A joint learning approach for genomic prediction in polyploid grasses.

Poaceae, among the most abundant plant families, includes many economically important polyploid species, such as forage grasses and sugarcane (Saccharum spp.). These species have elevated genomic complexities and limited genetic resources, hindering the application of marker-assisted selection strategies. Currently, the most promising approach for increasing genetic gains in plant breeding is genomic selection. However, due to the polyploidy nature of these polyploid species, more accurate models for incorporating genomic selection into breeding schemes are needed. This study aims to develop a machine learning method by using a joint learning approach to predict complex traits from genotypic data. Biparental populations of sugarcane and two species of forage grasses (Urochloa decumbens, Megathyrsus maximus) were genotyped, and several quantitative traits were measured. High-quality markers were used to predict several traits in different cross-validation scenarios. By combining classification and regression strategies, we developed a predictive system with promising results. Compared with traditional genomic prediction methods, the proposed strategy achieved accuracy improvements exceeding 50%. Our results suggest that the developed methodology could be implemented in breeding programs, helping reduce breeding cycles and increase genetic gains.

An Overview of the Genetics and Genomics of the Urochloa Species Most Commonly Used in Pastures.

Pastures based on perennial monocotyledonous plants are the principal source of nutrition for ruminant livestock in tropical and subtropical areas across the globe. The Urochloa genus comprises important species used in pastures, and these mainly include Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, and Urochloa ruziziensis. Despite their economic relevance, there is an absence of genomic-level information for these species, and this lack is mainly due to genomic complexity, including polyploidy, high heterozygosity, and genomes with a high repeat content, which hinders advances in molecular approaches to genetic improvement. Next-generation sequencing techniques have enabled the recent release of reference genomes, genetic linkage maps, and transcriptome sequences, and this information helps improve our understanding of the genetic architecture and molecular mechanisms involved in relevant traits, such as the apomictic reproductive mode. However, more concerted research efforts are still needed to characterize germplasm resources and identify molecular markers and genes associated with target traits. In addition, the implementation of genomic selection and gene editing is needed to reduce the breeding time and expenditure. In this review, we highlight the importance and characteristics of the four main species of Urochloa used in pastures and discuss the current findings from genetic and genomic studies and research gaps that should be addressed in future research.

A Semi-Automated SNP-Based Approach for Contaminant Identification in Biparental Polyploid Populations of Tropical Forage Grasses.

Artificial hybridization plays a fundamental role in plant breeding programs since it generates new genotypic combinations that can result in desirable phenotypes. Depending on the species and mode of reproduction, controlled crosses may be challenging, and contaminating individuals can be introduced accidentally. In this context, the identification of such contaminants is important to avoid compromising further selection cycles, as well as genetic and genomic studies. The main objective of this work was to propose an automated multivariate methodology for the detection and classification of putative contaminants, including apomictic clones (ACs), self-fertilized individuals, half-siblings (HSs), and full contaminants (FCs), in biparental polyploid progenies of tropical forage grasses. We established a pipeline to identify contaminants in genotyping-by-sequencing (GBS) data encoded as allele dosages of single nucleotide polymorphism (SNP) markers by integrating principal component analysis (PCA), genotypic analysis (GA) measures based on Mendelian segregation, and clustering analysis (CA). The combination of these methods allowed for the correct identification of all contaminants in all simulated progenies and the detection of putative contaminants in three real progenies of tropical forage grasses, providing an easy and promising methodology for the identification of contaminants in biparental progenies of tetraploid and hexaploid species. The proposed pipeline was made available through the polyCID Shiny app and can be easily coupled with traditional genetic approaches, such as linkage map construction, thereby increasing the efficiency of breeding programs.

Corrigendum: Genetic Mapping With Allele Dosage Information in Tetraploid Urochloa decumbens (Stapf) R. D. Webster Reveals Insights Into Spittlebug (Notozulia entreriana Berg) Resistance.

[This corrects the article DOI: 10.3389/fpls.2019.00092.].

Genomic Selection with Allele Dosage in Panicum maximum Jacq.

Genomic selection is an efficient approach to get shorter breeding cycles in recurrent selection programs and greater genetic gains with selection of superior individuals. Despite advances in genotyping techniques, genetic studies for polyploid species have been limited to a rough approximation of studies in diploid species. The major challenge is to distinguish the different types of heterozygotes present in polyploid populations. In this work, we evaluated different genomic prediction models applied to a recurrent selection population of 530 genotypes of Panicum maximum, an autotetraploid forage grass. We also investigated the effect of the allele dosage in the prediction, i.e., considering tetraploid (GS-TD) or diploid (GS-DD) allele dosage. A longitudinal linear mixed model was fitted for each one of the six phenotypic traits, considering different covariance matrices for genetic and residual effects. A total of 41,424 genotyping-by-sequencing markers were obtained using 96-plex and Pst1 restriction enzyme, and quantitative genotype calling was performed. Six predictive models were generalized to tetraploid species and predictive ability was estimated by a replicated fivefold cross-validation process. GS-TD and GS-DD models were performed considering 1,223 informative markers. Overall, GS-TD data yielded higher predictive abilities than with GS-DD data. However, different predictive models had similar predictive ability performance. In this work, we provide bioinformatic and modeling guidelines to consider tetraploid dosage and observed that genomic selection may lead to additional gains in recurrent selection program of P. maximum.

