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Genetic Diversity and Relationships in Jute (corchorus Spp.) Revealed by Ssr Markers

SAAIMATUL HUQ
Bangladesh Jute Research Institute, Dhaka,Bangladesh.

MD. SHAHIDUL ISLAM
Bangladesh Jute Research Institute, Dhaka,Bangladesh.

SAMIUL HAQUE
Bangladesh Jute Research Institute, Dhaka, Bangladesh.

ABU ASHFAQUR SAJIB
Department of Genetic Engineering and Biotechnology, University of Dhaka, Bangladesh.

NADIM ASHRAF
University of Nottingham, U.K.

HASEENA KHAN*
Plant Molecular Biology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Characterization of sixteen jute genotypes, from Corchorus olitorius L. and Corchorus capsularis L. using jute specific SSR marker attained a high polymorphism value of 92.20%. A total of 171 different alleles were amplified by 27 primer pairs with a mean of 6.33 ± 2.04 alleles per locus. The genetic diversity was also relatively high (0.81 ± 0.06). The Un-weighted Pair-group Method with Arithmetic averages (UPGMA) cluster analysis of the 16 jute genotypes produced a dendogram, which was in concordance with known information. The study reinforces the utility of SSR primers for providing useful and high levels of markers for individual plant genotypes even with a narrow genetic base.

Keywords: Jute; Genetic diversity; SSR; Genotypes; Polymorphism

Location: Bangladesh Jute Research Institute (BJRI), Dhaka

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Jute

Objectives:

The present study was undertaken to determine the genetic diversity and genetic relationships using SSR markers among 16 elite jute genotypes collected from BJRI.

Methodology:

Plant materials: The gene bank of Bangladesh Jute Research Institute (BJRI) has one of the world’s largest jute and allied fibre (JAF) germplasm collection of about 6000 accessions. Eleven popular jute varieties (four from C. olitorius and seven from C. capsularis) and 5 advanced lines (these will be released within couple of years as varieties for commercial cultivation) used in this study were selected for their superior fibre yield, quality and other characteristics, collected from the Physiology Department, Bangladesh Jute Research Institute (BJRI), Dhaka. Details of the genotypes along with their breeding pedigree and geographic origins are given in Table 1. DNA extraction: 1g of four-day old seedlings were ground in presence of liquid nitrogen. DNA was extracted from the ground sample using the CTAB following a protocol modified from Doyle and Doyle’s method (1990). DNA quality and quantity were checked following agarose gel electrophoresis (0.8% gel containing 0.5 µg/ml ethidium bromide) using known concentrations of uncut lambda DNA as standard. The final DNA concentration of each template stock was adjusted to 50 ng/µl. Designing of SSR primers: Twenty seven primer pairs, each flanking specific jute SSR sequences were used. The primers were designed from a SSR specific genomic library (developed by Vizon Sci. Inc., Canada) generated from an elite Bangladeshi jute variety O-4 that belongs to the species C. olitorius. Primers were designed following standard rules: 20-25 nucleotides in length, 40-60% GC content, and non-complementary 3´ nucleotides. Annealing temperatures for the primers were between 55ºC-65ºC. PCR amplification: PCR was performed in GeneAmp® PCR system 9700 (Applied Biosystems). Each of the 25µl reaction mixtures contained 50 ng of jute genomic DNA, 10 µM of each primer pair, 1× PCR buffer, 2.5 mM MgCl₂, 2 mM dNTPs, 1 unit Taq DNA polymerase (Invitrogen, India). The thermal cycler was programmed as follows: preheating for 5 min at 95°C; 35 cycles, each for 30 s at 95ºC (denaturation), 40 s at the annealing temperature of a particular primer pair, and 30 s at 72ºC (extension) and a final extension at 72ºC for 5 min, followed by cooling at 4ºC for infinite period. Separation and staining of PCR products: Before loading in gels, PCR products were denatured by adding equal volume of tracking dye (95% formamide, 10 mM EDTA, 0.23% bromophenol blue and 0.23% xylene cyanol) and heating at 94ºC for 5 min. Amplified products were separated in 8% denaturing polyacrylamide gel in 1×TBE buffer adjusted to pH 8.3 at 2000 V for 2.5-3 h, using sequencing gel apparatus (Adjustable nucleic acid sequencing unit, Model SG-400-33, C.B.S. Scientific Co. Del Mar, California). Silver staining of the gels was performed according to Streiff et al. (1998). Fragment sizes were calculated using the computer programme SEQAID II (Fragment Sizer) by comparing with fragments of 1kb-plus ladder marker DNA (Invitrogen, India). Data analysis: Amplified PCR products were scored to create a binary matrix. Effective allele per locus (Aep) were calculated according to Weir (1990) using formula 1/(1 - Hep), where Hep (the genetic diversity per locus) is equal to 1 – Σ Pi 2 and Pi is equal to the frequency of the ith allele of this locus. Shannon’s diversity index was calculated according to Martynov et al. (2003) with the formula H = -∑Pi ln (Pi), where Pi is the frequency of a particular allele i and ln is the symbol of natural logarithm. A pair-wise distance matrix was generated based on total and mean character differences in phylogenetic analysis (Swofford 2002). A dendrogram was constructed to identify genetic relatedness among the cultivars based on the distance matrix by applying the Un-weighted Pair-group Method with Arithmetic averages (UPGMA) cluster analysis using the software STATISTICA.

Source of Information: Bangladesh J. Bot. 38(2): 153-161, 2009 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

These SSR markers showed great promise as tools for collection and preservation strategies concerning various jute cultivars in situ. The ease of detection and the success of this study with SSR markers to elucidate the diversity among 16 cultivars may encourage the use of SSR markers to characterize all the germplasms (nearly 6000) at BJRI. This will surely reduce duplication in the collection, if any, and will be more useful to the user community. The information on genetic relatedness and characterization of cultivars will also help in making informed choices on selection of parental material based on genetic diversity. In addition, these SSR markers may be positioned on genetic maps, which may further facilitate plant breeders and geneticists to localize agronomically/horticulturally significant genes or gene complexes to specific sites on the genome (Liu et al. 1996). These traits could be introduced into desired cultivars by marker-assisted selection or map-based cloning.

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