Melina Cristina Mancini

SNP genotyping allows an in-depth characterisation of the genome of sugarcane and other complex autopolyploids

Many plant species of great economic value (e.g., potato, wheat, cotton, and sugarcane) are polyploids. Despite the essential roles of autopolyploid plants in human activities, our genetic understanding of these species is still poor. Recent progress in instrumentation and biochemical manipulation has led to the accumulation of an incredible amount of genomic data. In this study, we demonstrate for the first time a successful genetic analysis in a highly polyploid genome (sugarcane) by the quantitative analysis of single-nucleotide polymorphism (SNP) allelic dosage and the application of a new data analysis framework. This study provides a better understanding of autopolyploid genomic structure and is a sound basis for genetic studies. The proposed methods can be employed to analyse the genome of any autopolyploid and will permit the future development of high-quality genetic maps to assist in the assembly of reference genome sequences for polyploid species.

De Novo Assembly and Transcriptome Analysis of Contrasting Sugarcane Varieties

Sugarcane is an important crop and a major source of sugar and alcohol. In this study, we performed de novo assembly and transcriptome annotation for six sugarcane genotypes involved in bi-parental crosses. The de novo assembly of the sugarcane transcriptome was performed using short reads generated using the Illumina RNA-Seq platform. We produced more than 400 million reads, which were assembled into 72,269 unigenes. Based on a similarity search, the unigenes showed significant similarity to more than 28,788 sorghum proteins, including a set of 5,272 unigenes that are not present in the public sugarcane EST databases; many of these unigenes are likely putative undescribed sugarcane genes. From this collection of unigenes, a large number of molecular markers were identified, including 5,106 simple sequence repeats (SSRs) and 708,125 single-nucleotide polymorphisms (SNPs). This new dataset will be a useful resource for future genetic and genomic studies in this species.

Functional markers for gene mapping and genetic diversity studies in sugarcane.

BackgroundThe database of sugarcane expressed sequence tags (EST) offers a great opportunity for developing molecular markers that are directly associated with important agronomic traits. The development of new EST-SSR markers represents an important tool for genetic analysis. In sugarcane breeding programs, functional markers can be used to accelerate the process and select important agronomic traits, especially in the mapping of quantitative traits loci (QTL) and plant resistant pathogens or qualitative resistance loci (QRL). The aim of this work was to develop new simple sequence repeat (SSR) markers in sugarcane using the sugarcane expressed sequence tag (SUCEST database).FindingsA total of 365 EST-SSR molecular markers with trinucleotide motifs were developed and evaluated in a collection of 18 genotypes of sugarcane (15 varieties and 3 species). In total, 287 of the EST-SSRs markers amplified fragments of the expected size and were polymorphic in the analyzed sugarcane varieties. The number of alleles ranged from 2-18, with an average of 6 alleles per locus, while polymorphism information content values ranged from 0.21-0.92, with an average of 0.69. The discrimination power was high for the majority of the EST-SSRs, with an average value of 0.80. Among the markers characterized in this study some have particular interest, those that are related to bacterial defense responses, generation of precursor metabolites and energy and those involved in carbohydrate metabolic process.ConclusionsThese EST-SSR markers presented in this work can be efficiently used for genetic mapping studies of segregating sugarcane populations. The high Polymorphism Information Content (PIC) and Discriminant Power (DP) presented facilitate the QTL identification and marker-assisted selection due the association with functional regions of the genome became an important tool for the sugarcane breeding program.

GBS-based single dosage markers for linkage and QTL mapping allow gene mining for yield-related traits in sugarcane

BackgroundSugarcane (Saccharum spp.) is predominantly an autopolyploid plant with a variable ploidy level, frequent aneuploidy and a large genome that hampers investigation of its organization. Genetic architecture studies are important for identifying genomic regions associated with traits of interest. However, due to the genetic complexity of sugarcane, the practical applications of genomic tools have been notably delayed in this crop, in contrast to other crops that have already advanced to marker-assisted selection (MAS) and genomic selection. High-throughput next-generation sequencing (NGS) technologies have opened new opportunities for discovering molecular markers, especially single nucleotide polymorphisms (SNPs) and insertion-deletion (indels), at the genome-wide level. The objectives of this study were to (i) establish a pipeline for identifying variants from genotyping-by-sequencing (GBS) data in sugarcane, (ii) construct an integrated genetic map with GBS-based markers plus target region amplification polymorphisms and microsatellites, (iii) detect QTLs related to yield component traits, and (iv) perform annotation of the sequences that originated the associated markers with mapped QTLs to search putative candidate genes.ResultsWe used four pseudo-references to align the GBS reads. Depending on the reference, from 3,433 to 15,906 high-quality markers were discovered, and half of them segregated as single-dose markers (SDMs) on average. In addition to 7,049 non-redundant SDMs from GBS, 629 gel-based markers were used in a subsequent linkage analysis. Of 7,678 SDMs, 993 were mapped. These markers were distributed throughout 223 linkage groups, which were clustered in 18 homo(eo)logous groups (HGs), with a cumulative map length of 3,682.04 cM and an average marker density of 3.70 cM. We performed QTL mapping of four traits and found seven QTLs. Our results suggest the presence of a stable QTL across locations. Furthermore, QTLs to soluble solid content (BRIX) and fiber content (FIB) traits had markers linked to putative candidate genes.ConclusionsThis study is the first to report the use of GBS for large-scale variant discovery and genotyping of a mapping population in sugarcane, providing several insights regarding the use of NGS data in a polyploid, non-model species. The use of GBS generated a large number of markers and still enabled ploidy and allelic dosage estimation. Moreover, we were able to identify seven QTLs, two of which had great potential for validation and future use for molecular breeding in sugarcane.

