Microsatellite loci and genetic structure of artificial populations of Cotesia flavipes (Hymenoptera, Braconidae).

Cotesia flavipes (Cameron) is a parasitoid wasp used in the biological control of the sugarcane borer (Diatraea saccharalis) (Fabr., 1794). Studies on the genetic diversity of C. flavipes are hampered by the lack of highly polymorphic molecular markers. In this report, a set of 11 microsatellite loci were developed from an enriched library of C. flavipes. Four microsatellite loci were polymorphic and were screened in 212 C. flavipes individuals (183 females and 29 males) that were randomly sampled from seven rearing laboratory populations. The number of alleles ranged from two to three. The average inbreeding coefficient (FIS) among all laboratory populations was 0.120, indicating an excess of homozygotes. The average genetic diversity within the laboratory populations was 0.292, which is lower than the values reported for wild Cotesia spp populations. Genetic diversity was most pronounced within laboratory populations (70 to 90%). Most of the observed alleles were fixed or close to fixation. This low overall genetic diversity may have originated from a founder effect, i.e., the contribution of a small number of individuals (genes and alleles) to the formation of these populations. To our knowledge, this study is the first to provide microsatellite loci and an analysis of the genetic structure of C. flavipes. Our results suggest that new introductions of C. flavipes may increase genetic diversity and improve the efficiency of the biological control of D. saccharalis. In addition, population structure data could be used to estimate the minimum number of wasps to be imported.

Mendelian inheritance, genetic linkage, and genotypic disequilibrium at microsatellite loci in Genipa americana L. (Rubiaceae).

Genipa americana is a tropical tree species that is widely distributed in the humid tropical and subtropical regions of Central and South America. This study investigated Mendelian inheritance, genetic linkage, and genotypic disequilibrium at six microsatellite loci developed for G. americana. Adult trees (188) and regenerants (163) were sampled and genotyped in a fragmented population of the species. We also genotyped open-pollinated seeds from 12 seed-trees during reproductive events in 2010 and 2011. Significant deviations from the expected 1:1 Mendelian segregation were detected in 29.5% of the tests. Significant genetic linkage between pairwise loci was detected in 54.4% of the tests, but no genotypic disequilibrium was detected between pairwise loci for adult trees and regenerants. Overall, the results indicate that the six loci analyzed may be used in studies of G. americana's genetic diversity and structure, its mating system, and in parentage analyses.

Genetic diversity among Puccinia melanocephala isolates from Brazil assessed using simple sequence repeat markers.

Brown rust (causal agent Puccinia melanocephala) is an important sugarcane disease that is responsible for large losses in yield worldwide. Despite its importance, little is known regarding the genetic diversity of this pathogen in the main Brazilian sugarcane cultivation areas. In this study, we characterized the genetic diversity of 34 P. melanocephala isolates from 4 Brazilian states using loci identified from an enriched simple sequence repeat (SSR) library. The aggressiveness of 3 isolates from major sugarcane cultivation areas was evaluated by inoculating an intermediately resistant and a susceptible cultivar. From the enriched library, 16 SSR-specific primers were developed, which produced scorable alleles. Of these, 4 loci were polymorphic and 12 were monomorphic for all isolates evaluated. The molecular characterization of the 34 isolates of P. melanocephala conducted using 16 SSR loci revealed the existence of low genetic variability among the isolates. The average estimated genetic distance was 0.12. Phenetic analysis based on Nei's genetic distance clustered the isolates into 2 major groups. Groups I and II included 18 and 14 isolates, respectively, and both groups contained isolates from all 4 geographic regions studied. Two isolates did not cluster with these groups. It was not possible to obtain clusters according to location or state of origin. Analysis of disease severity data revealed that the isolates did not show significant differences in aggressiveness between regions.

Mendelian inheritance, genetic linkage, and genotypic disequilibrium at nine microsatellite loci of Cariniana legalis (Mart.) O. Kuntze.

Cariniana legalis is one of the largest tropical trees with a wide distribution in the Brazilian Atlantic rainforest. We investigated the Mendelian inheritance, genetic linkage, and genotypic disequilibrium at seven microsatellite loci specifically isolated for C. legalis, and at two previously developed heterologous microsatellite loci. Forty to 100 open-pollinated seeds were collected from 22 seed-trees in two populations. Using the Bonferroni correction, no remarkable deviations from the expected Mendelian segregation, linkage, or genotypic disequilibrium were detected in the nine loci studied. Only 3.7% of the tests were significant for investigations of the Mendelian proportions. On the other hand, only 2.8% of tests for linkage detection showed significance. In addition, among all pairwise tests used for investigating linkage disequilibrium, significance was found in 9.7% of the locus pairs. Our results show clear evidence that the nine simple sequence repeat loci can be used without restriction in genetic diversity, mating system, and parentage analyses.

Characterization of chloroplast DNA microsatellites from Saccharum spp and related species.

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.