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DNA Fingerprinting and Diversity analysis of BRRI hybrid varieties and Their corresponding Parents

M.M. Islam
Biotechnology Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh.

M.E. Hoque
Biotechnology Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh.

S.M.H.A. Rabbi
Biotechnology Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh.

M.S. Ali
Biotechnology Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh.

Abstract/ Executive Summary:

DNA fingerprinting and genetic diversity of four Bangladesh Rice Research Institute (BRRI) hybrid varieties and their parental lines were carried out. A total of 73 microsatellite markers were tested for screening the genotypes. Among the 73 amplified products, 37% had polymorphic bands showing 81 alleles. The number of alleles per locus ranged from two (RM10) to eight (RM327), where average allele number was 4.333. The Polymorphism Information Contents (PIC) lied between 0.337 (RM10) and 0.852 (RM327). RM327 was the most robust marker providing the highest PIC value (0.852). Pair- wise genetic dissimilarity coefficient interaction showed that BRRI hybrids two was the most genetically distant from each other whereas BRRI hybrids one, three, four and their respective parents were very close. Cluster analysis based on Dice’s similarity coefficient UPGMA system grouped BRRI hybrid and their parental lines into four major clusters at 0.41 cut off similarity coefficient. Four BRRI hybrid varieties grouped into four distinct clusters along with their component lines indicating their genetic closeness.

Keywords: Hybrid rice, Diversity analysis, Microsatellite markers, DNA fingerprinting

Location: Bangladesh Rice Research Institute

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To assessing genetic diversity and DNA fingerprinting of four BRRI hybrid varieties and their corresponding parents using SSR markers across chromosomes 1-12.

Methodology:

Sixteen genotypes, including four cytoplasmic male-sterile (CMS) lines (A line), four restorer lines (R line), four maintainer lines (B line) and four of their hybrid combinations were (F_1s) used for the present study. Seeds were collected from BRRI. Fresh leaf samples of 21-day- old rice seedlings were used as the source of genomic DNA. DNA was isolated following CTAB method with minor modifications described. At first leaf tissue were cut into small pieces, homogenized and digested with extraction buffer (1M Tris, 0.5M EDTA, 5M NaCl and 20% SDS, pH 8.0). After incubation for 20 min at 65°C with intermittent swirling, the mixture was emulsified with chloroform: IAA mix (24: 1 mixture of chloroform and isoamyl alcohol). After centrifugation, the upper aqueous layer was removed into a different tube and cold ethanol was added. After centrifugation a small pellet was visible. The pellets were then washed with 70% ethanol, dried by a concentrator and resuspended in an appropriate volume of TE buffer (1M Tris, 0.5M EDTA, pH 8.0). DNA quality was checked by agarose gel electrophoresis with lambda DNA (50 ng/μl) and quantification was done using a spectrophotometer. PCR was carried out in 10 μ l reactions volume containing 1 μl of MgCl₂ free 10 × PCR buffer with (NH₄) ₂SO₄, 1.2 μl of 25 mM MgCl₂, 0.2 μ l of 10 mM dNTPs, 0.2 μl of 5 U/μ l Taq DNA polymerase, 0.5 μl of 10 μM forward and reverse primers and 3 μl (10 ng) of DNA using a 96 well thermal cycler. Amplification were carried out in a thermal cycler (G- strom, GSI, England) with the following program: 94°C for 5 min (initial denaturation) followed by 35 cycles of 94°C for 1 min (denaturation), 55°C for 1 min (annealing), 72°C for 2 min (extension) with a final extension for 7 min at 72°C.The annealing temperatures were adjusted based on the specific requirements of each primer combination. After amplification, PCR products were mixed with gel loading dye (bromophenol blue, xylene cyanol and sucrose), and electrophoresed using vertical polyacrylamide gels (8% denatured poly ? acrylamide gel containing 19 : 1 acrylamide: bhisacrylamide) for manual genotyping. Four μl of the amplification products were resolved by running the gel in 1 × TBE buffer for 1.5 to 2.5 hrs (depending on the allele size) at around 90 volts and 500 mA electricity. The gels were stained in 1 μg/ml ethidium bromide and documented using UVPRO (Uvipro Platinum, EU) gel documentation unit. A total of 27 SSR markers (distributed across the 12 chromosomes) with clear amplifications were selected for genetic diversity analysis of four BRRI hybrids and their component lines. Size for each amplified allele was measured in base pair using Alpha? Ease FC 5.0 software (Alpha Innotech, USA). The summary statistics including the number of alleles per locus, major allele frequency, gene diversity, Polymorphism Information Content (PIC) values were determined using Power Marker version 3.25. The allele frequency data from Power Marker was used to export in binary format (allele presence = 1 and allele absence = 0) for analysis with NTSYS?pc version 2.1. The Excel file containing the binary data was imported into NT? Edit of NTSYS? pc. The similarity matrix was used to calculate similarity as DICE co ?efficient using SIMQUAL sub routine in SIMILARITY routine. The resultant similarity matrix was employed to construct dendrograms using Sequential Agglomerative Hierarchical Nesting (SAHN) based Unweighted Pair Group Method with Arithmetic means (UPGMA).

Source of Information: Plant Tissue Cult. & Biotech. 21(2): 189? 198, 2011 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In this study, the larger range of similarity values for genotypes indicated by microsatellite markers provides greater confidence for the assessments of genetic diversity and relationships, which can be used in future hybrid breeding programs.

Knowledge Management: Journal