Characterization of the Genetic Variability of a Sugarcane Commercial Cross Through Yield Components and Quality Parameters

The phenotypic characterization as well as the knowledge of the correlation among traits, is the first step to quantify the potential of a cross for further QTL (quantitative trait loci) detection. The present work aimed to evaluate the yield components and quality parameters variability of a mapping population derived from a bi-parental cross between IACSP95-3018 and IACSP93-3046 at plant cane and ratoon cane as well as to estimate the heritabilities and pair-wise correlation among the traits evaluated. The progeny clones differed significantly for the traits measures indicating the existence of significant amount of variability among them as also as the presence of transgressive clones. Broad-sense heritabilities values were generally high for stalk diameter, stalk weight, stalk height, Brix and Pol%Cane in plant cane and ratoon cane. Tones of sugarcane per hectare (TCH) were significantly correlated with stalk weight and stalk number in both years. Regarding to all the yield components, stalk number together with stalk weight were the most important components in the determination of TCH. While fiber and Pol%Cane were negative correlated showing that they are inversely correlated traits.

Genetic variation in a complex polyploid: unveiling the dynamic allelic features of sugarcane

Background 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 (approximately 10 Gb) and a high content of repetitive regions. Gene expression mechanisms are poorly understood in these cultivars. An approach using genomic, transcriptomic and genetic mapping can improve our knowledge of the behavior of genetics in sugarcane. Results The hypothetical HP600 and centromere protein C (CENP-C) genes from sugarcane were used to elucidate the allelic expression and genomic and genetic behavior of this complex polyploid. The genomically 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 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 duplications of HP600 and CENP-C (paralogs). This duplication occurred before the Saccharum genus formation and after the separation of sorghum and sugarcane, resulting in a nonexpressed HP600 pseudogene and a recombined fusion version of CENP-C and orthologous gene Sobic.003G299500 with at least two chimerical gene haplotypes expressed. The genetic map construction supported the difficulty of mapping markers located in duplicated regions of complex polyploid genomes. Conclusion All these findings describe a low synteny region in sugarcane, formed by events occurring in all members of the Saccharum genus. Additionally, evidence of duplicated and truncate gene expression and the behavior of genetic markers in a duplicated region was found. Thus, we describe the complexity involved in sugarcane genetics and genomics and allelic dynamics, which can be useful for understanding the complex polyploid genome.

“Targeted Sequencing by Gene Synteny,” a New Strategy for Polyploid Species: Sequencing and Physical Structure of a Complex Sugarcane Region

Sugarcane exhibits a complex genome mainly due to its aneuploid nature and high ploidy level, and sequencing of its genome poses a great challenge. Closely related species with well-assembled and annotated genomes can be used to help assemble complex genomes. Here, a stable quantitative trait locus (QTL) related to sugar accumulation in sorghum was successfully transferred to the sugarcane genome. Gene sequences related to this QTL were identified in silico from sugarcane transcriptome data, and molecular markers based on these sequences were developed to select bacterial artificial chromosome (BAC) clones from the sugarcane variety SP80-3280. Sixty-eight BAC clones containing at least two gene sequences associated with the sorghum QTL were sequenced using Pacific Biosciences (PacBio) technology. Twenty BAC sequences were found to be related to the syntenic region, of which nine were sufficient to represent this region. The strategy we propose is called “targeted sequencing by gene synteny,” which is a simpler approach to understanding the genome structure of complex genomic regions associated with traits of interest.

New Developments in Sugarcane Genetics and Genomics

Modern sugarcane cultivars (Saccharum spp.) are derived from an interspecific hybridization between Saccharum officinarum and Saccharum spontaneum and pose a significant challenge for both genotyping and data analysis. Due to their large (estimated at approximately 10 Gb) and complex genome, which can include variable ploidy levels and aneuploidy, studies involving molecular markers for genetic and quantitative trait locus (QTL) mapping are extremely laborious. Several advances in the genetics and genomics of sugarcane have recently become possible with the emergence of new sequencing technologies, the use of several types of marker systems, and the use of genotyping data analysis software. Molecular markers and comparative genomics are powerful resources that allow us to explore allelic variation and to thus understand the genome organization of sugarcane. This chapter provides an overview of what is known about the genetic structure and the genomics of sugarcane as well as the main genomics strategies developed for sugarcane. Among the strategies discussed are the use of single-nucleotide polymorphisms (SNPs) and bacterial artificial chromosome (BAC) libraries and the analysis of the syntenic relationships with related species (maize, sorghum, and rice).