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Molecular Assessment of Maize Inbred Lines (zea Mays L.) Using Microsatellite Markers

Maniruzzaman
Senior Scientific Officer
Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701, Bangladesh

M. G. Azam
Scientific Officer
Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701, Bangladesh

S. Islam
Scientific Officer
Biotechnology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701, Bangladesh

M. G. Hossain
Scientific Officer
Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701, Bangladesh

M. M. Rohman
Senior Scientific Officer
Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701, Bangladesh

Abstract/ Executive Summary:

Genetic diversity analysis and germplasm characterization are essential steps in plant breeding and molecular markers are proved tool to accomplish. The present study was undertaken at the Molecular Breeding Lab of Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI) to determine the genetic relatedness and molecular characterization of 15 maize inbred lines of BARI. In present study, genetic diversity analysis was performed by using 10 SSR primers to evaluate the polymorphisms, among them six primers showed distinct polymorphism between the maize inbred lines. The maize genotypes E81, E144, E08, E167, E102, E142 and E121 were found more diverged (0.9003) compared to other inbred lines. On the other hand, the lowest genetic distance values (0.1501) were found between the genotype E140 and genotype E80 followed by genotype E126 and genotype E140; genotype E140 and genotype E65; genotype E65 and genotype E80 values were identical (0.4502). The genotypes viz. E81, E144, E08, E167, E102, E142 and E121 were found far away from centroid of the cluster and rest of the genotypes were placed around the centroid. The Principal Coordinate Analysis (PCO) helped to visualize four major clusters and showed that seven maize inbred lines (E81, E58, E08, E167, E102, E142 and E121) were far away from the other genotypes. In conclusion, SSR markers enabled discrimination among accessions and provided valuable information for future use in improvement of these genomic resources.

Keywords: Molecular Diversity, Microsatellite Marker, Inbred Maize

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Maize

Objectives:

The objective of the present study was to use microsatellite markers for assessment of genetic diversity among the maize variety and inbred lines.

Methodology:

A total of 15 inbred lines of maize were randomly selected from BARI maize inbred lines. Seeds were grown in plastic pots. Then the pots were kept in the net house. After fifteen to twenty days (3 or 4 leaf stage) the fresh leaf was used for DNA isolation. Total DNA was isolated by CTAB method with slight modifications according to Maaß and Klass (1995). After treatment with 10µg/ml RNase A for half an hour at 37ºC, the DNA was purified with propanol. The purified DNA was dissolved in TE buffer and stored at -20ºC and the concentration was determined fluorometrically (Nano drop).
Ten SSR primer pairs were chosen (p-umc1354,p-umc1566,p-umc1292,p- bnlg1124,p-bnlg1179,phi002,phi037,phi038, phi039and bnlg565) to evaluate the polymorphism among the inbred lines. PCR conditions were optimized according to Hoxha et al. (2003). Here amplifications were performed in 20μl volumes containing 100ng genomic DNA, 2.5 mMdNTPs, 1.5mM MgCl2 , 10 pmol each forward and reverse primers, 3U TaqDNA polymerase and 10X PCR buffer (Genei). Thermal cycling consisted of initial denaturation at 95ºC for 3min, 30 cycles of 95ºC for 1 min, annealing temperature 55ºC for 1 min and 72ºC for 1 min, followed by a final extension at 72ºC for 5 min. PCR products were stored at 4ºC until use. The PCR products were visualized in Polyacrylamide gel electrophoresis (PAGE).
Molecular weight for each amplified allele was measured in base pair using Alpha-Ease FC 5.0 software. The allele frequency data from Power Marker version 3.25 (Liu and Muse, 2005) was used to export the data in binary format (allele presence="I" and allele absence = "0") for analysis with NTSYS-pc version 2.2 (Rohlf, 2002).
The summary statistics including the number of alleles per locus, major allele frequency, gene diversity, polymorphism information content (PIC) values were determined using Power Marker version 3.25 (Liu and Muse, 2005). A similarity matrix was calculated with the Simqual subprogram using the Dice coefficient, followed by cluster analysis with the SAHN subprogram using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering method as implemented in NTSYS-pc was used to construct a dendrogram showing relationship among the genotypes. The similarity matrix was also used for principal coordinate analysis (PCA) with the D Center, Output, and MXPlot subprograms in computer program Numerical Taxonomy and Multivariate Analysis System (NTSYS-pc).

Source of Information: Bangladesh J. Agril. Res. 43(4): 533-542, December 2018 ISSN 0258-7122 (Print), 2408-8293 (Online)

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The two dimensional graphical view of Principal Coordinate Analysis (PCO) showed the spatial distribution of the 15 maize inbred lines along the two principal axes. The genotypes viz. E81, E144, E08, E167, E102, E142 and E121 were found far away from centroid of the cluster and rest of the genotypes were placed around the centroid. The genotypes were placed far away from the centriod were more genetically diverged compared to the genotypes placed near the centriod were likely to be genetically more similar. However, centriod may be defined as the vector representing the middle point of the cluster which contained at least one number for each variable. The connecting line between the each genotype and the centriod represented eigen vectors for the respective genotypes.

Knowledge Management: Journal