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.

Genome-wide approaches for the identification of markers and genes associated with sugarcane yellow leaf virus resistance.

Sugarcane yellow leaf (SCYL), caused by the sugarcane yellow leaf virus (SCYLV) is a major disease affecting sugarcane, a leading sugar and energy crop. Despite damages caused by SCYLV, the genetic base of resistance to this virus remains largely unknown. Several methodologies have arisen to identify molecular markers associated with SCYLV resistance, which are crucial for marker-assisted selection and understanding response mechanisms to this virus. We investigated the genetic base of SCYLV resistance using dominant and codominant markers and genotypes of interest for sugarcane breeding. A sugarcane panel inoculated with SCYLV was analyzed for SCYL symptoms, and viral titer was estimated by RT-qPCR. This panel was genotyped with 662 dominant markers and 70,888 SNPs and indels with allele proportion information. We used polyploid-adapted genome-wide association analyses and machine-learning algorithms coupled with feature selection methods to establish marker-trait associations. While each approach identified unique marker sets associated with phenotypes, convergences were observed between them and demonstrated their complementarity. Lastly, we annotated these markers, identifying genes encoding emblematic participants in virus resistance mechanisms and previously unreported candidates involved in viral responses. Our approach could accelerate sugarcane breeding targeting SCYLV resistance and facilitate studies on biological processes leading to this trait.

The Wild Sugarcane and Sorghum Kinomes: Insights Into Expansion, Diversification, and Expression Patterns.

The protein kinase (PK) superfamily is one of the largest superfamilies in plants and the core regulator of cellular signaling. Despite this substantial importance, the kinomes of sugarcane and sorghum have not been profiled. Here, we identified and profiled the complete kinomes of the polyploid Saccharum spontaneum (Ssp) and Sorghum bicolor (Sbi), a close diploid relative. The Sbi kinome was composed of 1,210 PKs; for Ssp, we identified 2,919 PKs when disregarding duplications and allelic copies, and these were related to 1,345 representative gene models. The Ssp and Sbi PKs were grouped into 20 groups and 120 subfamilies and exhibited high compositional similarities and evolutionary divergences. By utilizing the collinearity between the species, this study offers insights into Sbi and Ssp speciation, PK differentiation and selection. We assessed the PK subfamily expression profiles via RNA-Seq and identified significant similarities between Sbi and Ssp. Moreover, coexpression networks allowed inference of a core structure of kinase interactions with specific key elements. This study provides the first categorization of the allelic specificity of a kinome and offers a wide reservoir of molecular and genetic information, thereby enhancing the understanding of Sbi and Ssp PK evolutionary history.

Sugarcane mosaic virus mediated changes in cytosine methylation pattern and differentially transcribed fragments in resistance-contrasting sugarcane genotypes.

Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it is mainly controlled by using resistant cultivars. Studies on the changes in sugarcane transcriptome provided the first insights about the molecular basis underlying the genetic resistance to SMD; nonetheless, epigenetic modifications such as cytosine methylation is also informative, considering its roles in gene expression regulation. In our previous study, differentially transcribed fragments (DTFs) were obtained using cDNA-amplified fragment length polymorphism by comparing mock- and SCMV-inoculated plants from two sugarcane cultivars with contrasting responses to SMD. In this study, the identification of unexplored DTFs was continued while the same leaf samples were used to evaluate SCMV-mediated changes in the cytosine methylation pattern by using methylation-sensitive amplification polymorphism. This analysis revealed minor changes in cytosine methylation in response to SCMV infection, but distinct changes between the cultivars with contrasting responses to SMD, with higher hypomethylation events 24 and 72 h post-inoculation in the resistant cultivar. The differentially methylated fragments (DMFs) aligned with transcripts, putative promoters, and genomic regions, with a preponderant distribution within CpG islands. The transcripts found were associated with plant immunity and other stress responses, epigenetic changes, and transposable elements. The DTFs aligned with transcripts assigned to stress responses, epigenetic changes, photosynthesis, lipid transport, and oxidoreductases, in which the transcriptional start site is located in proximity with CpG islands and tandem repeats. Real-time quantitative polymerase chain reaction results revealed significant upregulation in the resistant cultivar of aspartyl protease and VQ protein, respectively, selected from DMF and DTF alignments, suggesting their roles in genetic resistance to SMD and supporting the influence of cytosine methylation in gene expression. Thus, we identified new candidate genes for further validation and showed that the changes in cytosine methylation may regulate important mechanisms underlying the genetic resistance to SMD.

