Allelic data revealing interrelatedness in rice species ( Oryza sativa, Oryza glaberrima, Oryza barthii ) and the interspecific hybrids (NERICA)

The current level of genome coverage provided by microsatellite markers in rice is sufficient for DNA fingerprinting, providing Information on diversity and population structure which is expected to assist plant breeders by providing a more rational basis for expanding the gene pool and for identifying material that harbours alleles of value for plant improvement. Allelic data from the species studied revealed that rice interspecific hybrids (NERICA) were more closely related to Oryza sativa than to Oryza glaberrima which was closely associated to Oryza barthii than to O sativa . Comparative polymorphism between species showed that O. sativa, Nerica, Oryza barthii and O. glaberrima produced 51, 45, 40 and 36 alleles with 1461, 238, 305 and 445 polymorphic bands respectively with 17 SSR markers. Alleles ranged from 2 to 4 in O. sativa and nerica; from 2 to 3 in O. glaberrima and 1 to 4 in O. barthii . The average polymorphic information content and resolving power was highest in O. sativa (0.53, 0.63) , followed by nerica (0.52, 0.62), next was O. barthi (0.29, 0.30) and least in O. glaberrima (0.22, 0.22) . Alleles at RM508 were most reliable in profiling O. barthii genotypes. Definitive markers (RM240, RM488) for O. glaberrima and O. sativa (RM587) were obtained. Cluster analysis grouped the rice genotypes into ten clusters at a similar coefficient of 68% with genotypes of the same genetic similarity clustering together. Based on this study therefore, molecular fingerprinting and crop improvement could be advanced for breeding programmes.


INTRODUCTION
Genomic relationship among Oryza, an agronomically important genus with basic chromosome number of twelve has been delineated by various kinds of genetic and cytogenetic analysis including chromosome pairing (Katayama, 1982), morphology (Morishma and Oka, 1970), isozymes (Glaszman, 1987) and DNA polymorphism (Ren et al., 2003). Oryza sativa and Oryza glaberrima are AA genome species with minor sub *Corresponding author. E-mail: s.ramatu@gmail.com. genomic differences that evolved by independent and parallel evolutionary process in Asia and Africa (Ghesquiere et al., 1977). The knowledge of the extent and structure of genetic diversities and species relationships in the genus Oryza is essential not only for understanding the process of evolution but also for the development of appropriate and efficient strategies for the collection, conservation and introgression of useful genes to cultivated crops.
Our hypothesis is that the African rice gene pool is still underutilized. Therefore, the objectives of this study were to evaluate the genetic diversity of rice genotypes based on microsatellite markers and to determine differences in pattern of diversity among and within species. Information was also provided about rare alleles that can be used in cultivar advancement.

MATERIALS AND METHODS
One hundred and fifty (150)

DNA extraction and SSR markers
Genomic DNA was extracted from young leaf tissues obtained from the sreenhouse following the method described by Dellaporta et al. (1982), using 5 mg of leaf tissue from at least three seedlings. Thirty two microsatellite markers were evaluated of which Seventeen (17) polymorphic rice microsatellite markers were obtained. The original sources and motifs for these markers were found in Temnykh et al. (2000) and rice gene database (http:llars.genome.cornell.edu/rice) verified in October 2, 2001). The nucleic acid concentration was quantified using ND-1000 spectrophotometer (Nano-Drop Technologies inc. 2007. www. Nanodrop.com)

Polymerase chain reaction
Polymerase chain reaction was run using 10 µl reaction mixture containing 1 µl 10* PCR buffer, 0.5 µl of 25 m MgCl2, 0.2 µl of 10 mM DNTP's, 0.25 µl of 20 pm primer pair, 0.1 µl of Taq polymerase, 2.10 µl milu-Q water and 5 µl of l0 ng/µl DNA. The reaction was run using Perkin-Elmer thermocycler according to the following amplification procedure: 2 min pre heating at 94°C, followed by 34 cycles of 30 s denaturing at 94°C, 30 s annealing at 55 and 67°C depending on the primer pair used, 30 s initial extension time at 72°C and final extension of 2 min at 72°C. The amplified products were subjected to electrophoresis using a 2% agarose gel in 1* TBE buffer at 78 to 80 volts for 2 to 3 h for good band separation. The gel was stained with 0.5 µl/L ethidium bromide for 5 min and visualized under ultra-violet light using a gel documentation system.

