Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.

Cultivated peanut (Arachis hypogaea L), an important agronomic crop, exhibits a considerable amount of variability for morphological traits and for resistance'to diseases and pests. In contrast, molecular marker assays have detected little variation at the nucleic acid level. Identification of molecular markers would be of great help to peanut breeders, geneticists, and taxonomists. The objectives of this work were to identify simple sequence repeat (SSR) markers in cultivated peanut and to test these markers for their ability to discriminate among accessions. Peanut total genomic DNA libraries were constructed and screened with 3~p-labeled dinucleotide repeats, (GT)I0 and (CTh0. DiNA sequences were obtained from the SSR-containing clones and, when possible, primer pairs were designed on the basis of DNA sequences flanking the repeat motif. Primer pairs were tested in polymerase chain reaction (PCR) assays using a collection of 22 peanut DNAs, representing both cultivated peanut and wild species. In all, six SSR markers, five from the library screening procedure and one additional marker obtained from a search of publicly available DNA sequences, detected polymorphisms among the peanut DNAs. Discrimination power was high among the cultivated peanuts, with 17 unique genotypes represented among the 19 accessions tested. From two to 14 DNA fragments were amplified per SSR marker, and as a group, the six markers may amplify up to 10 putive SSR loci. The SSR markers identified in this study were more effective in detecting molecular variation in cultivated peanut than all other DNA-based markers evaluated to date. C ULTIVATED PEANUT is an important crop internationally for both direct human consumption and as an oilseed crop. Peanuts were domesticated in South America, probably in Southern Bolivia or Northern Argentina (Gregory et al., 1980; Kochert et al., 1996). Peanut culture was subsequently dispersed to many areas of South America, Mexico, and the Caribbean islands. The crop was unknown to the Europeans when they arrived in the New World early in the 16th century. The value of peanuts was quickly recognized, however, and the crop was later distributed to Africa and Asia by the Portuguese and Spanish. Peanuts are now widely grown in both the New and the Old World. China and India are the largest producers. The genus Arachis contains about 70 taxa, all of which are native to South America (Krapovickas and Gregory, 1994). Nearly all Arachis species are diploid, but cultivated peanut is an allotetraploid. Evidence from molecular marker studies of genetic variation suggests that

phisms among 36 sorghum lines were equally low for profiles obtained by means of 30 RAPD primers or 29 DNA markers are being increasingly utilized in cultivar develop- RFLP probes. Vierling et al. (1994) found 73 RAPD ment, quality control of seed production, measurement of genetic diversity for conservation management, varietal identity, and to assist primers discriminated among sorghum lines but those in maintenance of intellectual property protection (IPP). The use of data did not allow lines to be associated into groupings simple sequence repeats (SSRs) for variety profiling can provide high that reflected pedigrees. Associations among 34 lines discrimination, with excellent reproducibility at less cost than for determined by 19 RFLP probes, 21 RAPD primers, and restriction fragment length polymorphisms (RFLPs). The objective 41 ISSRs were markedly different and dependent on of this study was to evaluate the potential utility of SSR technology the source of molecular profile data (Yang et al., 1996). for applications in research, product development, seed production, Ahnert et al. (1996) reported a study of 105 sorghum and genetic resource conservation management for sorghum. Fifty inbreds that used 104 RFLP probes which showed genetically diverse elite sorghum [Sorghum bicolor (L.) Moench]higher levels of polymorphism and associations of lines inbreds were used to compare the discrimination abilities of 15 SSR that were congruent with pedigree information and primers with 104 RFLPs and to compare the associations among lines revealed by these molecular data and by pedigrees. RFLP data allowed

BackgroundThe genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.ResultsA total of 67 new microsatellite markers (mainly TTG motif) were developed for Arachis. Only three of these markers, however, were polymorphic in cultivated peanut. These three new markers plus five other markers characterized previously were evaluated for number of alleles per locus and gene diversity using 60 accessions of A. hypogaea. Genetic relationships among these 60 accessions and a sample of 36 wild accessions representative of section Arachis were estimated using allelic variation observed in a selected set of 12 SSR markers. Results showed that the Brazilian peanut germplasm collection has considerable levels of genetic diversity detected by SSR markers. Similarity groups for A. hypogaea accessions were established, which is a useful criteria for selecting parental plants for crop improvement. Microsatellite marker transferability was up to 76% for species of the section Arachis, but only 45% for species from the other eight Arachis sections tested. A new marker (Ah-041) presented a 100% transferability and could be used to classify the peanut accessions in AA and non-AA genome carriers.ConclusionThe level of polymorphism observed among accessions of A. hypogaea analyzed with newly developed microsatellite markers was low, corroborating the accumulated data which show that cultivated peanut presents a relatively reduced variation at the DNA level. A selected panel of SSR markers allowed the classification of A. hypogaea accessions into two major groups. The identification of similarity groups will be useful for the selection of parental plants to be used in breeding programs. Marker transferability is relatively high between accessions of section Arachis. The possibility of using microsatellite markers developed for one species in genetic evaluation of other species greatly reduces the cost of the analysis, since the development of microsatellite markers is still expensive and time co...

Plant genetic resources conservation is a challenge based on the need to balance acquisition priorities with the constrained level of financial support that is dedicated to long-term maintenance of collections. As such, understanding how variation is partitioned among accessions is essential for effective management. It is generally recognized that most large collections exhibit varying levels of genetic redundancy. To address this issue, all of the sorghum [Sorghum color (L.) Moench] accessions identified as 'Orange' presently maintained by the U.S. National Plant Germplasm System (NPGS) were assayed with 15 simple sequence repeat (SSR) markers. Genotyping was performed with fluorescent primers with five primer sets in each of three multiplex polymerase chain reactions (PCRs) and automated allele sizing. A total of 96 individuals were analyzed, five plants from each of 19 Orange accessions and one individual from an elite inbred line, 'RTx430'. The SSR markers provided substantial genetic resolution among the Orange entries. Average heterozygosity estimates were low, and phenetic analyses (neighbor-joining dendograms) were generally consistent with known historical relationships among accessions. Most accessions were genetically distinct, but two redundant groups (involving a total of five entries) were found among the 19 Orange accessions evaluated. The molecular variance analysis (AMOVA) showed that 90% of the total genetic variation was partitioned among accessions, while one-tenth of the variation resulted from genetic differences between individual plants within accessions. The variance analysis also indicated that it should be possible to reduce the number of Orange accessions held by NPGS by almost half without seriously jeopardizing the overall amount of genetic variation contained in these holdings. This study demonstrated that a limited number of $SR markers can be used in a cost-efficient manner to rapidly assess variation in accessions of Orange sorghum.