Effect of thermotherapy, Leifsonia xyli subsp. xyli titres, sugarcane genotype and diagnostic techniques on ratoon stunt control in Brazil.

AIMS: To examine the interaction of diagnostic techniques, initial titres of Leifsonia xyli subsp. xyli (Lxx), sugarcane genotype and thermotherapy on ratoon stunt (RSD) control. METHODS AND RESULTS: Single buds of RB867515, RB92579 and RB966928 were submitted to 50°C/2 h or 52°C/30 min under factorial block design and five replications; results were checked 9 months later by serological (DBI) and molecular (PCR) techniques. A 10,000 bootstrapping simulations were performed to infer the best plot size based on the experimental coefficient of variation. Analysis of variance showed significance only on initial Lxx titres and RSD control. Despite the absence of significance in the overall analysis, minor differences in control success with different methods and cultivars are predicted to have a major epidemiological impact on RSD, considering successive harvests and vegetative increase. According to an epidemiological interpretation, the 50°C/2 h treatment was more effective, cultivar RB966928 was the most susceptible and the PCR-based method was the most sensitive for pathogen detection. The minimum required plants per plot was 15, indicating high precision of our experiment CONCLUSIONS: Data interpretation considered both the statistical analysis and the epidemiology aspect of RSD in order to improve RSD management. The Brazilian sugarcane industry will benefit from this approach since it is not using it. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that examined multiple factors that affect RSD control. Our work pinpointed the importance of the thermotherapy, its best combination as well as the diagnostic test. Also, the effect of the cultivar to respond to management strategies. Because the epidemiological aspect of RSD was taken into consideration, results of our work can have an impact on RSD control in the field.

Novel Tools for Adjusting Spatial Variability in the Early Sugarcane Breeding Stage.

The detection of spatial variability in field trials has great potential for accelerating plant breeding progress due to the possibility of better controlling non-genetic variation. Therefore, we aimed to evaluate a digital soil mapping approach and a high-density soil sampling procedure for identifying and adjusting spatial dependence in the early sugarcane breeding stage. Two experiments were conducted in regions with different soil classifications. High-density sampling of soil physical and chemical properties was performed in a regular grid to investigate the structure of spatial variability. Soil apparent electrical conductivity (ECa) was measured in both experimental areas with an EM38-MK2® sensor. In addition, principal component analysis (PCA) was employed to reduce the dimensionality of the physical and chemical soil data sets. After conducting the PCA and obtaining different thematic maps, we determined each experimental plot's exact position within the field. Tons of cane per hectare (TCH) data for each experiment were obtained and analyzed using mixed linear models. When environmental covariates were considered, a previous forward model selection step was applied to incorporate the variables. The PCA based on high-density soil sampling data captured part of the total variability in the data for Experimental Area 1 and was suggested to be an efficient index to be incorporated as a covariate in the statistical model, reducing the experimental error (residual variation coefficient, CVe). When incorporated into the different statistical models, the ECa information increased the selection accuracy of the experimental genotypes. Therefore, we demonstrate that the genetic parameter increased when both approaches (spatial analysis and environmental covariates) were employed.

Assessment of Gene Flow to Wild Relatives and Nutritional Composition of Sugarcane in Brazil.

The commercial release of genetically modified organisms (GMO) requires a prior environmental and human/animal health risk assessment. In Brazil, the National Biotechnology Technical Commission (CTNBio) requires a survey of the area of natural occurrence of wild relatives of the GMO in the Brazilian ecosystems to evaluate the possibility of introgressive hybridization between sexually compatible species. Modern sugarcane cultivars, the focus of this study, derive from a series of hybridization and backcrossing events among Saccharum species. The so-called "Saccharum broad sense" group includes around 40 species from a few genera, including Erianthus, found in various tropical regions, particularly South-Eastern Asia. In Brazil, three native species, originally considered to belong to Erianthus, were reclassified as S. angustifolium (Nees) Trin., S. asperum (Nees) Steud., and S. villosum Steud., based on inflorescence morphology. Thus, we have investigated the potential occurrence of gene flow among the Brazilian Saccharum native species and commercial hybrids as a requisite for GMO commercial release. A comprehensive survey was carried out to map the occurrence of the three native Saccharum species in Brazil, concluding that they are sympatric with sugarcane cultivation only from around 14°S southwards, which precludes most Northeastern sugarcane-producing states from undergoing introgression. Based on phenology, we concluded that the Brazilian Saccharum species are unable to outcross naturally with commercial sugarcane since the overlap between the flowering periods of sugarcane and the native species is limited. A phylogenomic reconstruction based on the full plastid genome sequence showed that the three native Saccharum species are the taxa closest to sugarcane in Brazil, being closer than introduced Erianthus or Miscanthus. A 2-year study on eight nutritional composition traits of the 20 main sugarcane cultivars cultivated in Brazil was carried out in six environments. The minimum and maximum values obtained were, in percent: moisture (62.6-82.5); sucrose (9.65-21.76); crude fiber (8.06-21.03); FDN (7.20-20.68); FDA (4.55-16.90); lipids (0.06-1.59); ash (0.08-2.67); and crude protein (0.18-1.18). Besides a considerable amount of genetic variation and plastic responses, many instances of genotype-by-environment interaction were detected.

Expression Quantitative Trait Loci (eQTL) mapping for callose synthases in intergeneric hybrids of Citrus challenged with the bacteria Candidatus Liberibacter asiaticus.

