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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

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

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

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

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

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Program Area: Knowledge Management

Commodity: Rice

Objectives:

The present investigation was carried out with the objectives of 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 (F₁s) 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 by Zheng et al. (1995). 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 (Nano drop 1000 V3.6, USA). PCR was carried out in 10 μl reactions volume containing 1 μl of MgCl2 free 10 × PCR buffer with (NH4)2SO4, 1.2 μl of 25 mM MgCl₂, 0.2 μl of 10 mM dNTPs, 0.2 μl of 5 U/μl Taq DNA polymerase, 0.5 μl of 10 μM forward and reverse primers (Promega corporation, USA) 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.

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

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Achievement/Findings:

Pair‐wise genetic dissimilarity coefficient was measured among the test entries. The highest and lowest dissimilarity values were 0.8889 and 0.1844, respectively which was found in several pair interactions. Pair‐wise genetic dissimilarity coefficient interaction showed that BRRI hybrid two was the most genetically distant from each other whereas BRRI hybrid one, three, four and their respective parents were very close. To get maximumheterosis, diverged genotypes should be exploited in any hybrid breeding program. But from the present study it reveals that BRRI hybrids did not show highest heterosis due to not using the distant parents. It can be concluded that hybrids having highest heterosis could be obtained if the divergent parents (BHR1A, BHR2A, BHR2B, BHR4A, BHR4B BHR4R) were used for crossing. Therefore, in future, attempts should be taken to make crosses between the most diverse parents to get hybrid varieties with highest heterosis. Cluster analysis was used to group the BRRI hybrid genotypes and their parents to construct a dendogram. UPGMA based dendogram obtained from the binary data deduced from the DNA profiles of the analyzed samples. A total of four distinct groups resulted out of analysis of pooled SSR marker data at a cut‐off similarity coefficient 0.41, UPGMA clustering system of the four hybrids and their parental lines revealed that, they have very strong parental linkage. This fingerprinting data will identify the genotypes very easily and the information generated from the study could be used in further molecular characterization with other hybrid genotypes. 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