Genetic Mapping With Allele Dosage Information in Tetraploid Urochloa decumbens (Stapf) R. D. Webster Reveals Insights Into Spittlebug (Notozulia entreriana Berg) Resistance.

Urochloa decumbens (Stapf) R. D. Webster is one of the most important African forage grasses in Brazilian beef production. Currently available genetic-genomic resources for this species are restricted mainly due to polyploidy and apomixis. Therefore, crucial genomic-molecular studies such as the construction of genetic maps and the mapping of quantitative trait loci (QTLs) are very challenging and consequently affect the advancement of molecular breeding. The objectives of this work were to (i) construct an integrated U. decumbens genetic map for a full-sibling progeny using GBS-based markers with allele dosage information, (ii) detect QTLs for spittlebug (Notozulia entreriana) resistance, and (iii) seek putative candidate genes involved in defense against biotic stresses. We used the Setaria viridis genome a reference to align GBS reads and selected 4,240 high-quality SNP markers with allele dosage information. Of these markers, 1,000 were distributed throughout nine homologous groups with a cumulative map length of 1,335.09 cM and an average marker density of 1.33 cM. We detected QTLs for resistance to spittlebug, an important pasture insect pest, that explained between 4.66 and 6.24% of the phenotypic variation. These QTLs are in regions containing putative candidate genes related to defense against biotic stresses. Because this is the first genetic map with SNP autotetraploid dosage data and QTL detection in U. decumbens, it will be useful for future evolutionary studies, genome assembly, and other QTL analyses in Urochloa spp. Moreover, the results might facilitate the isolation of spittlebug-related candidate genes and help clarify the mechanism of spittlebug resistance. These approaches will improve selection efficiency and accuracy in U. decumbens molecular breeding and shorten the breeding cycle.

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

BACKGROUND: Forage 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. FINDINGS: Microsatellite 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. CONCLUSIONS: The 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.

Chromosomal distribution and evolution of abundant retrotransposons in plants: gypsy elements in diploid and polyploid Brachiaria forage grasses.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

Variabilidade genética de acessos da cultivar ‘BRS Bela’ de Stylosanthes guianensis usando marcadores moleculares RAPD / Genetic variability of accessions of ‘BRS Bela’ cultivar of Stylosanthes guianensis using RAPD markers

Taxonomicamente, Stylosanthes guianensis está dividida em quatro variedades botânicas: var. guianensis, var. pauciflora, var. canescens e var. microcephala. A cultivar 'BRS Bela' é uma das duas cultivares dessa leguminosa forrageira registradas no Brasil e é composta por uma mistura física de sementes de quatro acessos pertencentes a variedades botânicas ainda desconhecidas. O objetivo deste trabalho foi caracterizar a variabilidade genética entre os quatro acessos que compõem esta cultivar e determinar suas inter-relações com acessos de variedades botânicas conhecidas, usando a técnica de polimorfismos de DNA amplificados ao acaso (RAPD). Foram analisados 10 primers decâmeros em 36 acessos, do banco de germoplasma da Embrapa gado de Corte, de S. guianensis: 14 da variedade botânica pauciflora, 11 da var. guianensis, quatro da var. canescens, três da var. microcephala e os quatro acessos da cultivar 'BRS Bela'. As bandas amplificadas foram analisadas como dados binários e uma matriz de similaridade genética foi gerada, usando coeficiente de Jaccard. Com base na dissimilaridade genética, os acessos foram agrupados pelos métodos de ligação média entre grupos (UPGMA) e Tocher. A média de similaridade genética dentro das variedades botânicas foi de 0,72 para var. pauciflora, 0,63 para var. microcephala, 0,62 para var. canescens e 0,46 para var. guianensis. Entre os quatro acessos da cultivar 'BRS Bela', esta média foi de 0,62. Ambos os métodos de agrupamento geraram resultados similares e tenderam a agrupar os acessos da mesma variedade botânica. Os acessos da cultivar 'BRS Bela' foram agrupados com acessos var. guianensis. Os resultados mostram que há variabilidade genética dentro das variedades botânicas de S. guianensis e entre os acessos da cultivar 'BRS Bela' e que existe uma tendência ao agrupamento por variedade botânica.

