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Research Detail

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Fakhrul Islam Monshi
Department of Genetics and Plant Breeding, Faculty of Agriculture, Sylhet Agricultural University, Sylhet, 3100, Bangladesh

Mohammed Shafi Ullah Bhuiyan
Department of Genetics and Plant Breeding, Faculty of Agriculture, Sylhet Agricultural University, Sylhet, 3100, Bangladesh

Rehenuma Tabassum
Department of Crop Botany and Tea Production Technology, Faculty of Agriculture, Sylhet Agricultural University, Sylhet, 3100, Bangladesh

Ten germplasm of litchi were collected from different eco-geographic regions of Bangladesh to evaluate their adaptation in hilly areas and screening the genetic variability both in morphological and molecular characteristics by RAPD markers. The morphological data was conducted in Randomized Complete Block Design (RCBD) with three replications and found the significant differences at all stages. Among the germplasm, the maximum number of branches per plant (14.33) and number of leaves per plant (66.33) were found in Mongalbaria which were statistically similar to BARI litchi-2 13.67 and 61.67, respectively. The germplasm, Malaysian recorded the highest plant height (68.40 cm) and the lowest trunk diameter (1.77 cm), while China-3 gave the highest trunk diameter (3.23 cm). So, by assessing morphological parameters, Mongalbaria and BARI litchi-2 have a bright prospect for growing under the hilly areas in Sylhet region. In Random Amplified Polymorphic DNA (RAPD) markers analysis, primer OPB-04 showing good technical resolution and sufficient variation among germplasm and produced a total of 7 RAPD markers of which 6 (85.71%) were considered as polymorphic loci. The highest genetic distance (1.9459) was observed in Bombai vs. Malaysian varietals pair whereas, the lowest genetic distance (0.1234) was estimated in BARI litch-2 vs. BARI litch-3 varietals pair. The UPGMA dendrogram showed that 10 germplasm of litchi could be classified into different groups or clusters according to their genetic similarities and dissimilarities. Two germplasm Bombai and Malaysian were found the highest genetic distance than the others that ultimately helpful for further hybridization program.

  Germplasm adaptation, hilly area, genetic variability, RAPD markers
  Sylhet Agricultural University
  00-00-2013
  00-00-2014
  Variety and Species
  Litchi

To find out the adaptability (growing performance) and the genetic variation among the litchi germplasm in hilly areas of Sylhet Agricultural University, Sylhet, Bangladesh.