Machine learning approaches reveal genomic regions associated with sugarcane brown rust resistance.

Sugarcane is an economically important crop, but its genomic complexity has hindered advances in molecular approaches for genetic breeding. New cultivars are released based on the identification of interesting traits, and for sugarcane, brown rust resistance is a desirable characteristic due to the large economic impact of the disease. Although marker-assisted selection for rust resistance has been successful, the genes involved are still unknown, and the associated regions vary among cultivars, thus restricting methodological generalization. We used genotyping by sequencing of full-sib progeny to relate genomic regions with brown rust phenotypes. We established a pipeline to identify reliable SNPs in complex polyploid data, which were used for phenotypic prediction via machine learning. We identified 14,540 SNPs, which led to a mean prediction accuracy of 50% when using different models. We also tested feature selection algorithms to increase predictive accuracy, resulting in a reduced dataset with more explanatory power for rust phenotypes. As a result of this approach, we achieved an accuracy of up to 95% with a dataset of 131 SNPs related to brown rust QTL regions and auxiliary genes. Therefore, our novel strategy has the potential to assist studies of the genomic organization of brown rust resistance in sugarcane.

Gene Duplication in the Sugarcane Genome: A Case Study of Allele Interactions and Evolutionary Patterns in Two Genic Regions.

Sugarcane (Saccharum spp.) is highly polyploid and aneuploid. Modern cultivars are derived from hybridization between S. officinarum and S. spontaneum. This combination results in a genome exhibiting variable ploidy among different loci, a huge genome size (~10 Gb) and a high content of repetitive regions. An approach using genomic, transcriptomic, and genetic mapping can improve our knowledge of the behavior of genetics in sugarcane. The hypothetical HP600 and Centromere Protein C (CENP-C) genes from sugarcane were used to elucidate the allelic expression and genomic and genetic behaviors of this complex polyploid. The physically linked side-by-side genes HP600 and CENP-C were found in two different homeologous chromosome groups with ploidies of eight and ten. The first region (Region01) was a Sorghum bicolor ortholog region with all haplotypes of HP600 and CENP-C expressed, but HP600 exhibited an unbalanced haplotype expression. The second region (Region02) was a scrambled sugarcane sequence formed from different noncollinear genes containing partial duplications of HP600 and CENP-C (paralogs). This duplication resulted in a non-expressed HP600 pseudogene and a recombined fusion version of CENP-C and the orthologous gene Sobic.003G299500 with at least two chimeric gene haplotypes expressed. It was also determined that it occurred before Saccharum genus formation and after the separation of sorghum and sugarcane. A linkage map was constructed using markers from nonduplicated Region01 and for the duplication (Region01 and Region02). We compare the physical and linkage maps, demonstrating the possibility of mapping markers located in duplicated regions with markers in nonduplicated region. Our results contribute directly to the improvement of linkage mapping in complex polyploids and improve the integration of physical and genetic data for sugarcane breeding programs. Thus, we describe the complexity involved in sugarcane genetics and genomics and allelic dynamics, which can be useful for understanding complex polyploid genomes.

Unraveling the genetic structure of Brazilian commercial sugarcane cultivars through microsatellite markers.

The Brazilian sugarcane industry plays an important role in the worldwide supply of sugar and ethanol. Investigation into the genetic structure of current commercial cultivars and comparisons to the main ancestor species allow sugarcane breeding programs to better manage crosses and germplasm banks as well as to promote its rational use. In the present study, the genetic structure of a group of Brazilian cultivars currently grown by commercial producers was assessed through microsatellite markers and contrasted with a group of basic germplasm mainly composed of Saccharum officinarum and S. spontaneum accessions. A total of 285 alleles was obtained by a set of 12 SSRs primer pairs that taken together were able to efficiently distinguish and capture the genetic variability of sugarcane commercial cultivars and basic germplasm accessions allowing its application in a fast and cost-effective way for routine cultivar identification and management of sugarcane germplasm banks. Allelic distribution revealed that 97.6% of the cultivar alleles were found in the basic germplasm while 42% of the basic germplasm alleles were absent in cultivars. Of the absent alleles, 3% was exclusive to S. officinarum, 33% to S. spontaneum and 19% to other species/exotic hybrids. We found strong genetic differentiation between the Brazilian commercial cultivars and the two main species (S. officinarum: [Formula: see text] = 0.211 and S. spontaneum: [Formula: see text] = 0.216, P<0.001), and significant contribution of the latter in the genetic variability of commercial cultivars. Average dissimilarity within cultivars was 1.2 and 1.4 times lower than that within S. officinarum and S. spontaneum. Genetic divergence found between cultivars and S. spontaneum accessions has practical applications for energy cane breeding programs as the choice of more divergent parents will maximize the frequency of transgressive individuals in the progeny.

