L. Jungmann

Genetic Diversity and Population Structure of the Brachiaria brizantha Germplasm

Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. (syn. Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster) is a species used primarily as forage in tropical America and Southeast Asia. B. brizantha has been extensively researched since the 1980s with the initiation of the Tropical Forages Breeding Program conducted by the Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária; EMBRAPA), holding one of the largest germplasm collections in the world. This work has identified 15 new microsatellite markers for this species, which have been used in addition to five previously reported markers, to estimate the genetic similarities among 172 accessions and six cultivars of this species. Similarity index values ranged from 0.40 to 1.00. Two duplications were found in the germplasm. A Bayesian analysis performed using the STRUCTURE 2.3.3 program revealed the presence of three clusters with different allelic pools. This analysis is valuable for the performance of crosses to explore heterosis; however, the mode of reproduction of the accessions and ploidy barriers must be observed for effective exploration. A grouping analysis using the neighbor-joining method was consistent with the STRUCTURE analysis, and a combination approach suggested that this germplasm collection does not exhibit considerable genetic variability despite the presence of three distinct allelic pools. The lack of correlation between the genetic and geographic distances is also discussed.

Development of microsatellite markers for Brachiaria humidicola (Rendle) Schweick

We describe the first panel of nuclear simple sequence repeats (SSRs) loci for Brachiaria humidicola (Rendle) Schweick., a warmseason grass with facultative apomixis, variation in ploidy levels (6X–9X), and important forage grass species in the Tropics. Of 38 pairs of primers obtained by using an enriched-library methodology, 27 revealed polymorphism in 58 accessions of the Germplasm Collection of B. humidicola held at Embrapa Beef Cattle, Brazil. Eleven loci amplified in B. dictyoneura, a closely related species with unclear taxonomic boundaries with B. humidicola. Transferability to other three Brachiaria species was also evaluated. The developed microsatellites are potentially useful for genetic studies of B. humidicola, as well as phylogenetic evaluations, conservation and breeding applications.

Isolation and characterization of microsatellite loci in the tropical forage legume Stylosanthes guianensis (Aubl.) Sw.

Stylosanthes guianensis is an important tropical pasture legume. Knowledge of genetic diversity and structure of S. guianensis populations is of great importance for the conservation and germplasm management of this species. Thus, 20 microsatellite markers were developed from a S. guianensis enriched library. The microsatellites were characterized in 20 accessions from the germplasm collection of the Empresa Brasileira de Pesquisa Agropecuária (Embrapa). The average number of alleles per locus varied from 2 to 7, with an average of 4 alleles per locus. The observed and expected heterozygosities ranged from 0 to 0.60 and 0.10 to 0.85, respectively. This new set of microsatellites will contribute towards studies of genetic diversity and conservation of S. guianensis.

New microsatellite markers developed from Urochloa humidicola (Poaceae) and cross amplification in different Urochloa species

BackgroundUrochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.FindingsMicrosatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccards coefficient ranged from 0.000 to 0.913.ConclusionsThis work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap

The first set of nuclear simple sequence repeat (SSR) loci for Brachiaria brizantha (Hochst. ex A. Rich.) Stap is described. A microsatellite-enriched library was constructed and 19 loci were characterized. About 13 SSR loci were found to be polymorphic and across-taxa amplification tests showed that six of them can be transferred to four other species of Brachiaria. This new SSR resource will be a powerful tool for population genetic studies of B. brizantha, for interspecific genetic studies within the genus Brachiaria, for mapping and for marker assisted selection in breeding.

Polymorphic microsatellite loci for Stylosanthes macrocephala Ferr. et Costa, a tropical forage legume

Microsatellites were developed for Stylosanthes macrocephala, aiming at developing tools for studying the genetic diversity of this species. A total of 13 polymorphic microsatellite markers were isolated from a S. macrocephala enriched genomic library. The isolated microsatellites were characterized in 20 accessions of the S. macrocephala germplasm collection belonging to the Empresa Brasileira de Pesquisa Agropecuária (Embrapa) Cerrados. The number of alleles per locus varied from 2 to 11, with an average of 4 alleles per locus. The observed and expected heterozygosities ranged from 0 to 0.25 and 0.05 to 0.90, respectively. Cross-amplification of the S. macrocephala polymorphic microsatellites was evaluated in three other Stylosanthes species. The microsatellites reported herein are the first set of microsatellite markers developed for S. macrocephala and are potentially useful for further studies on genetic diversity, conservation and breeding of this species.

