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Research Detail

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I AHMED
Plant Genetic Resources Centre, BARI, Joydebpur, Gazipur

M R MOLLA
Plant Genetic Resources Centre, BARI, Joydebpur, Gazipur

S RAHMAN
Plant Genetic Resources Centre, BARI, Joydebpur, Gazipur

M A HOSSAIN
Plant Genetic Resources Centre, BARI, Joydebpur, Gazipur

This experiment was carried out with 47 germplasm of chilli at Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur. An extensive research programme has been initiated for the estimation of genetic diversity, identification and discrimination of chilli germplasm. The modified SDS and phenol: chloroform: IAA protocol found to be cost-effective and latex-free high-quality genomic DNA was obtained successfully. A total of 115 collected germplasm and about 50 microsatellite markers will be used in this experiment. Initially, twenty primer pairs with clear and expected amplified product sizes were selected. This work is a progress report of the utilization of one microsatellite marker CL-CAMS-351. The marker was found to be polymorphic. Using this primer across 47 germplasm and a total of 7 alleles size ranging from 191 bp to 211 bp was detected. Forty-seven germplasm were separated into 14 groups based on different allele size. Germplasm collected from the same area were drawn together in the same group. Both homozygosity and heterozygosity were found across the 47 germplasm. Variation was observed in SSR banding patterns and it could be possible due to different geographical sources and also different morphological traits. After using a higher number of microsatellite markers selected from the different chromosome, it could be possible to produce unique DNA profiles of chilli germplasm and estimate genetic variation among the germplasm.

  Molecular characterization, Genetic diversity, Chilli, SSR markers
  Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur.
  00-00-2014
  00-00-2015
  Conservation and Biodiversity
  Plant, Chilli

The present investigation was undertaken to characterize and give robust genetic diversity estimates in cultivated chilli peppers using SSR markers.

The study was carried out at Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur, Bangladesh. One hundred and fifteen collected germplasm were planted for morphological characterization in the year 2014-15. Young leaves sample has been collected from 90-95 days mature plants based on their morphological traits. As dissimilar or different plant characters were observed in the same collected accession. For this reason, a collection of leaf sample was made from the morphologically homozygous plant. Bulked DNA was isolated from 4-5 fresh leaves using following the protocol described by Saghai-Maroof et al. (1984) and also used by Rahman et al. (2007) with some modifications. Excluding usage of CTAB and liquid nitrogen, the modified protocol included digestion with homogenization buffer (Solution: Tris-50 mM, EDTA-25 mM, NaCl-300 mM, 1% SDS and deionized water) at 65ºC for 30 min, extraction with phenol: chloroform: isoamyl alcohol (25:24:1), precipitation with ice-cold and extra pure isopropyl alcohol and purification with absolute ethanol (Plus sodium acetate, 3M) and 70% ethanol chronologically. Finally, DNA sample of each chilli germplasm dissolving in 30-40 μl of TE buffer within 1.5 ml eppendorf tube was preserved separately at -20ºC. The presence of genomic DNA was confirmed on 1% agarose gel qualitatively. The gels were visualized under UV light and photographed using a photo documentation system (UV Transilluminator, Uvitec, UK). The Polymerase chain reactions was set up 10 μl volumes containing 75 ng template DNA, 0.1 U/μl Taq DNA polymerase, 0.4 mM each of the dNTPs, 1.25 μM of each of primer, 4 mM MgSO4, 20 mM KCl, 16 mM (NH4)2SO4 , 20 mM Tris-HCl, pH8.8 and a suitable amount of sterile deionized water. The reaction was performed in an oil-free eppendorf Mastercycler® nexus Gradient thermal cycler. SSRs were amplified under the following “touchdown” PCR conditions: 94oC/3 min denaturation, 11 cycles of  94oC/0.5 min,  58-60oC/1 min, decreasing by 1oC per cycle, and 72oC/1 min; 30 cycles of  94oC/0.5 min,  52-55-oC/1 min and 72oC/1 min; a final extension for 5 min. The success of the reaction product following 2% agarose gel electrophoresis was determined. PCR-products were electrophoresed on a 6% denaturing polyacrylamide gel containing 19:1 acrylamide: bis-acrylamide and 8M urea. Electrophoresis was done using the SequiGen GT Sequencing Cell (BIO-RAD Laboratories, Hercules, CA, USA) electrophoresis system. A pre-run of the gel for 30 minutes at 120W was followed by a final run at 60W and 50ºC upon loading of denatured PCR products for a specified period of time depending on the size of amplified DNA fragment (usually 1 hour for 100 bp). After completion of electrophoresis, the DNA fragments were visualized following the Promega (Madison, WI) silver-staining protocol. The bands representing particular alleles at the microsatellite loci were scored manually and designated the bands as A, B, C, etc. from the top to the bottom of the gel. The genotypes of different individuals were hypothetically scored as AA, BB, CC, etc. for homozygous or as AB, AC, BC etc. for heterozygous. The software DNA FRAG version 3.03 was used to estimate allelic length (Nash, 1991).

