Materials and Methods

Seeds were introduced from Bangalore (India) and Pakokku (Myanmar). Leaf samples were collected from one-year-old plants with both germplasms in a small experimental population domesticated at Yuanjiang County in Yunnan Province, China (23°32.580′ N, 102°03.350′ E, elevation 610 m). Genomic DNA samples were extracted using a DP320 DNAsecure Plant kit (Tiangen, China). The isolation of microsatellite loci was performed according to a modified enrichment protocol of fast isolation by AFLP of sequences containing repeats (FIASCO) (Zane et al., 2002). Briefly, about 250 ng of genomic DNA was completely digested with 5 U of MseI restriction enzyme (Fermantas, Canada) in a 20 μL volume, and then 15 μL of digested DNA was ligated to 1 μm MseI amplified fragment length polymorphism (AFLP) adaptor pair (5′-TACTCAGGACTCAT-3′/5′-GACGATGAGTCCTGAG-3′) (Vos et al., 1995) with 2 U of T4 DNA ligase (Fermantas, Canada) in a volume of 30 μL. In succession, 5 μL of 1:10 diluted ligation products acting as templates were amplified with AFLP adaptor-specific primers MseI-N (5′-GATGAGTCCTGAGTAAN-3′) in a 20 μL reaction containing MgCl₂ 1.5 mm, 1.25 μm MseI-N primers, 0.2 mm dNTPs, and 1 U of Taq DNA polymerase (Biomed, China). The polymerase chain reaction (PCR) was performed following the procedure: 95 °C for 3 min, 20 cycles of 94 °C for 30 s, 53 °C for 60 s, 72 °C for 60 s, followed by 72 °C for 5 min.

For enrichment of the fragments containing SSRs, the PCR products, with a size range of 200 to 1000 bp, were denatured at 95 °C for 5 min and were then hybridized with 5′-biotinylated probe (AG)₁₅ in a 250-μL solution (4.16 × SSC and 0.07% SDS) at 48 °C for 2 h. Hybridization products were selectively captured with 600 μL of Streptavidin MagneSphere® Paramagnetic Particles (Promega, Madison, WI), which were prewashed in 150 μL of TEN₁₀₀ (10 mm Tris-HCl, 1 mm EDTA, and 100 mm NaCl, pH 7.5) for three times. The mixture was incubated at room temperature for 30 min with constant gentle agitation. Subsequently, the complex of streptavidin-coated beads and DNA hybridized to the biotinylated probe was separated from nonspecific DNA in a magnetic field with two washing steps, including three times in 500 μL of TEN₁₀₀₀ (10 mm Tris-HCl, 1 mm EDTA, and 1 M NaCl, pH 7.5) for 8 min, and then three times in 500 μL of high stringency buffer (0.2 × SSC and 0.1% SDS) for 5 min. The separated target DNA fragments were released by incubating at 95 °C for 10 min twice, first in 100 μL of TE (10 mm Tris-HCl and 1 mm EDTA, pH 8.0), second in 50 μL of denaturing solution (12 μL of 10 M NaOH, 11.5 μL of acetic acid, and 26.5 μL of TE).

Recovered DNA was subjected to a second round of PCR with MseI-N primers according to the same procedure as the first round of PCR described above. The amplification products, after being purified with DP1502 multifunctional DNA purification and the recirculation kit (BioTeke, China), were directly ligated into PMD18-T vector (Takara, Japan) and then transformed into competent cells of Escherichia coli, strain DH5α (Biomed, China). Clones were selected using blue/white selection after growth on IPTG-XGal media. The white clones were picked out and tested by means of PCR with a primer combination of (AG)₁₀ and M13⁺/M13^– universal primers, for further identifying positive ones containing microsatellites. The insert fragments of positive clones were sequenced using an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA). After sequencing, primer pairs were designed based on the sequences flanking microsatellite repeats with Primer Premier 5.0 (Clarke and Gorley, 2001).

