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Evaluation of Morpho-molecular Diversity and Pod Borer Resistance in Country Bean

Muhammad Shahidul Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Tajul Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Nihar Ranjan Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Abdul Hannan
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Monirul Islam
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh

Abstract/ Executive Summary:

Country bean is a nutritious vegetable with commercial importance. Pod borer is a major pest of country beans in Bangladesh. Assessment of genetic variation among germplasm is a prerequisite for improvement of any crop. The present study was conducted to evaluate the yield potential, pod borer resistance and genetic diversity of 20 country bean varieties. At first, a field experiment was conducted with twenty cultivars to evaluate yield contributing characters and pod borer resistance. The data revealed that the variety Kaloputi showed the highest yield (2111.09 g) of pods plant^–1 . This variety (Kaloputi) had the highest number of pods plant^–1 (271), lowest percentage (3.08%) of infestation, highest number (1086.0) of seeds plant^–1 and highest dry seed yield over the other varieties. The variety BARI seem 5 showed the least performance in most of the parameters. Another experiment was conducted to find the genetic diversity among the 20 cultivars using SSR markers. The cluster analysis showed that the genotypes were divided into four groups. The cluster 1 was composed of seven BARI released varieties, cluster 2 was composed of two modern (IPSA seem 2 and BARI seem 8) and three local varieties (Khisamoti, Rifa and Goalgada), cluster 3 was constructed by six local and one modern variety (BARI seem 9) and Cluster 4 consisted of only cultivar Mostofa. The wide variations in phenotypic and genotypic level and pod borer resistance observed among the genotypes can be potentially used for future improvement of country bean.

Keywords: Genetic diversity, L. purpureus, Microsatellite, Resistance, Yield, Country bean

Location: Plant Genetic Resource Centre (PGRC), Bangladesh Agricultural Research Institute (BARI), Gazipur, Dhaka and local cultivars were collected from different parts of Bangladesh.

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Pulses

Objectives:

To evaluate the diversity of country bean at a phenotypic level using yield contributing traits and pod borer resistance and at the DNA level using SSR markers

Methodology:

Plant materials: Twenty genotypes were originally collected from Plant Genetic Resource Centre (PGRC), Bangladesh Agricultural Research Institute (BARI), Gazipur, Dhaka and local cultivars were collected from different parts of Bangladesh. Morphological characterization: The experiment on morphological characterization was carried out at the Entomology Experimental Field, Bangladesh Agricultural University, Mymensingh, during the rabi season. The land was ploughed and cross ploughed several times with power tiller to obtain good tilth. The Experimental plots were fertilized with cow dung @10 t/ha, Urea @50 kg/ha, TSP @150 kg/ha and MoP @150 kg/ha (Rashid, 1999). The experiment was laid out in a randomized complete block design with three replications. The unit plot size was 1.3 x 1.3 m maintaining 0.25 m distance between the plots. Bamboo sticks were used to facilitate the climbing of the plants upwards. Data were recorded on vein and leaf colour, length, width and shape of leaf, leaflet length, growth habit, number of primary and secondary branches plant^{– 1} , flower buds raceme^–1and raceme plant^–1 , days to 50% flowering, length, width and colour of pod, number of healthy and infested pods plant^–1 , infestation percentage, yield of green pods plant^–1 and yield of dry seeds plant^–1 from each plant of each plot. Statistical analysis: The data were subjected to analysis of variance (ANOVA) and the treatment means were separated by Duncan’s Multiple Range Test using MSTAT-C software. Molecular diversity analysis Total DNA was extracted from the leaves by Cetyl Trimethyl Ammonium Bromide (CTAB) method (Saghai-Maroof et al., 1984). Thirteen microsatellite primers were selected for the present study based on genetic diversity experiment (Shivachi et al., 2012). Out of thirteen primers, nine primers showed clear amplified bands. The details of the selected primers are given in Table 2. The PCR cocktail for SSR analysis had a total volume of 10 μl/reaction, including DNA. It was composed of 1.0 μl genomic DNA, 5 μl PCR master mix (Go-taq green master mix, Promega corporation, U.S.A), 1 μl forward primer, 1 μl reverse primer, and 3 μl nuclease-free water. Samples were subjected to the following thermal profile for amplification in a thermocycler: The reaction mix was preheated at 94ºC for 3 min followed by 35 cycles of 3 min, denaturation at 94ºC, 1 min annealing at 55-65 ºC (based on the annealing temperature of the individual primer) and elongation at 72ºC for 1 min. After the last cycle, a final step was maintained at 72ºC for 5 min to allow complete extension of all amplified fragments followed by holding at 4ºC until electrophoresis. SSR data analysis Molecular weights for SSR products were estimated with AlphaEase 4C software. The individual fragments were assigned as alleles of the appropriate microsatellite loci. Molecular weight data of alleles were used to determine the genetic distance for phylogeny reconstruction based on the neighbor-joining method (Saitou and Nei, 1986). The 20 genotypes were clustered based on the matrix of genetic similarities using the Unweighted Pair Group Method with arithmetic averages (UPGMA). The cluster analysis and dendrogram construction were performed using NTSYSPC (version 2.1).

Source of Information: J Bangladesh Agril Univ 18(1): 25–33, 2020

Article Link (Online Journal): https://doi.org/10.5455/JBAU.73722

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The wide range of dissimilarity values found in the molecular study suggests that the germplasm collection represents a genetically diverse population. The results show that rich diversity exists between the germplasm collections from different geographical regions of the country. The morphological study confirmed that the local cultivar Kaloputi showed higher plant growth and yield. This variety showed higher resistance to pod borer infestation. Similar performance was shown by the cultivar Mostafa. The findings suggest that some of these genotypes, especially two local varieties, kaloputi and Mostafa, are promising for using as parents for the development of new variety. Further studies are necessary for the development of pod borer resistant variety with a higher yield.

Knowledge Management: Journal