Development And Characterization Of Microsatellites In Cultivated Peanut (Arachis Hypogaea L)

Cultivated peanut (Arachis hypogaea L.) is unusual among crop plants because of its extremely low variability for DNA markers although wide differences exist for morphological characters among closely-related genotypes. Microsatellites or single sequence repeats (SSR) were proved to be highly abundant and highly polymorphic DNA markers in both animal and plant genomes and they have been recommended as standard technology for constructing molecular maps. Microsatellites were traditionally isolated by screening partial genomic library with radio-labeled SSR probes, which was inefficient and less useful when dealing with taxa with relatively low frequency of microsatellites such as plants, or when a large number of microsatellites is required for map construction.In the present study, microsatellites were enriched by hybridizing biotin-labeled SSR probes with AFLP fragments and captured the hybridized SSR-containing fragments by magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNAs were largely enriched for microsatellites. Two kinds of enrichment methods were adopted. In the first method, DNAs that used for enrichment were selectively (by three-selective base primer pairs) amplified AFLP fragments and the enriched products were not subject to cloning but recovering directly from polyacrymide gel. A collection of PCR products amplified by 235 AFLP primer pairs were enriched with ten SSR probes and 155 specific bands were recovered and 121 of them were sequenced. Only 9 of them contain non-perfect microsatellite and nine primers were designed based on the sequence data. Seven of them produced PCR products with expected size, but none of them detected polymorphism among 22 cultivated peanut genotypes. Nevertheless, one primer, Pm-8, generate a band that is specific only to two accessions, PI 576613 and 576617. This marker is an STS and may be useful in genotype identification.In the second method, microsatellites were enriched from pre-amplified (single-selective base primer pairs) AFLP fragments with 5 SSR probes [(GA)is, (GT)i5,in(AT)15, (GGC)15, (ATT)15]. The AFLP primers are Hind lll-A/Mse I-N and Mse I-N. After enrichment, the eluted DNAs were cloned into plasmid and recovered by PCR using T3/T7primer. A total of 208 cloned fragments were sequenced and 14 sequences were found to contain microsatellites when the redundant clones were excluded. Fourteen primer pairs including three from minisatellite sequences were designed and were used to amplify 44 cultivated peanut accessions. One of them, Pm-15, detected 4 alleles among the panel of peanut genotypes. The results indicated that GA/CT repeats were the most abundant microsatellites in peanut, followed by GGC/CCG repeats. The polymorphism in peanut detected by SSR primers isolated in this study, as with other molecular markers, was relatively lower than that in most other plant species. Of the two enrichment methods, the second one was proved to be reliable although a few modifications are needed for it to be a routine approach, while the first one is hardly applicable unless the non-specific binding can be avoided or reduced and the DNA recoverage from the gel bands can be improved.