Genetic-genomic studies in Urochloa decumbens : an important tropical forage grass

Orientador: Anete Pereira de SouzaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Urochloa decumbens (sinonímia Brachiaria decumbens) é uma gramínea forrageira de origem africana, predominantemente tetraploide e apomítica, que desempenha papel relevante na pecuária brasileira. Apesar da enorme importância, estudos em nível molecular são limitados devido à complexidade genética da espécie, falta de investimentos, além do programa de melhoramento ser muito recente. Nesse contexto, aplicações práticas de ferramentas genômicas estão notadamente atrasadas, em contraste com outras espécies que já avançam para seleção assistida por marcadores e seleção genômica. Esse trabalho objetivou contribuir para um melhor entendimento genético-genômico de U. decumbens, a fim de fornecer informações úteis aos programas de melhoramento genético da espécie. Para tanto, um conjunto de marcadores microssatélites foi desenvolvido a partir de uma biblioteca genômica enriquecida em motivos repetitivos. Cento e treze locos microssatélites foram desenvolvidos, dos quais 89 foram polimórficos entre os 34 genótipos analisados. A transferibilidade dos locos foi avaliada nas espécies U. dictyoneura, U. humidicola, U. brizantha e U. ruziziensis, sendo que as maiores taxas de amplificação foram observadas nessas duas últimas, confirmando a proximidade genética com U. decumbens reportada em trabalhos anteriores. Um mapa genético integrado foi construído utilizando-se marcadores SNPs (Single Nucleotide Polymorphisms) oriundos da técnica de GBS (Genotyping-by-Sequencing) e locos relacionados à resistência à cigarrinha-das-pastagens (Notozulia entreriana) foram mapeados. Para a descoberta dos marcadores SNPs foram utilizados cinco pseudo-referências: genoma de Panicum hallii, genoma de P. virgatum, genoma de Setaria viridis, genoma de S. italica e um transcriptoma de U. decumbens. A ploidia e a dosagem de cada loco bi-alélico foi estimada pelo software SuperMASSA. Após as filtragens, o alinhamento que possibilitou a identificação do maior número de marcadores SNPs foi com o pseudo-referência S. viridis. O Software TetraploidSNPMap permitiu mapear 1000 marcadores com diferentes dosagens alélicas, distribuídos em nove grupos de homologia. A extensão total do mapa foi 1335,2 cM com densidade de um marcador a cada 1,3 cM. O grupo de homologia 9 (GH9) apresentou 187 marcadores SNPs, sendo o grupo mais saturado, enquanto que o GH6 foi o que apresentou menos marcadores, 63. Utilizando o mapeamento por intervalo (IM), três QTLs para resistência as cigarrinhas-das-pastagens foram mapeados. A proporção da variação explicada pelo QTL variou de 4,66% a 6,24%. Este estudo é o primeiro a reportar o desenvolvimento de marcadores microssatélites específicos para U. decumbens, a construção de um mapa genético intraespecífico para a espécie com SNPs com diferentes dosagens alélicas, bem como o mapeamento de QTLs. Essas ferramentas poderão auxiliar na elaboração de estratégias a serem introduzidas nos programas de melhoramento, de forma a aumentar a eficiência dos processos de seleção e acelerar o desenvolvimento de novas cultivaresAbstract: Urochloa decumbens (syn. Brachiaria decumbens) is a forage grass of African origin, predominantly tetraploid and apomictic, that plays a relevant role in Brazilian livestock production. Despite the enormous importance, studies at the molecular level are limited due to the genetic complexity of the species, lack of investments, and the breeding program is very recent. In this context, practical applications of genomic tools are markedly delayed, in contrast to other species that are already advancing for marker-assisted selection and genomic selection. This work aimed to contribute to a better genetic-genomic understanding of U. decumbens, in order to provide useful information to the breeding program of the species. For this, a set of microsatellite markers was developed from a genomic library enriched in repetitive motifs. One hundred and thirteen microsatellite loci were developed, of which 89 were polymorphic among the 34 genotypes analyzed. The transferability of the loci was evaluated in the species U. dictyoneura, U. humidicola, U. brizantha and U. ruziziensis, and the highest amplification rates were observed in the latter two, confirming the genetic proximity with U. decumbens, already reported in previous studies. An integrated genetic map was constructed using SNPs (Single Nucleotide Polymorphisms) from the GBS (Genotyping-by-Sequencing) technique and loci related with spittlebug (Notozulia entreriana) resistance were mapped. For the discovery of the SNPs markers four pseudo-references were used: Panicum hallii genome, P. virgatum genome, Setaria viridis genome, S. italica genome and a U. decumbens transcriptome. Ploidy and dosage of each bi-allelic loci was estimated using the SuperMASSA software. After the filtrations, the alignment that allowed the identification of a greater number of SNPs markers was the genome of S. viridis. TetraploidSNPMap Software allowed mapping of 1000 markers, with different allelic dosages, distributed in nine homology groups. The total length of the map was 1335,2 cM with density of one marker every 1.3 cM in average. Homology group nine (G9) presented 187 SNPs markers, the most saturated group, whereas HG6 was the one with the lowest markers, 63. Using interval mapping (IM), three QTLs for resistance to spittlebug were mapped. The proportion of variation explained by QTL ranged from 4.66% to 6.24%. This study is the first to report the development of specific microsatellite markers to U. decumbens, as well as to construct an intraspecific genetic map for the species with the SNPs with different allelic dosages as well as the mapping of QTL. These tools can be helpful to establish strategies in breeding programs and increase the efficiency of selection processes and accelerate the development of new cultivarsDoutoradoGenetica Vegetal e MelhoramentoDoutora em Genética e Biologia Molecular02008/52197-4CAPESFAPES

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

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