Polymorphic information content (PIC)
The value of a marker for detecting polymorphism (Polymorphic Information Content (PIC) between genotype for each primer combination depending on the number of detectable alleles and the distribution of their frequency was calculated following the formula proposed by Anderson et al. (1993).
where Pij is the frequency of the j th allele for marker 'i' and summation extends over n alleles. Aliyu et al. 13 Band Informativeness: Ib The ability of the primer to distinguish between accessions was assessed by calculating their resolving power (Rp) as Rp=∑Ib where Ib is band informativeness, Ib = 1-[2×(0.5-Pi) ] and Pi is the proportion of accession containing band I (Prevost and Wilkinson, 1999).

Cluster analysis
The number of repeats for each allele was determined by comparing the size of the PCR products with that of IR36 whose repeat number was characterized by Temnykh et al. (2000). The estimated repeats amplified fragments was treated as a unit character and scored as a binary code 1 and 0 for presence and absence respectively. Only prominent and unambiguous bands were scored. Genetic similarities were evaluated using Jaccard similarity coefficient for pair-wise comparisons based on the proportion of shared bands produced by primers generated using 'SMQUAL' sub-program of NTSYS-pc software (Rohlf, 2000).

Comparative polymorphism between species
SSR Polymorphism showed that O. sativa genotypes produced 51 allele and a total of 1461 bands with an average allele per marker of 3, ranging from 2 to 4 (Table  1). Forty five (45) alleles were detected in interspecific hybrids with the number of alleles per marker ranging from 2 to 4 with an average of 2.65 (Table 2). In O. glaberrima genotypes, 36 alleles were obtained with 445 bands and an average allele per marker of 2.12 ranging from 1 to 3 (Table 3). O. barthii had a monomorphic allele at RM 216 with alleles per marker ranging from 1 to 4 and produced a total of 40 alleles with 305 bands with an average allele of 2.4 (   (Tables 1 to 4).

Clustering of rice genotypes
The dendrogram (Figure 1) at 68.5% similarity coefficient