Experimental materials: The present research study was carried out during the year 2013-2014 for collection and adaptation of litchi germplasm and find out the genetic diversity in different morphological characters and also for genetic traits among the germplasm by RAPD markers. Total ten varieties of Litchi chinensis were collected from two different geographical locations of Bangladesh. BARI litchi-2 and BARI litchi-3 were collected from BARI (Bangladesh Agricultural Research Institute) regional sub-station at Akbarpur, Moulvibazar and China-3, Bombai, Mongalbaria, Malaysian, Bedana, BAU litchi-1, BAU litchi-2 and BAU litchi-3 from Germplasm Center, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh. Litchi germplasm were planted on 4th November 2013 in slightly hilly areas of Sylhet Agricultural University region in Randomized Complete Block Design (RCBD) with three replications. Each treatment comprised of three, thus involving total of thirty genotypes in the experiment. The research study was carried out in two different patterns such as morphological characteristics and molecular analysis of litchi germplasm. Morphological characteristics: The experiment was laid out in a randomized complete block design with three replications. Spacing between rows was 2 and 1.7 m between plants in a row. Recommended production packages were followed to ensure normal plant growth and development. Data on various characters, such as; plant height, number of branches per plant, number of leaves per plant and trunk diameter was taken from 3 selected plants from each genotype. For all parameters, data was recorded in four occasions such as first day of planting, 90, 180 and 270 Days After Planting (DAP). Diameter of the trunk was measured in centimeter from three portion of the each plant viz. base, middle and top at the same occasion of data collection using Vernier Scalipers. Statistical analysis: Appropriate statistical analyses (computer programme MSTAT-C v.2.10) were performed with the mean data of each character. Least Significant Difference (LSD) test at 5% probability level was applied to compare the differences among treatments means. Molecular analysis of litchi germplasm: Ten germplasm of litchi viz BARI litchi-2, BARI litchi-3, Malaysian, Mongalbaria, China-3, Bedana, BAU litchi-1, BAU itchi-2, BAU litchi-3 and Bombai were used in the study. Fresh and young leaf samples were collected from one out of three replications of each litchi germplasm and used as the source of genomic DNA. Extraction of genomic DNA: Genomic DNA was extracted from the young leaf tissues following standard procedures at the Genetic Engineering Laboratory, Department of Genetics and Plant Breeding, Sylhet Agricultural University, Sylhet, Bangladesh. Approximately 0.3 g of leaf tissues was cut into small pieces, homogenized and digested in extraction buffer [50 mM Tris-HCl, 25 mM Ethylene Diamine Tetra Acetic Acid (EDTA), 300 mM NaCl and 1% SDS (Sodium Dodecyl Sulphate), pH = 8.0]. The DNA was purified by successive extraction with Phenol: Chloroform: Isoamyl alcohol = 25:24:1 (v/v/v); pH near 8.0 with TEN buffers. The DNA was precipitated first absolute (100%) ethanol, pelleted by centrifugation, then reprecipitated in 70% ethanol with 20 μL 3 M sodium acetate. The pellets were air-dried and re-suspended in 50 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0). PCR amplification: Polymerase Chain Reaction (PCR) reactions were performed on each DNA sample in a 10 μL reaction mix containing 1 μL of 10X Ampli Taq polymerase buffer, 0.25 μL of 0.4 μM Primer, 1 μL of 250 μL dNTPs, 0.5 unit of 0.2 μL Ampli Taq DNA polymerase (Takara, Japan) and 4 μL of 50 ng μLG1 plant genomic DNA and a suitable amount of sterile deionized water. The DNA amplification was performed in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf). The reaction mix was preheated at 94°C for 3 min followed 44 cycles of 1 min denaturation at 94°C, 1 min annealing at 34°C and elongation or extension at 72°C for 2 min. After the last cycle, a final step of 5 min at 72°C was added to allow complete extension of all amplified fragments. The PCR products from each sample were confirmed by running 1.5% agarose gel containing 5 μL Ethidium bromide in 1X TBE buffer at 120 V for 1 h. Loading dye (2.5 μL) was added to the PCR products and loaded in the wells. Molecular weight marker DNA (1000 bp DNA ladder on right side) was also loaded on the one side of the gel. The RAPD bands were observed under ultra violet light on a transilluminator and documented by taking photograph using a Gel documentation system. RAPD data analysis: Data from molecular marker techniques requires detailed analysis to establish a genetic relationship among the litchi germplasm. For each primer, polymorphic bands are scored for their presence (1) or absence (0) in all the accessions by visually assessing photographs of the gels. The size of amplification product was determined by comparison with gene ruler (1000 bp DNA ladder). Only distinct, reproducible, well-resolved fragments, in the size range from 200-600 bp, were scored as discrete variables for the 10 accessions. Bands or RAPD markers not identified by both the persons and readers were considered as non-scorable. The scores obtained using all primers in the RAPD analysis were then combined to create a single data matrix. From the band data, monomorphic and polymorphic bands were identified for each type of cultivar. This was used for estimating polymorphic loci (Nei, 1973) gene diversity, genetic identity, genetic distance and constructing a Unweighted Pair Group Method of Arithmetic (UPGMA) means dendrogram among the populations using computer program POPGENE (Version 1.31).

  International Journal of Plant Breeding and Genetics 9 (4): 218-227, 2015; ISSN 1819-3595
  DOI: 10.3923/ijpbg.2015.218.227
Funding Source:
1.   Budget:  
  

It could be concluded that by assessing morphological characteristics among the litchi germplasm, Mongalbaria and BARI litchi-2 have a bright prospect for growing under the hilly areas in Sylhet, Bangladesh. On the other hand, two germplasm Bombai and Malaysian have the highest genetic distance than the others by using RAPD markers. The RAPD markers are the reliable and most effective tools for the plant breeders to reveal the genetic variation among the germplasm that ultimately helpful for further hybridization program. So, to get more clear findings, this experiment should be repeated for further verification up to the fruit settings of litchi germplasm.

  Journal
  


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