Genotypic Diversity Among Wild Populations of Buffelgrass (Cenchrus ciliaris L. Link) in Al-Qassim Region

MATERIALS AND METHODS

Population sampling: Five populations of C. ciliaris located in Al-Qassim region (The Central of Saudi Arabia), were sampled during September of 2007. Sampling tried to cover the current distribution of buffelgrass (wild populations) in Al-Qassim region (Buraydah-1, Buraydah-2, Al-Molida, AL-Qaraa and Al-Shamasia). Leaf tissue was collected from 50 plants, using 10 individuals per population. To increase the likelihood of picking leaves from different clones, leaf samples were taken from individuals at least 10 m apart. Samples were immediately stored in liquid nitrogen and once in the lab, they were transferred to an ultra cold freezer (-80°C). In this study, populations were defined as discrete groups of plants covering an area of at least 1 ha.

DNA extraction: Bulk leaf samples from 10 plants of each of buffelgrass population were used. The bulk sample of leaves was first ground into fine powder with liquid nitrogen. Genomic DNA was extracted adding 260 μL of CTAB extraction buffer and 975 μL of STE buffer. The mixture was then agitated and subsequently centrifuged at 12,000 rpm for 8 min. The supernatant was eliminated and the precipitated was resuspended in 250 μL of CTAB buffer and 750 μL of STE buffer. Samples were centrifuged at 12,000 rpm for 8 min, the supernatant was eliminated and the precipitated was resuspended in 600 μL of 2x CTAB buffer. The samples were treated with ribonuclease 7000 μ mL^-1 (4 μL sample^-1) at 37°C for 20 min to digest RNA. After this treatment, 25 μL of proteinase-K (20 mg mL^-1) was added and incubated at 65°C for 30 min. The samples were placed on ice for 15 min. DNA was isolated using chloroform: octanol 24:1 separation (adding 600 μL sample^-1 and homogenising), centrifuged at 9000 rpm for 12 min and the supernatant was transferred to a fresh centrifuge tube. DNA was precipitated with 600 μL of cold isopropanol (-20°C) and maintained for 12 h at -20°C. After that time, samples were centrifuged at 12,500 rpm for 7 min and the supernatant was eliminated. The pellet was washed agitating gently for 5 min with 70% ethanol (-20°C). The pellet was dried, resuspended in 100 μL of PCR grade water and stored at 4°C. DNA concentration of each extract was quantified using an Eppendorf biophotometer.

RAPD analysis: RAPD analysis was run in a thermal cycler (Thermolyne Amplitron).The reaction mixture (25 μL) will be contained 1x PCR buffer with Mg Cl₂ (50 mM K Cl, 10 mM Tris-HCl (pH 9.0), 2 mM Mg Cl₂ and Triton X-100), 200 μM each of dATP, dCTP, dGTP and dTTP, 30 ng template DNA, 50 pM of oligonucleotide primer (Operon Tech., CA, USA) and 1.5 unit of Taq polymerase. The mixtures was subjected to the following conditions: hold at 94°C for 2 min, followed by 40 cycles of 94°C for 1 min, 36°C for 1 min and 72°C for 2 min and a final hold at 72°C for 5 min. PCR products will be visualized along with a DNA marker on 2% agarose gel with 1X TAE buffer and was detected by staining with the ethidium bromide.

ISSR assay: The ISSR-PCR method was carried out according to Nagaoka and Ogihara (1997). Amplification was carried out in 25 μL reaction volumes, containing 1X Taq polymerase buffer (50 mM KCl, 10 mM Tris, pH 7.5, 1.5 mM Mg Cl₂) and 1 unit of Taq polymerase (Pharmacia Biotech, Germany) supplemented with 0.01% gelatin, 0.2 mM of each dNTPs (Pharmacia Biotech, Germany), 50 pmol of ISSR primers and 50 ng of total genomic DNA. Amplification was performed in a thermal cycler (Thermolyne Amplitron).

Data analysis: RAPD and ISSR data were scored for presence (1), absence (0) or as a missing observation (9) and each band was regarded as a locus. Two matrices, one for each marker, were generated. The Genetic Similarities (GS) were calculated according to Nei and Li (1979):

where, Nij is the number of bands present in both genotypes i and j, Ni is the number of bands present in genotypes i and Nj is the number of bands present in genotype j. Based on the similarity matrix, a dendrogram showing the genetic relationships between genotypes, was constructed using the unweighted pairgroup method with arithmetic average (UPGMA) though the software NTSYS-pc version 2.11 (Rohlf, 2000).