Evidence of Allopolyploidy in Urochloa humidicola Based on Cytological Analysis and Genetic Linkage Mapping

The African species Urochloa humidicola (Rendle) Morrone & Zuloaga (syn. Brachiaria humidicola (Rendle) Schweick.) is an important perennial forage grass found throughout the tropics. This species is polyploid, ranging from tetra to nonaploid, and apomictic, which makes genetic studies challenging; therefore, the number of currently available genetic resources is limited. The genomic architecture and evolution of U. humidicola and the molecular markers linked to apomixis were investigated in a full-sib F1 population obtained by crossing the sexual accession H031 and the apomictic cultivar U. humidicola cv. BRS Tupi, both of which are hexaploid. A simple sequence repeat (SSR)-based linkage map was constructed for the species from 102 polymorphic and specific SSR markers based on simplex and double-simplex markers. The map consisted of 49 linkage groups (LGs) and had a total length of 1702.82 cM, with 89 microsatellite loci and an average map density of 10.6 cM. Eight homology groups (HGs) were formed, comprising 22 LGs, and the other LGs remained ungrouped. The locus that controls apospory (apo-locus) was mapped in LG02 and was located 19.4 cM from the locus Bh027.c.D2. In the cytological analyses of some hybrids, bi- to hexavalents at diakinesis were observed, as well as two nucleoli in some meiocytes, smaller chromosomes with preferential allocation within the first metaphase plate and asynchronous chromosome migration to the poles during anaphase. The linkage map and the meiocyte analyses confirm previous reports of hybridization and suggest an allopolyploid origin of the hexaploid U. humidicola. This is the first linkage map of an Urochloa species, and it will be useful for future quantitative trait locus (QTL) analysis after saturation of the map and for genome assembly and evolutionary studies in Urochloa spp. Moreover, the results of the apomixis mapping are consistent with previous reports and confirm the need for additional studies to search for a co-segregating marker.

Microsatellite markers in tropical legume (Centrosema pubescens Benth): development, characterization, and cross-species amplification in Centrosema sp

Centrosema pubescens Benth is a forage legume widespread in tropical America. Twenty-six polymorphic microsatellite markers were isolated and characterized in 15 genotypes of C. pubescens from the Cerrados Research Center Germplasm Bank of the Brazilian Agricultural Research Corporation (Embrapa). The number of alleles observed for each locus ranged from 2 to 5, with an average of 3 alleles per locus. The polymorphism information content (PIC) varied between 0.39 and 0.86 (average 0.57) and the discriminating power (D) ranged from 0.45 to 0.98 (average 0.68). The observed heterozygosity (Ho) and the expected heterozygosity (He) were 0.01–0.81 and 0.10–0.86, respectively. A cross-amplification test in 11 Centrosema species suggested potential transferability of these microsatellites. The data indicated that the polymorphic microsatellite markers developed in this work should be useful for assessing genetic diversity in further breeding programs and germplasm conservation.

Development of Microsatellite Markers for Brachiaria brizantha and Germplasm Diversity Characterization of this Tropical Forage Grass

Grasses of the African genus Brachiaria are the most widely planted forages in Brazil. We previously reported the construction of microsatellite-enriched libraries for five Brachiaria species. Now the development of microsatellite markers for B. brizantha and their use for the genetic characterization of morphologically divergent accessions of the germplasm collection of Embrapa are presented. Fifteen pairs of primers were designed and assayed on 23 genotypes. From the fifteen loci evaluated, ten were polymorphic. The divergence between genotypes was estimated using Jaccards coefficient of similarity and UPGMA method was used for clustering genotypes. The results showed that microsatellites are powerful tools for characterizing genetic diversity of Brachiaria species.