Taxonomically Stylosanthes guianensis is divided in four botanical varieties: var. guianensis, var. pauciflora, var. canescens and var. microcephala. The 'BRS Bela' cultivar is one of the two cultivars of this forage legume registered on Brazil, it is made up of a physical mixture of seeds of four accessions of unknown botanical varieties. The objective in this research was to characterize the genetic variability among four accessions of the cultivar 'BRS Bela' and to determine its genetic relationship with others accessions of known botanical varieties, using the random amplified polymorphic DNA (RAPD) technique. Ten decamer primers were evaluated in 36 accessions, from the germoplasm bank of Embrapa Beef Cattle, of S. guianensis: 14 of the botanical variety pauciflora, 11 of var. guianensis, four of var. canescens, three of var. microcephala and the four accessions of cultivar 'BRS Bela'. The amplified bands were analyzed as binary data and a matrix of genetic similarity was generated using the coefficient of Jaccard. Genetic dissimilarity data were utilized for clustering the accessions by not weighted pair-group method with arithmetical average (UPGMA) and Tocher methods. The mean genetic similarity within of the botanic varieties was of 0.72 for var. pauciflora, 0.63 for var. microcephala, 0.62 for var. canescens and 0.46 for var. guianensis. Among the four accessions of cultivar 'BRS Bela' this mean was 0.62. Both methods of clustering generated similar results and showed a tendency to cluster the accessions of the same botanical variety. The accessions of cultivar 'BRS Bela' were grouped with accessions of botanical variety guianensis. The results show that there is genetic variability within of the botanical varieties of S. guianensis and among the accessions of the cultivar 'BRS Bela' and that there is a tendency to the clustering by botanical variety.

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 H(O) and H(E) 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.

Diversidade genética molecular entre cultivares e híbridos de Brachiaria spp. e Panicum maximum / Molecular genetic diversity among cultivars and hybrids of Brachiaria spp. and Panicum maximum

A utilização de marcadores moleculares pode servir para direcionar cruzamentos, confirmar novos híbridos e identificar genótipos para fins comerciais. Nesse contexto, o objetivo deste trabalho foi analisar a diversidade genética entre cultivares e híbridos de Brachiaria spp. e Panicum maximum usando marcadores moleculares do tipo RAPD (Polimorfismos de DNA amplificados ao acaso). Foram 22 genótipos analisados com 10 primers, os quais amplificaram 178 fragmentos polimórficos de DNA, que foram usados para estimar a similaridade genética por meio do coeficiente de Jaccard. Os valores de similaridade obtidos variaram de 0,066 a 0,841. A estrutura genética entre todos os genótipos estudados foi estimada pelo método UPGMA (Método de agrupamento com médias aritméticas não ponderadas), revelando três grupos distintos com altos valores de bootstrap (>89 por cento). Os resultados demonstraram que a técnica RAPD oferece uma maneira rápida, relativamente barata e útil para a caracterização da diversidade genética entre as diferentes cultivares e híbridos de Brachiaria ssp. e P. maximum analisados.

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 percent). 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.

Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine.

Schistosomiasis is a major public health problem that affects mainly developing countries. There are 200 million people worldwide infected with schistosomes resulting in more than 250,000 deaths per year. Although schistosomicidal drugs exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study we isolated a cDNA clone encoding the Schistosoma mansoni lung-stage Sm22.6 protein, which is 100% and 79% identical with the 22.6 kDa adult worm tegument antigen of S. mansoni and S. japonicum, respectively. Further, we produced recombinant (r) Sm22.6 and constructed an Sm22.6 DNA vaccine. Western blot analysis confirmed the identity of purified MBP-Sm22.6 fusion protein using anti-MBP (maltose binding protein) and anti-rSm22.6 antibodies. Additionally, C57BL/6 mice were immunized and specific anti-Sm22.6 IgG responses were produced when both vaccination strategies were used. Importantly, only rSm22.6 vaccine provided levels of protection against challenge infection (34.5%). Mice immunized with rSm22.6 induced production of IgG1 and IgG2a and synthesis of IFN-gamma and IL-4 in cultured mouse splenocytes. Finally, rSm22.6 vaccination induced a Th0 type of immune response and protective immunity that suggests Sm22.6 as a potential candidate to compose an anti-schistosome vaccine.

Genetic variability in five species of anostomidae (ostariophysi - characiformes)

A variabilidade genética de 5 espécies da família Anostomidae pertencentes aos gêneros Schizodon (S. intermedius e S. nasutus) e Leporinus (L. friderici, L. elongatus e L. obtusidens) coletadas em uma localidade do rio Tibagi (Paraná, Brasil) foi analisada comparativamente utilizando dados protéicos de 7 sistemas que codificam 19 locos no fígado, músculo e coraçäo. Dos locos identificados, 9 säo polimórficos, com valores estimados de proporçäo de locos polimórficos (P) que variaram de 16.7 por cento em S. intermedius a 36.85 por cento em L. friderici, e a heterozigosidade média observada (Ho) foi de 0.0027 ñ 0.015 e 0.109 ñ 0.042, nessas mesmas espécies. O valor estimado de identidade genética (I) entre L. friderici e S. intermedius (0.749) e S. nasutus (0.787) sugere que estas säo espécies congenéricas. As características morfológicas determinam que estas espécies pertencem a gêneros distintos, no entanto os dados de identidade e distância genética obtidos demonstram que essas três espécies, no nível genético-bioquímico, têm uma maior similaridade.