RESULTS

Identification and evaluation of RAPD and ISSR markers for diversity estimates: In RAPD analysis, fifteen primers of arbitrary nucleotide sequence were used to amplify DNA segments from five populations of buffelgrass. The number of amplification bands per primer varied between 0 and 10. Ten primers out of 15 were selected for further analysis based on the intensity, size and number of amplified products (Table 1). Analysis of the 10 selected primers among the five buffelgrass populations included in this study generated 75 bands, 11 of which were polymorphic (14.6%).

Table 1:	RAPD primers with the number of amplified products, polymorphic fragments among buffelgrass populations

Fig. 1:	Polymorphism revealed using RAPD primers (OPA-10, OPA-16 and OPA-17) to amplify genomic DNA purified from buffelgrass populations (left to right) Buraydah-1(C1) and Buraydah-2 (C2) Al-Molida (C3), AL-Qaraa (C4) and Al-Shamasia (C5). The first lane is 1 kbp ladder DNA marker

Table 2:	ISSR primers with the number of amplified products and polymorphic fragments

Examples of polymorphism are shown in Fig. 1. Polymorphism between populations can arise through nucleotide changes that prevent amplification by introducing a mismatch at one priming site; deletion of a priming site; insertions that render priming sites too distant to support amplification; and insertions or deletions that change the size of the amplified product (Williams et al., 1990).

In ISSR analysis, a total of 34 bands were observed, with 5.6 bands per primer (Table 2). Six out of 34 bands (17.6%) were polymorphic. The number of amplification bands per primer varied between 0 and 11. The trinucleotide repeats (CTC)n primer had more bands than (GAG)n and (CAC)n primers and dinucleotide repeats (CA)n and (GA)n primers (Table 2). On the other hand, tetranucleotide repeats did not amplify with DNA of buffelgrass populations. This might indicate that di- and trinucleotide-based ISSR-PCR markers could provide potential marker in buffelgrass genome.

Genetic diversity: The pair-wise genetic distance estimates of the buffelgrass populations in this study were analyzed and are given in Table 3. The genetic similarity coefficients among all buffelgrass ranged from 0.89 to 0.97. Maximum similarity was observed between: Buraydah-1 and Buraydah-2 populations (0.95) and Buraydah-2 and Al-Shamasia populations (0.97). Buraydah-2 population was quite distinct from AL-Qaraa population. Cluster analysis was conducted to generate a dendogram elucidating for relationships among buffelgrass populations. The dendogram constructed with UPGMA analysis revealed two clusters (Fig. 2). Cluster I contained Buraydah-1 (C1) and Buraydah-2 (C2) and Al-Shamasia (C5) populations.

Table 3:	Simple matching coefficients of similarity determined from analysis using RAPD and ISSR loci

Fig. 2:	Dendrogram constructed from similarity coefficients and showing the clustering of buffelgrass populations

Cluster II contained Al-Molida (C3) and AL-Qaraa (C4) populations.

DISCUSSION

The molecular data from this study indicate that buffelgrass is generally genetically invariant in all five populations studied. The low level of genetic variation is perhaps not surprising given the presumed apomictic breeding system of buffelgrass (Poulin et al., 2005). Most apomictic species, however, have some sexual capability and this breeding system is usually associated with populations of coexisting, genetically variable clones (Richards, 1997). The molecular data presented here indicate that genetic factors are unlikely to be driving the variation in buffelgrass populations in the central of Saudi Arabia. Populations of buffelgrass are most likely completely apomictic and somatic mutations are probably rare, although rare sexual recombination or somatic mutation cannot be ruled out (Poulin et al., 2005). Also, Gutierrez-Ozuna et al. (2009) found that the levels and distribution of genotypic and genetic diversity among pasture and roadside populations fall within the range.

Several factors may affect levels of genotypic diversity among populations of buffelgrass (Gutierrez-Ozuna et al., 2009). Founder events during colonization and the frequency of sexual reproduction (Hignight et al., 1991) may affect levels of variation within populations while patterns of migration may affect levels of differentiation. Gutierrez-Ozuna et al. (2009) estimated the genetic diversity among Buffelgrass (C. ciliaris) populations. They found that 27 reproducible bands generated with three primers, 16 (59.26%) were polymorphic. While, in the current study reported that 17.6% of bands were polymorphic. Similar to my results were reported by Poulin et al. (2005) using Inter-Simple Sequence Repeat markers (ISSRs) to examine genetic variation in Pennisetum setaceum populations from the three areas (Arizona, California and Hawaii). They found that screening of 16 primers revealed no genetic variation within any population or between any geographical areas. The current study reported that the trinucleotide repeats (CTC)n primer had more bands than (GAG)n and (CAC)n primers and dinucleotide repeats (CA)n and (GA)n primers. However, the repeats (GA)_n and (CA)_n were the most abundant in rice (Nagaraju et al., 2002) and date palm (Trifi et al., 2000) genomes.