The amount of genomic DNA was quantified at 260nm spectrophotometrically (Spectronic GENESYS™ 10 Bio). Using the absorbance reading obtained for DNA sample of each chilli germplasm, the original DNA concentrations were determined. About fifty microsatellite primer pairs were identified and those are located at 12 chromosomes from a different publication. Preliminarily, twenty primer pairs of the different chromosome were tested for their better responsiveness in amplifying the target genomic region of template DNA and to check the expected PCR product sizes in base pairs. All twenty primer pairs with clear and expected amplified product sizes were selected and one pair were used for microsatellite analysis in the present study. The rest of the 30 primer pairs will be tested for their better responsiveness in amplifying the target genomic region of template DNA and will check the expected PCR product sizes in base pairs.

  Annual Research Report 2014-2015, Plant Genetic Resources Centre, BARI, Joydebpur, Gazipur
  
Funding Source:
1.   Budget:  
  

Two standard protocols have been tested for chilli genomic DNA isolation of which one was SDS (Sodium dodecyl sulfate) and phenol: chloroform: IAA followed by alcohol precipitation and another one CTAB (Cetyl trimethylammonium bromide)  Quality and quantity of DNA was authenticated by gel electrophoresis and spectrophotometry. If a single high-molecular-weight band was formed, the DNA quality was considered good, and if it formed a smear, the DNA quality was considered to be poor. The modified SDS and phenol: chloroform: IAA protocol found to be cost-effective and latex-free high-quality genomic DNA was obtained successfully compared to the CTAB DNA isolation procedure. All of the DNA samples were found to be of good quality in this study. The electropherogram of DNA of 24 individuals (12 of SDS and 12 of CTAB protocol) from 12 chilli germplasm. Standard PCR protocol was used for the first 20 primer amplification following the protocol described by Minamiyama et al. (2006) with some modification. If the primer was shown good band resolution intensity, less searing, amplifying the target genomic region of template DNA, the PCR protocol considered to be correct. All 20 primers pairs were shown better responsiveness with clear and expected amplified product sizes and one pair were used for microsatellite analysis in the present study. The electropherogram of PCR products of 20 primer pairs in two chilli germplasm. All 47 chilli germplasm were successfully amplified with the CL02-CAMS-351 microsatellite primer pairs. This microsatellite marker was found to be polymorphic, revealing a total of 07 alleles with sizes range from 170 bp to 198 bp. Banding patterns generated by these primer pairs in 47 chilli germplasm. Allele sizing technologies are well established and can be readily used to size microsatellite alleles from any organism (Song et al., 1999). SSR genotypic data from a number of loci have the potential to provide unique allelic profiles or DNA fingerprints for precisely establishing genotypic identity. SSR band positions against one SSR marker used in this study. Forty-seven germplasm were separated into 14 groups based on different allele sizes. Among 14 groups, three germplasm of group 01, five germplasm of group 02, two germplasm of group 06 and four germplasm of group 09 showed homozygosity and it was FF, AA, BB and GG respectively. On the other hand, heterozygosity was found in 34 germplasm which was formed, other 10 groups. Group 02 contained the highest number of germplasm which was 12, whereas AM-18, RISA-55, RISA-89 and RT-05 had a distinct status in this study and which could be explained by dissimilar SSR banding patterns due to their different geographical sources. Unique allele was detected in these four germplasm. In our study, most of the germplasm collected from similar areas drawn together in the same group. It implies that genetic makeup of the locus for which the primer is responsible to distinguish along with low variation also in the morphological traits or geographical proximity or same origin.

The information generated through this study on the genetic difference and unique SSR band profiles indicates that it could distinguish and estimate genetic diversity among the germplasm used in this study. The present results are an indication indicated of a comprehensive study. After using a higher number of microsatellite markers selected from the different chromosomes, it could be possible to produce unique DNA profiles of chilli genotypes and analysis of genetic variation among the different genotypes. For this reason, an extensive research programme has been started and basic parts have been completed. Within a couple of months, the rest of the study will be completed to identify more genetically diverse germplasm of chilli with desired agronomic traits which could be effectively utilized in a quality improvement breeding program that will be helpful to develop high yielding varieties with better quality traits.

   

  Report/Proceedings
  


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