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Genetic Diversity Among Rice Cultivars

Analysis of Genetic Diversity Among Rice Cultivars in Ebonyi State Using Inter Simple Sequence Repeat (ISSR) Marker


The genetic diversity and similarity among 38 different Abakaliki rice cultivars cultivated in Ebony State was studied using ISSR ( UBC 835) marker. Seeds of the different rice cultivars were collected from four different local Government areas of Ebonyi State and raised in pot soil in EBSU Biotechnology Research and Development Centre green house for 3 weeks. Young leaves of the rice plants were collected for genomic DNA extraction using CTAB method (Cethyltrimethyl ammonium bromide). The   extracted DNAs from the different accession were amplified with UBC 835 primer using PCR procedure. The PCR products were separated into distinct DNA bands of different sizes in 1.5% agarose gel by electrophoresis. The gel bands were scored for presence or absence of specific bands using Microsoft excel and ladder DNA. The gel result revealed a total of 49 bands and the presence of 6 distinct alleles ranging in size from 450 bp to 1800 bp across the different cultivars. The allele with maximum occurrence was 550 bp which was amplified in 17 accessions while the allele with the minimum occurrence was 1800 bp which occurred only in one accession (19, mkpuru mkpuru). However, the rice cultivars with the highest number of alleles was accession (17, 22, 24, 25 and 28) which had three alleles.  The result also showed the PIC value and percentage polymorphism of the marker to be 0.591 and 100% respectively. This study revealed that the rice cultivars cultivated in Abakaliki are genetically diverse.


  1. Background of the Research

Rice is one of the world’s most important food crops, providing food for more than one third of the world’s population and that it is one of the most important staple food crops of Africa, Asia and South America ( Londo et al, 2006). It is no longer a luxury food but has become the cereal that constitutes a major source of calories for the urban and the rural populations (Sasaki and Burr, 2000). There are two major species of rice, Oryza sativa (Asian rice) and Oryza glaberrima (African rice) (Lin, 2005). Several other minor rice types have been identified with genetic markers (Fukuoka et al., 2001). Rice is the main source of the 35-60% dietary calories consumed by more than 3 billion people, and it is considered as the world’s most diverse crop and probably, the most versatile crop (Li et al., 2003).

Nigeria plays a prominent role in rice production and consumption in West African sub-region, with production statistics of about 50% (Ogunbayo et al., 2005). Ebonyi State, with its capital city- Abakaliki, can be called the home of local rice production in Nigeria. It is made up of savanna and semi tropical vegetation, humid, sandy and marshy soil. It is also adorned with moisture soil for growing of varieties of cash and food crops including rice, yam, cassava and cocoyam (Nwanze et al., 2006). The production of Abakaliki rice, as reported by (Udemeze, 2010), has made Ebonyi State a commercial hub for the agric-sub sector. Moreover, 80 percent of Abakaliki rice is grown organically against the use of synthetic chemical fertilizers, because of the wide distribution of the fertile soil in the State (Imolehin and Wada, 2000).

Molecular markers help to achieve the best understanding of this genetic variation in the different species. Therefore, molecular characterization could reveal their phylogeny and this information would be quite useful in utilizing these germplasms in genetic improvement of the existing rice varieties. Molecular marker based genetic diversity analysis (MMGDA) has the potential for assessing changes in genetic diversity over time and space (Duwick, 1984). Molecular markers, especially DNA-based markers, have been used extensively for the study of genetic diversity, unambiguous identification of germplasm and their protection under the trade related intellectual property rights (TRIPS) of the World Trade Organization (WTO). Mackill (Hammat et al., 1994).The estimation of genetic diversity between different genotypes is the first and foremost process in plant breeding (Rajesh et al, 2012). Among numerous techniques available for assessing the genetic variability and relatedness among crop germplasm, DNA based markers provide very effective and reliable tools for studying genetic diversity in crop germplasm and evolutionary relationships ((Akinwale et al, 2009)). Compared to morphological analysis, molecular markers can reveal differences among the genotypes at molecular level. They provide the information that helps in deciding the distinctiveness of species and their ranking according to the number of close relatives and phylogenetic position (Rahman et al, 2007). Molecular markers also serve as a valuable tool to assess the genetic variation, varietal classification and germplasm identification of rice. A successful breeding program depends on the genetic diversity of a crop for achieving the goals of improving the crop and producing high yielding varieties. Rice plant height is a polygenic measurable trait, likely influenced by environment and determined by the extent and type of its genetic variability (Akinwale et al, 2009).

Inter-simple sequence repeats ISSR are a class of molecular markers based on inter-tandem repeats of short DNA sequences. These regions lie within the microsatellite repeats and offer great potential to determine intra-genomic and inter-genomic diversity compared to other arbitrary primers, since they reveal variation within unique regions of the genome at several loci simultaneously. They exhibit specificity of sequence-tagged-site markers, but need no sequence information for primer synthesis enjoying the advantage of random markers (Zietkiewicz et al., 1994; Goodwin et al., 1997). The primers used in ISSR analysis can be based on any of the SSR motifs (di-, tri-, tetra- or penta-nucleotides) found at microsatellite loci, giving a wide array of possible amplification products, and can be anchored to genomic sequences making either side of the targeted simple sequence repeats (Zietkiewicz et al., 1994). The ISSR method was proven especially useful in the Poaceae family for the analysis of nearly isogenic lines (Akagi et al, 1996) and in differentiation of rice varieties (Parsons et al, 1997). The ISSR markers based on AG, GA and (GATA)n repeats have been reported to be very informative and cost-effective in determining genetic relationships among diverse accessions of rice germplasm (Joshi et al, 2000; Sarla et al, 2005; Reddy et al, 2009).

1.2 Aim and Objectives

The aim of this study was to determine the genetic diversity among rice varieties cultivated in Abakaliki and some other elite lines using Inter Simple Sequence Repeat (ISSR) marker.

The specific objectives of the study are as follow

a. To extract DNA from the different rice varieties.

b. To amplify the specific locus of the extracted DNA and separate of the PCR products by electrophoresis.

c. To estimate genetic diversity among different rice genotype based on the ISSR data.

                                         CHAPTER TWO

2.1 History of Rice

Rice is the main staple food for more than half of the world’s population. It is the world’s most diverse cereal crop and it is cultivated in five ecosystems including irrigated, rainfed lowland, upland, deep-water and tidal wetlands (Khush and Virk, 2000). It is grown as far north as Manchuria in China (50°N) and as far south as Uruguay and New South Wales, Australia (around 35°S) (Khush and Virk, 2000).


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