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Molecular Characterization of Rapeseed-mustard Germplasm Using Ssr Marker (2019--2020)

M R ISLAM
Plant Genetic Resources Center, BARI, Joydebpur, Gazipur

R AFROZ
Plant Genetic Resources Center, BARI, Joydebpur, Gazipur

M S UDDIN
Plant Genetic Resources Center, BARI, Joydebpur, Gazipur

M N ISLAM
Plant Genetic Resources Center, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

Brassica mustard species represent one of the most important oilseed crops in Bangladesh. A better understanding of their genetic diversity is essential for the proper utilization of genotypes in breeding programs. In this study, 120 genotypes were taken to characterize with the help of SSR markers to reveal the genetic diversity among genotypes of oilseed Brassica species. All 10 microsatellite markers were found to be polymorphic conferring to the previous study. Among the primers Ra2-A11, Ra2-D04, Ra2-E03, Ra2-F11, and Ra2-G09 have been responded to the SSR marker selection study. This experiment is being continued in the molecular breeding laboratory, Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur.

Keywords: SSR markers, Genetic diversity, Mustard, Microsatellite markers

Location: Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur.

Start Date: 00-00-2019

End Date: 00-00-2020

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To characterizes the collected rapeseed-mustard genotypes applying SSR markers to assess the level of genetic diversity and relationships within genotypes among economically important oilseed Brassica species including B. rapa, B. napus and B. juncea. This information of molecular characterization of rapeseed-mustard germplasm will make a benefit for the future breeding program

Methodology:

The study was carried out at Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur. A total of 120 rapeseed-mustard germplasm including3 released varieties and117 landraces/local cultivars were selected for the present study genomic DNA was isolated from young actively growing fresh leaf tissues following SDS extraction, phenol: chloroform: isoamyl alcohol purification, and ethanol precipitation method. DNA quality was checked by electrophoresis in a mini-gel and quantified using a spectrophotometer (Spectronic Genesis^TM, New York, USA). All of the DNA samples were found to be of good quality in this study. A total of 10 SSR primers were selected for the genetic diversity analysis on the basis of various published articles on oilseed Brassica diversity (Tommasiniet al., 2003; Lowe et al., 2004, Turi et al., 2012). Of these, 5 produced unsatisfied results; therefore they were not included in the further study. SSR analysis was performed following the protocol of Turi et al., 2012 with minor modifications. PCR amplification reactions were carried out in a total volume of 10μl containing 5X Green GoTaq Reaction Buffer (Promega, USA), 25 mM MgCl₂,1.25 U Taq DNA polymerase (Thermo Scientific, USA), 0.4 mM each of the dNTPs (NEB, USA), 10 μM forward and reverse primers and 50 ng template DNA. The mixtures were prepared at 0°C and transferred to the thermal cycler. The reaction was carried out in Mastercycler nexus Gradient thermal cycler (Eppendorf, Germany). The thermal cycler was programmed to 1 cycle of 3 min at 94°C as an initial hot start and strand separation step. This was followed by 35 cycles of 1 min at 94°C for denaturation, 1 min for annealing temperature depending on the marker used (45°C-54°C), and 1 min at 72° C for primer elongation. Finally, 1 cycle of 10 min at 72°C was used for the final extension. Amplified products were stored at 4°C until further use.

Source of Information: Annual Research Report 2019-2020, Plant Genetic Resources Center, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

According to the DNA amplification patterns of the previous report, all ten microsatellite markers used in this analysis were found to be polymorphic. SSR marker selection agarose gel photograph shown in Figure 1. Result unveiled that, primer Ra2-A11, Ra2-D04, Ra2-E03, Ra2-F11, and Ra2-G09 has been responded and exhibited polymorphic banding pattern. However, the rest of the primers did not respond in SSR analysis. Two genotypes were used in this test, one traditional cultivar BD-9323 and one released variety BARI sarisha-14. Nevertheless, further study like PCR, polyacrylamide gel electrophoresis of remaining genotypes is still going on. Half of the selected primers respond in SSR marker analysis and another half did not. Responded markers are Ra2-A11, Ra2-D04, Ra2-E03, Ra2-F11, and Ra2-G09. Studies with these SSR markers and selected genotypes are still in progress.

Knowledge Management: Report/Proceedings