DISCUSSION
Simple sequence repeat (SSR) markers are PCR based markers that can detect a significantly higher degree of polymorphism in rice (Yang et al., 1994) and are suitable for evaluating genetic diversity among closely related rice cultivars (Akagi et al., 1997). In rice, molecular markers have been used to identify accessions (Olufowote et al., 1997), determine genetic structure and pattern of diversity for cultivars of interest (Akagi et al., 1997), and optimize assembly of core collection (Schoen and Brown, 1993). The higher number of alleles observed in O. sativa could result from the marker specificity or might be attributed to natural and human selection, leading to high genetic diversity of cultigens (Semon et al., 2005). The relatively low genetic variation of the cultivated O. glaberrima, compared to O. barthii was also noted by other studies using RFLP (Wang et al., 1992), AFLP (Aggarwal et al., 1999) and SSR (Joshi et al., 2000) analysis that the wider geographical distribution of the Asian taxa compared to others may have contributed to the higher genetic variation (Vaughan, 1994). Selection after domestication has led to the immense diversity in varieties that characterizes many domesticated plant species which as Darwin pointed out can exceed the range of phenotypic variation in their wild ancestors (Rindos, 1984). The presence of low genetic variation in species as observed between O. glaberrima and O. barthii implies that these species may have undergone a relatively recent differentiation (Vaughan, 1994). Specie relatedness as depicted by shared alleles indicated the trend of evolution and progenitors of the species. Following this trend therefore, O. glaberrima was most closely related to O. barthii. Porterez (1970) and Oka (1988) reported that O. barthii is a progenitor of O. glaberrima. The alleles shared between the interspecifics and O. sativa also indicated that the interspecifics might be closer to O. sativa than to O.glaberrima . The larger number of alleles shared between the interspecifics and O. sativa than between the interspecifics and O. glaberrima could be attributed to gene silencing during molecular analysis or that the alleles were lost (Pham and Bougerol, 1993 Semon et al. (2005). There was no association between the PIC value and the number of alleles detected at a polymorphic locus. The observed pattern was consistent with the result obtained by Yang et al. (1994) using only 10 SSR loci in his work but varied with the report of Yu et al. (2003) based on larger sample size. The highest Rp value was also detected in O. sativa and lowest in O. glaberrima. O. sativa had more informative primers than other species studied. Higher informative primers obtained with the interspecifics than O. glaberrima was attributed to the interspecifics possessing more O. sativa alleles. A positive correlation was observed for PIC and Rp value. The presence of rare alleles seemed positively associated with the number of genotypes studied. Rare alleles generated were larger with O. sativa genotypes followed by O. glaberrima. The presence of rare alleles in these species indicated that these species may be useful to plant breeders and geneticists as a rich source of genetic material. Markers associated with rare alleles could also be utilized in marker assisted selection programmes.
Genotypes derived from genetically similar background cluster together as species exhibit a spatial structure of genetic variation (Ren et al., 2003). The different level of diversity is attributed to the rate of mutation, migration, dispersal mechanisms, biotic and abiotic selection intensities which are determined by location, climate and soil (Kork et al., 1999). The grouping of the genotypes could be attributed to geographical regions or distinct ecotypes that have evolved over the course of rice cultivation. Domestication is a complex evolutionary process in which human use of plant and animal species lead to morphological and physiological changes that distinguish domesticated taxa from their wild ancestors (Hancock, 2005). The cluster obtained was consistent with work on genetic variation in Prunus Africana showing that genetic distinctness and differentiation of populations may arise from geographic and ecological isolation (Dawson and Powell, 1999).
Clustering of the interspecifics with O. sativa suggested that molecular analysis was unable to identify the parental allele of O. glaberrima in these genotypes. Another possibility is that O. glaberrima genes were not integrated in the genetic material of the interspecifics because of preferential allele associations (Pharm and Bougerol, 1993) or that the marker was able to identify more of the O. sativa alleles in the interspecifics as the markers were specie specific. Clustering of O. sativa with O. glaberrima have been reported by Semon et al. (2005) that major portions of chromosomes of O. glaberrima could not be distinguished from O. sativa. The position and organization of two ribosomal RNA gene clusters (45sr DNA and 5sr DNA) were found to be similar in the chromosome of the two species (Ohimido and Fukui, 1995). Today, both O. glaberrima and O. sativa are commonly grown in mixtures by farmers in upland and rainfed environments. Natural intermediates between the two species have been reported, but the outbreeding rate is estimated to be low (between 2 and 5%) (Jones et al., 1997). Artificial selection tends to enhance population structure and contribute undoubtedly to sub groupings identified in this study. Many accessions of O. glaberrima have also been reported to carry genetic evidence of some level of admixture with O. sativa which could be accounted for by the introduction of O. sativa into West Africa between the 15th and 17th centuries by the Arab traders and Portuguese navigators off the Atlantic Coast (Porteres, 1970).
The genetic profile of O. glaberrima is also consistent with the cultural history of rice cultivation pattern in West Africa where O. glaberrima is often grown in mixture with O. sativa. The low level of diversity within O. glaberrima may be attributed uniformly to environmental variables (Buso et al., 1998). As a self pollinating specie that have undergone founder effect, the degree of variation between O. glaberrima is expected to exceed the variability observed within species, thus O. glaberrima offers an unusual opportunity to detect and characterize the nature of emerging population structure (Jones et al., 1997).
The intra population diversity observed in O. barthii were expected since the species is predominatly autogamous (Bezancon, 1977). Accessions of O. barthii that out-grouped from other O. barthii accessions to cluster with O. glaberrima may require further classifications. However, O. barthii are progenitors of O. glaberrima (Porteres, 1970) and genetic evidence points to a common ancestral gene pool.