Identification, classification and transcriptional profiles of dirigent domain-containing proteins in sugarcane.

Dirigent (DIR) proteins, encoded by DIR genes, are referred to as "dirigent" because they direct the outcome of the coupling of the monolignol coniferyl alcohol into (+) or (-) pinoresinol, the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIR domain-containing or DIR-like proteins are, thus, termed for not having a clear characterization. A transcriptome- and genome-wide survey of DIR domain-containing proteins in sugarcane was carried out, in addition to phylogenetic, physicochemical and transcriptional analyses. A total of 120 non-redundant sequences containing the DIR domain were identified and classified into 64 groups according to phylogenetic and sequence alignment analyses. In silico analysis of transcript abundance showed that these sequences are expressed at low levels in leaves and genes in the same phylogenetic clade have similar expression patterns. Expression analysis of ShDIR1-like transcripts in the culm internodes of sugarcane demonstrates their abundance in mature internodes, their induction by nitrogen fertilization and their predominant expression in cells that have a lignified secondary cell wall, such as vascular bundles of young internodes and parenchymal cells of the pith of mature internodes. Due to the lack of information about the functional role of DIR in plants, a possible relationship is discussed between the ShDIR1-like transcriptional profile and cell wall development in parenchyma cells of sugarcane culm, which typically accumulates large amounts of sucrose. The number of genes encoding the DIR domain-containing proteins in sugarcane is intriguing and is an indication per se that these proteins may have an important metabolic role and thus deserve to be better studied.

Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content.

Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.

Lignification in sugarcane: biochemical characterization, gene discovery, and expression analysis in two genotypes contrasting for lignin content.

Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-high-performance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and form the baseline for the rational metabolic engineering of sugarcane feedstock for bioenergy purposes.

Composição bromatológica e digestibilidade de cana-de-açúcar colhida em duas épocas do ano / Chemical composition and digestibility of sugarcane harvested at two periods of the year

Objetivou-se com este estudo avaliar o efeito da época de corte sobre a composição bromatológica e digestibilidade in vitro de genótipos de cana-de-açúcar, comparar a composição bromatológica e digestibilidade in vitro das frações colmo e folhas, e estabelecer possíveis correlações entre composição química e digestibilidade in vitro da planta inteira de genótipos da cana-de-açúcar. Nove genótipos foram colhidos em maio e setembro de 2006. Em maio, somente a fração planta inteira foi analisada, em setembro foram separados em colmos, folhas ou planta inteira para determinação da composição bromatológica, sacarose (POL) e digestibilidade in vitro. Colmos apresentaram menor teor de fibra em detergente neutro (FDN) e lignina na MS, e maior digestibilidade in vitro da matéria seca (DIVMS) do que folhas. Em contrapartida, apresentaram menor digestibilidade in vitro da FDN (DIVFDN), maior teor de lignina na FDN e menor concentração de proteína bruta (PB). Houve redução no teor e digestibilidade da FDN com o avanço da maturidade, enquanto que DIVMS, POL e lignina aumentaram. A DIVMS se mostrou negativamente correlacionada com FDN e FDN/POL, porém não houve correlação entre DIVFDN e FDN ou FDN/POL. Conclui-se que o avanço da maturidade promoveu redução na DIVFDN e aumento na DIVMS, não existindo correlação genética entre acúmulo de açúcar e digestibilidade in vitro da fibra. Os dados deste artigo indicam não ser esperado que a seleção de genótipos com maior DIVFDN do colmo altere o teor de açúcar do mesmo.

The objectives of this study were to evaluate the effect of time of harvest on chemical composition and in vitro digestibility of sugarcane genotypes, to compare chemical composition and in vitro digestibility of the stem and leaf fractions, and to determine possible correlations between chemical composition and in vitro digestibility of the whole plant in sugarcane genotypes. Nine genotypes were harvested in May and September of 2006. In May, only the whole-plant fraction was analyzed, in September the genotypes were separated in stems, leaves or whole-plant for determination of chemical composition, sucrose (POL) and in vitro digestibility. Stems had lower neutral detergent fiber (NDF) and lignin in the DM, and greater in vitro dry matter digestibility (IVDMD) than leaves. However, stems had lower in vitro NDF digestibility (IVNDFD), higher lignin in the NDF and lower crude protein (CP). The NDF and IVNDFD were reduced with advanced maturity, while IVDMD, POL and lignin were increased. IVDMD was negatively correlated with NDF and NDF/POL, however there was no correlation between IVFDND and NDF or NDF/POL. It can be concluded that with the advance in maturity the IVNDFD was reduced and IVDMD was increased, and there was no genetic correlation between accumulation of sugar and in vitro fiber digestibility. Data from this study indicate that it is not expected that selection of genotypes with greater stem IVNDFD would alter the sugar content of the plant.

