Project description:BackgroundUrochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X-9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species' genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species.FindingsA set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species.ConclusionsThis work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection.
Project description:This study aims to reveal genes related to lignin production in Urochloa humidicola through RNA-Seq. This species is widely used as forrage for cattle, being some species of Urochloa responsible for 85% of pastures in Brazil. Given this importance, lignin is a molecule directly related to low digestibility in cattle. Eight samples were chosen from previous lignin production and forage quality data; four samples had low lignin production, and four had high lignin production. The second extended leaves were collected for RNA extraction using RNeasy® Plant Mini Kit. The cDNA library preparation was generated according to Illumina TruSeq Stranded mRNA Sample Prep kit protocol, and RNA sequencing was performed using HiSeq 2500 sequencer. Quality control was measured by FastQC software v 0.11.8. As Urochloa humidicola does not have a sequenced genome, a transcriptome assembly was built by two approaches: through Trinity v 2.8.4 (Grabherr et al., 2011), and Stringtie v 2.0.4 software (Pertea et al., 2015), using Urochloa ruziziensis as reference genome. Then, the final transcriptome was assembled using those two de novo assembles by PASA v 2.2 (Haas et al., 2003). Salmon v 0.7.2 software (Patro et al., 2017) was used to quantify the sequenced reads. The DEG was identified using DESeq2 package, and genes functions were annotated through Trinotate software (Bryant et al., 2017). In total, approximately 123 million reads were sequenced. The final assembled transcriptome was formed by 48,695 transcripts. The differential expressed genes analysis revealed 258 significatively genes. Here, we highlighted genes related to flavonoid biosynthetic process, regulation of phenylpropanoid metabolic process, and Myb-like DNA-binding domain.
Project description:BACKGROUND: Urochloa 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. FINDINGS: Microsatellite 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 Jaccard's coefficient ranged from 0.000 to 0.913. CONCLUSIONS: This 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.
Project description: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.
| S-EPMC4785737 | biostudies-literature
Project description:Microbial diversity of Brachiaria silage
Project description:Background and aimsSignal grass (Urochloa decumbens) is a widely used pasture grass in tropical and sub-tropical areas due to its high aluminiun (Al) resistance. However, the underlying mechanisms conferring this resistance are not clearly understood.MethodsThe Al concentrations of bulk root tissues and the intracellular compartment were examined, including the impact of a metabolic inhibitor, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Next, we examined changes in the properties of signal grass root tissues following exposure to toxic levels of Al, including the cell wall cation exchange capacity (CEC), degree of methylation and concentrations of cell wall fractions.Key resultsAlthough signal grass was highly resistant to Al, there was a delay of 24-48 h before the expression of this resistance. We found that this delay in the expression of Al resistance was not related to the total Al concentration in the bulk apical root tissues, nor was it related to changes in the Al bound to the cell wall. We also examined changes in other properties of the cell wall, including the CEC, degree of methylation and changes in the concentration of pectin, hemicellulose and cellulose. We noted that concentrations of intracellular Al decreased by approx. 50 % at the same time that the root elongation rate improved after 24-48 h. Using CCCP as a metabolic inhibitor, we found that the intracellular Al concentration increased approx. 14-fold and that the CCCP prevented the subsequent decrease in intracellular Al.ConclusionsOur results indicate that the delayed expression of Al resistance was not associated with the Al concentration in the bulk apical root tissues or bound to the cell wall, nor was it associated with changes in other properties of the cell wall. Rather, signal grass has an energy-dependent Al exclusion mechanism, and this mechanism requires 24-48 h to exclude Al from the intracellular compartment.
Project description:BackgroundForage species of Urochloa are planted in millions of hectares of tropical and subtropical pastures in South America. Most of the planted area is covered with four species (U. ruziziensis, U. brizantha, U. decumbens and U. humidicola). Breeding programs rely on interspecific hybridizations to increase genetic diversity and introgress traits of agronomic importance. Knowledge of phylogenetic relationships is important to optimize compatible hybridizations in Urochloa, where phylogeny has been subject of some controversy. We used next-generation sequencing to assemble the chloroplast genomes of four Urochloa species to investigate their phylogenetic relationships, compute their times of divergence and identify chloroplast DNA markers (microsatellites, SNPs and InDels).ResultsWhole plastid genome sizes were 138,765 bp in U. ruziziensis, 138,945 bp in U. decumbens, 138,946 bp in U. brizantha and 138,976 bp in U. humidicola. Each Urochloa chloroplast genome contained 130 predicted coding regions and structural features that are typical of Panicoid grasses. U. brizantha and U. decumbens chloroplast sequences are highly similar and show reduced SNP, InDel and SSR polymorphism as compared to U. ruziziensis and U. humidicola. Most of the structural and sequence polymorphisms were located in intergenic regions, and reflected phylogenetic distances between species. Divergence of U. humidicola from a common ancestor with the three other Urochloa species was estimated at 9.46 mya. U. ruziziensis, U. decumbens, and U. brizantha formed a clade where the U. ruziziensis lineage would have diverged by 5.67 mya, followed by a recent divergence event between U. decumbens and U. brizantha around 1.6 mya.ConclusionLow-coverage Illumina sequencing allowed the successful sequence analysis of plastid genomes in four species of Urochloa used as forages in the tropics. Pairwise sequence comparisons detected multiple microsatellite, SNP and InDel sites prone to be used as molecular markers in genetic analysis of Urochloa. Our results placed the origin of U. humidicola and U. ruziziensis divergence in the Miocene-Pliocene boundary, and the split between U. brizantha and U. decumbens in the Pleistocene.
Project description: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.