oC and a final extension at 72^oC for 7 minutes. The SSR amplification products were separated through a vertical polya crylamide gel electrophoresis unit. DNA fragments were expressed using ethidium bromide staining procedure. The gels were stained for 20 minutes. After that gels were documented using gel documentation unit (UV-trans-illuminator). SSR data analysis: The size of most intensely amplified fragments was determined by comparing the migration distance of amplified fragments relative to the molecular weight of known size markers and 25 base pairs (bp) DNA ladder using AlfaView software. The number of alleles per locus, major allele frequency, gene diversity, PIC, Nei’s genetic identity and genetic distance values were calculated using Power Marker version 3.25. MEGA5.22 software was used to construct a UPGMA (Unweighted Pair Group Method with Arithmetic Averages) dendrogram showing the distance-based inter-relationship among the genotypes.

Source of Information: J. Bangladesh Agril. Univ. 12(2): 307–311, 2014

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

An SSR based screening to 34 rice genotypes using 3 markers selected a total of 29 alleles with an average of 9.67 alleles per locus. The highest PIC value was recorded for primer RM167 and that was the lowest for the primer RM147. Thirty four genotypes were grouped into 4 clusters where the genotypes of cluster 1 (8 genotypes including the earliest maturing genotype R7) were of shorter growth duration. SSR markers used in this study were convenient, neutral & co-dominant in nature.

Knowledge Management: Journal