Composição bromatológica e digestibilidade de cana-de-açúcar colhida em duas épocas do ano / Chemical composition and digestibility of sugarcane harvested at two periods of the year

Objetivou-se com este estudo avaliar o efeito da época de corte sobre a composição bromatológica e digestibilidade in vitro de genótipos de cana-de-açúcar, comparar a composição bromatológica e digestibilidade in vitro das frações colmo e folhas, e estabelecer possíveis correlações entre composição química e digestibilidade in vitro da planta inteira de genótipos da cana-de-açúcar. Nove genótipos foram colhidos em maio e setembro de 2006. Em maio, somente a fração planta inteira foi analisada, em setembro foram separados em colmos, folhas ou planta inteira para determinação da composição bromatológica, sacarose (POL) e digestibilidade in vitro. Colmos apresentaram menor teor de fibra em detergente neutro (FDN) e lignina na MS, e maior digestibilidade in vitro da matéria seca (DIVMS) do que folhas. Em contrapartida, apresentaram menor digestibilidade in vitro da FDN (DIVFDN), maior teor de lignina na FDN e menor concentração de proteína bruta (PB). Houve redução no teor e digestibilidade da FDN com o avanço da maturidade, enquanto que DIVMS, POL e lignina aumentaram. A DIVMS se mostrou negativamente correlacionada com FDN e FDN/POL, porém não houve correlação entre DIVFDN e FDN ou FDN/POL. Conclui-se que o avanço da maturidade promoveu redução na DIVFDN e aumento na DIVMS, não existindo correlação genética entre acúmulo de açúcar e digestibilidade in vitro da fibra. Os dados deste artigo indicam não ser esperado que a seleção de genótipos com maior DIVFDN do colmo altere o teor de açúcar do mesmo.

The objectives of this study were to evaluate the effect of time of harvest on chemical composition and in vitro digestibility of sugarcane genotypes, to compare chemical composition and in vitro digestibility of the stem and leaf fractions, and to determine possible correlations between chemical composition and in vitro digestibility of the whole plant in sugarcane genotypes. Nine genotypes were harvested in May and September of 2006. In May, only the whole-plant fraction was analyzed, in September the genotypes were separated in stems, leaves or whole-plant for determination of chemical composition, sucrose (POL) and in vitro digestibility. Stems had lower neutral detergent fiber (NDF) and lignin in the DM, and greater in vitro dry matter digestibility (IVDMD) than leaves. However, stems had lower in vitro NDF digestibility (IVNDFD), higher lignin in the NDF and lower crude protein (CP). The NDF and IVNDFD were reduced with advanced maturity, while IVDMD, POL and lignin were increased. IVDMD was negatively correlated with NDF and NDF/POL, however there was no correlation between IVFDND and NDF or NDF/POL. It can be concluded that with the advance in maturity the IVNDFD was reduced and IVDMD was increased, and there was no genetic correlation between accumulation of sugar and in vitro fiber digestibility. Data from this study indicate that it is not expected that selection of genotypes with greater stem IVNDFD would alter the sugar content of the plant.

Seleção de 39 variedades de cana-de-açúcar para a alimentação animal / Selection of 39 varieties of sugarcane for animal feeding

Foi desenvolvido no Instituto de Zootecnia, em Nova Odessa, SP, um experimento para avaliar 39 variedades de canas-de-açúcar, com vistas à alimentação animal. As canas foram avaliadas através de análise sob componentes principais e agrupamento. A análise de agrupamento foi efetuada pelo método UPGA (unweighted pair group method with arithimetic average), levando-se em conta, 4 caracteres, produção de matéria seca, teor de carboidratos totais não estruturais, teor de fibra insolúvel em detergente neutro e degradabilidade efetiva da matéria seca. Levando-se em conta apenas a qualidade e degradabilidade foram selecionados os grupos 6, 7, 8, 9 e 10. Incluindo na avaliação, a produção de matéria seca, os grupos selecionados foram os de número 6, 8 e 9.