To assess polymorphism of designed primer pairs, 24 M. oleifera individuals (12 each with germplasms of India and Myanmar) in the experimental population were collected to screen polymorphism. PCR reaction was done in a 20-μL volume using a PTC-100 thermal cycler (MJ Research, Cambridge, MA). Each reaction was performed using 25 ng of genomic DNA, 1 × buffer [with 20 mm Tris-HCl, pH 8.4, 20 mm KCl, 10 mm (NH₄)₂SO_4, and 1.5 mm MgCl₂], 0.25 mm dNTP each, 0.5 μm of each primer, and 1.5 U of Taq DNA polymerase (Biomed). The PCR programs took place as follows: initial denaturing at 95 °C for 5 min, followed by 30cycles of 94 °C for 30 s, primer-specific annealing temperature 55 to 61 °C for 30 s, 72 °C for 30 s and a final extension at 72 °C for 8 min and a hold at 4 °C. The amplified products were then electrophoresed in 8% polyacrylamide gels and visualized by silver staining. Electrophoretic patterns were scored and checked with a 20-bp DNA ladder marker (Takara, Tokyo) used to estimate allele sizes.

Population genetic parameters were finally assessed. Allele numbers, expected (H_E) and observed (H_O) heterozygosities, and Hardy-Weinberg equilibrium (HWE) were calculated by GENEPOP, version 3.4, on the web (http://genepop.curtin.edu.au/) (Raymond and Rousset, 1995). Tests for linkage disequilibrium between loci were run in FSTAT, version 2.9.3.2 (Goudet, 1995), with significance level adjusted by sequential Bonferroni corrections (Rice, 1989).

Results and Discussion

Screened by colony PCR, 210 of 288 randomly selected clones contained potential microsatellite motifs. After sequencing, 209 clones were successfully sequenced, and a total of 192 clones (67%) were found to contain SSRs and were subjected to primer designing. In all, 69 pairs of 20-base primers were designed. Of these primers designed for M. oleifera, 46 pairs successfully amplified target regions and, finally, 20 pairs displayed polymorphism. Characteristics of these 20 loci are shown in Table 1. The number of alleles per locus ranged from two to six, with an average of three. The expected (H_E) and observed (H_O) heterozygosities ranged from 0.3608 to 0.7606 (average of 0.5455) and from 0.0000 to 0.8750 (average of 0.4562), respectively. Significant departures from HWE were detected in all 20 loci, among which seven loci significantly deviated from HWE (P < 0.01), probably due to excess of homozygotes (suggesting inbreeding pressure) or the limitation of sample size. In addition, no locus pair showed significant linkage disequilibrium after Bonferroni correction.

Table 1.

Characteristics of 20 polymorphic microsatellite loci in Moringa oleifera.

View Table

The 20 pairs of polymorphic microsatellite primers presented here would be useful for aspects of detailed population genetic studies of M. oleifera. First, these markers are currently being used for the research of pollen-mediated gene flow within populations. Although outcrossing rate, an important parameter of mating systems, has been known for sure, randomization and minimum distance between related individuals needs to be worked out to maximize cross-fertilization among unrelated clones and to minimize selfing or mating among related ramets in designing M. oleifera seed orchards (Muluvi et al., 2004). With these microsatellite primers, paternity analysis of M. oleifera, involving pollen dispersal distance and gene flow pattern, would accumulate fundamental data for design and management of M. oleifera seed orchards. Moreover, these SSR primers would be helpful in revealing introduction traces and genetic components of introduced populations. Investigation by AFLP concluded that at least two sources of germplasm had been introduced to Kenya from India since the turn of 20th century (Muluvi et al., 1999). Now that M. oleifera has been used for over 100 years, information on genetic diversity levels and relatedness of introduced populations should be enriched more to further facilitate reasoned decisions on its selective breeding and conservation.