Lucimara Chiari

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.

Identification of Stylosanthes guianensis varieties using molecular genetic analysis

Molecular genetic diversity and population structure analysis were used to clarify the controversial botanical classification of Stylosanthes guianensis. The accessions were clustered in nine groups, each of which was mainly composed of only one of the four botanical varieties.

Using genetic diversity information to establish core collections of Stylosanthes capitata and Stylosanthes macrocephala.

Stylosanthes species are important forage legumes in tropical and subtropical areas. S. macrocephala and S. capitata germplasm collections that consist of 134 and 192 accessions, respectively, are maintained at the Brazilian Agricultural Research Corporation Cerrados (Embrapa-Cerrados). Polymorphic microsatellite markers were used to assess genetic diversity and population structure with the aim to assemble a core collection. The mean values of HO and HE for S. macrocephala were 0.08 and 0.36, respectively, whereas the means for S. capitata were 0.48 and 0.50, respectively. Roger’s genetic distance varied from 0 to 0.83 for S. macrocephala and from 0 to 0.85 for S. capitata. Analysis with STRUCTURE software distinguished five groups among the S. macrocephala accessions and four groups among those of S. capitata. Nei’s genetic diversity was 27% in S. macrocephala and 11% in S. capitata. Core collections were assembled for both species. For S. macrocephala, all of the allelic diversity was represented by 23 accessions, whereas only 13 accessions were necessary to represent all allelic diversity for S. capitata. The data presented herein evidence the population structure present in the Embrapa-Cerrados germplasm collections of S. macrocephala and S. capitata, which may be useful for breeding programs and germplasm conservation.

Isolation and characterization of microsatellite loci in tropical forage Stylosanthes capitata Vogel.

Stylosanthes capitata is an important tropical pasture legume. Knowledge of genetic diversity and structure of S. capitata populations is of great importance for the conservation and germplasm management of this species. Thus, eight microsatellite markers were developed from an S. capitata‐enriched library. They were characterized in 20 accessions from the germplasm collection of the Empresa Brasileira de Pesquisa Agropecuária (Embrapa). The observed and expected heterozygosities ranged from 0.16 to 0.85 and from 0.40 to 0.85, respectively. These microsatellites are the first set of molecular markers from this species and will contribute towards studies of genetic diversity, conservation and breeding of S. capitata.

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.

Diversidade genética molecular entre cultivares e híbridos de Brachiaria spp. e Panicum maximum

The use of molecular markers may serve to direct crossings, confirm new hybrids and identify new genotypes for commercial purposes. In that context, this research aimed to analyze the genetic diversity among cultivars and hybrids of Brachiaria spp. and P. maximum using molecular markers of the type RAPD (Random amplified polymorphic DNA). It was analyzed 22 genotypes with 10 primers, which amplified 178 DNA polymorphic fragments, which were used to estimate the similarity using Jaccard coefficient. The values of similarity ranged from 0.066 to 0.841. The genetic structure among genotypes was estimated by UPGMA (Unweighted pair-group method with arithmetical average) and revealed three distinct groups with high bootstrap values (>89%). The results showed that the RAPD is a fast, relatively inexpensive and useful technique for genetic divergence characterization between different cultivars and hybrids of Brachiaria spp. and P. maximum.

Aluminum tolerance on genotypes of signal grass

O objetivo neste trabalho foi avaliar a tolerância ao aluminio de cinco genotipos de Urochloa decumbens (D24, CD24-2, CD24-27, CD24-45 e D62) e cinco de Urochloa ruziziensis (R30, R44, R46, R50 e R125) em cultivo hidroponico. Desses genotipos, foram coletados perfilhos uniformes que foram transferidos para duas solucoes: solucao 1 (200 μM CaCl2, pH 4,2) e solucao 2 (200 μM CaCl2 + 200 μM AlCl3, pH 4,2). Apos 21 dias, as plantas tiveram suas raizes separadas, coradas e digitalizadas para analise do comprimento da raiz principal e do diâmetro da raiz principal. O experimento foi realizado em delineamento inteiramente casualizado, em fatorial (genotipos × doses) com tres repeticoes. Houve interacao significativa entre genotipo e dose para comprimento e diâmetro da raiz principal, cujos resultados apresentaram, na media, diferenca significativa para a maioria dos genotipos. As medias de comprimento da raiz principal na ausencia e na presenca de aluminio nao foram significativas para D62 (Urochloa decumbens cv. Basilisk), CD24-45 e R46. Para diâmetro da raiz principal, as medias nao foram significativas para CD24-2, CD24-27 e D62. O genotipo D62 foi o unico classificado como tolerante ao aluminio e o R50, o unico sensivel. A maior parte dos genotipos avaliados apresentou de media a baixa tolerância. Essa classificacao foi baseada nos intervalos de confianca (IC 99%) para a media dos indices de tolerância relativa estimados para comprimento e diâmetro da raiz principal. Esses resultados podem ser utilizados na escolha de genitores visando ao desenvolvimento de populacoes segregantes para estudos de heranca e mapeamento de genes e/ou locos relacionados a tolerância ao aluminio em Urochloa.

Microsatellite loci for Urochloa decumbens (Stapf) R.D. Webster and cross-amplification in other Urochloa species

BackgroundForage grasses of the African genus Urochloa (syn. Brachiaria) are the basis of Brazilian beef production, and there is a strong demand for high quality, productive and adapted forage plants. Among the approximately 100 species of the genus Urochloa, Urochloa decumbens is one of the most important tropical forage grasses used for pastures due to several of its agronomic attributes. However, the level of understanding of these attributes and the tools with which to control them at the genetic level are limited, mainly due to the apomixis and ploidy level of this species. In this context, the present study aimed to identify and characterize molecular microsatellite markers of U. decumbens and to evaluate their cross-amplification in other Urochloa species.FindingsMicrosatellite loci were isolated from a previously constructed enriched library from one U. decumbens genotype. Specific primers were designed for one hundred thirteen loci, and ninety-three primer pairs successfully amplified microsatellite regions, yielding an average of 4.93 alleles per locus. The polymorphism information content (PIC) values of these loci ranged from 0.26 to 0.85 (average 0.68), and the associated discriminating power (DP) values ranged from 0.22 to 0.97 (average 0.77). Cross-amplification studies demonstrated the potential transferability of these microsatellites to four other Urochloa species. Structure analysis revealed the existence of three distinct groups, providing evidence in the allelic pool that U. decumbens is closely related to Urochloa ruziziensis and Urochloa brizantha. The genetic distance values determined using Jaccard’s coefficient ranged from 0.06 to 0.76.ConclusionsThe microsatellite markers identified in this study are the first set of molecular markers for U. decumbens species. Their availability will facilitate understanding the genetics of this and other Urochloa species and breeding them, and will be useful for germplasm characterization, linkage mapping and marker-assisted selection.

De novo RNA sequencing and analysis of the transcriptome of signalgrass (Urochloa decumbens) roots exposed to aluminum.

Acidic soils occupy a vast area in the world, and the aluminum (Al) in these soils can directly interact with plant cells and tissues to inhibit their growth and reduce yields. The signalgrass Urochloa decumbens cv. Basilisk (syn. Brachiaria decumbens cv. Basilisk), a widely sown tropical forage grass, is recognized for its high productivity under intensive use, vigorous growth, ease of establishment, and good forage value throughout the year, as well as its exceptional adaptation to infertile acid soils. We sequenced the transcriptome from roots of U. decumbens cv. Basilisk under two conditions, with and without Al, using Illumina paired-end sequencing technology and performed de novo assembly of those reads, which yielded 164,920 transcripts. Of these transcripts, 113,918 were assigned a putative protein function through comparisons with different gene set databases. Additionally, 13,375 simple sequence repeat (SSR) markers were identified. Digital gene expression analyses were conducted to identify 6698 differentially expressed genes between treatments, revealing a great differences in the root transcriptional landscape when exposed to aluminum. An extensive annotation of the differentially expressed genes (DEGs), made possible to identify several transcripts with putative functions correlated to aluminum exposure, most belonging to vesicle transportation, cell wall modifications and metal handling ontologies. In this work, abundant, high-quality transcripts were obtained, providing a reference platform for future biotechnological studies and breeding programs for this species and its close relatives.

Mating systems in tropical forages: Stylosanthes capitata Vog. and Stylosanthes guianensis (Aubl.) Sw.

Melissa de Oliveira Santos-Garcia, Rosângela Maria Simeao Resende, Lucimara Chiari, Maria Imaculada Zucchi and Anete Pereira de SouzaStylosanthes capitata and S. guianensis are important forage legumes for tropical areas. The only available estimates of S. capitata and S. guianensis outcrossing rates were based on morphological markers, and the genus is considered as being mainly self-pollinated. Here we describe an estimation of the outcrossing rate in S. capitata and S. guianensis using microsatellite markers. The outcrossing rates were estimated in S. capitata and S. guianensis open-pollinated populations of 20 progenies consisting of ten individuals each. The multi locus outcrossing rate for S. capitata was estimated using 10 polymorphic loci, whereas five microsatellites were used for S. guianensis. The multi locus outcrossing rates for S. capitata and S. guianensis were 31 and 26%, respectively, suggesting a mixed mating system with predominance of autogamy. Comparison of single locus and multi locus estimates of outcrossing rates indicated that little inbreeding other than selfing occurred. The estimated Wright’s fixation index of the parental generation was lower than expected based on the multi locus outcrossing rate, possibly resulting from the use of some heterozygous breeding genotypes for the study. The data on the outcrossing rate described here are potentially useful for breeding programs and for maintenance of germplasm collections of these Stylosanthes species.