Citrus plants have been extremely affected by Huanglongbing (HLB) worldwide, causing economic losses. HLB disease causes disorders in citrus plants, leading to callose deposition in the phloem vessel sieve plates. Callose is synthesized by callose synthases, which are encoded by 12 genes (calS1- calS12)in Arabidopsis thaliana. We evaluated the expression of eight callose synthase genes from Citrus in hybrids between Citrus sunki and Poncirus trifoliata infected with HLB. The objective of this work was to identify possible tolerance loci combining the expression quantitative trait loci (eQTL) of different callose synthases and genetic Single-Nucleotide Polymorphism (SNP) maps of C. sunki and P. trifoliata. The expression data from all CscalS ranged widely among the hybrids. Furthermore, the data allowed the detection of 18 eQTL in the C. sunki map and 34 eQTL in the P. trifoliata map. In both maps, some eQTL for different CscalS were overlapped; thus, a single region could be associated with the regulation of more than one CscalS. The regions identified in this work can be interesting targets for future studies of Citrus breeding programs to manipulate callose synthesis during HLB infection.

QTL detection for growth and latex production in a full-sib rubber tree population cultivated under suboptimal climate conditions.

BACKGROUND: Rubber tree is cultivated in mainly Southeast Asia and is by far the most significant source of natural rubber production worldwide. However, the genetic architecture underlying the primary agronomic traits of this crop has not been widely characterized. This study aimed to identify quantitative trait loci (QTLs) associated with growth and latex production using a biparental population established in suboptimal growth conditions in Brazil. RESULTS: A full-sib population composed of 251 individuals was developed from crossing two high-producing Asiatic rubber tree cultivars, PR 255 and PB 217. This mapping population was genotyped with microsatellite markers from enriched genomic libraries or transcriptome datasets and single-nucleotide polymorphism (SNP) markers, leading to construction of a saturated multipoint integrated genetic map containing 354 microsatellite and 151 SNP markers. Height and circumference measurements repeated over a six-year period and registration of cumulative latex production during six consecutive months on the same individuals allowed in-depth characterization of the genetic values of several growth traits and precocious latex production. Growth traits, circumference and height, were overall positively correlated, whereas latex production was not correlated or even negatively correlated with growth traits. A total of 86 distinct QTLs were identified, most of which were detected for only one trait. Among these QTLs, 15 were linked to more than one phenotypic trait (up to 4 traits simultaneously). Latex production and circumference increments during the last wintering period were associated with the highest numbers of identified QTLs (eleven and nine, respectively), jointly explaining the most significantly observed phenotypic variances (44.1% and 44.4%, respectively). The most important QTL for latex production, located on linkage group 16, had an additive effect of the male parent PB 217 and corresponded to a QTL at the same position detected in a previous study carried out in Thailand for the biparental population RRIM 600 x PB 217. CONCLUSIONS: Our results identified a set of significant QTLs for rubber tree, showing that the performance of modern Asiatic cultivars can still be improved and paving the way for further marker-assisted selection, which could accelerate breeding programs.

QTL mapping for fruit quality in Citrus using DArTseq markers.

BACKGROUND: Citrus breeding programs have many limitations associated with the species biology and physiology, requiring the incorporation of new biotechnological tools to provide new breeding possibilities. Diversity Arrays Technology (DArT) markers, combined with next-generation sequencing, have wide applicability in the construction of high-resolution genetic maps and in quantitative trait locus (QTL) mapping. This study aimed to construct an integrated genetic map using full-sib progeny derived from Murcott tangor and Pera sweet orange and DArTseq™ molecular markers and to perform QTL mapping of twelve fruit quality traits. A controlled Murcott x Pera crossing was conducted at the Citrus Germplasm Repository at the Sylvio Moreira Citrus Centre of the Agronomic Institute (IAC) located in Cordeirópolis, SP, in 1997. In 2012, 278 F1 individuals out of a family of 312 confirmed hybrid individuals were analyzed for fruit traits and genotyped using the DArTseq markers. Using OneMap software to obtain the integrated genetic map, we considered only the DArT loci that showed no segregation deviation. The likelihood ratio and the genomic information from the available Citrus sinensis L. Osbeck genome were used to determine the linkage groups (LGs). RESULTS: The resulting integrated map contained 661 markers in 13 LGs, with a genomic coverage of 2,774 cM and a mean density of 0.23 markers/cM. The groups were assigned to the nine Citrus haploid chromosomes; however, some of the chromosomes were represented by two LGs due the lack of information for a single integration, as in cases where markers segregated in a 3:1 fashion. A total of 19 QTLs were identified through composite interval mapping (CIM) of the 12 analyzed fruit characteristics: fruit diameter (cm), height (cm), height/diameter ratio, weight (g), rind thickness (cm), segments per fruit, total soluble solids (TSS, %), total titratable acidity (TTA, %), juice content (%), number of seeds, TSS/TTA ratio and number of fruits per box. The genomic sequence (pseudochromosomes) of C. sinensis was compared to the genetic map, and synteny was clearly identified. Further analysis of the map regions with the highest LOD scores enabled the identification of putative genes that could be associated with the fruit quality characteristics. CONCLUSION: An integrated linkage map of Murcott tangor and Pera sweet orange using DArTseq™ molecular markers was established and it was useful to perform QTL mapping of twelve fruit quality traits. The next generation sequences data allowed the comparison between the linkage map and the genomic sequence (pseudochromosomes) of C. sinensis and the identification of genes that may be responsible for phenotypic traits in Citrus. The obtained linkage map was used to assign sequences that had not been previously assigned to a position in the reference genome.

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

BACKGROUND: Sugarcane (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. RESULTS: We 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. CONCLUSIONS: This 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.

Developing a common bean core collection suitable for association mapping studies.

Because of the continuous introduction of germplasm from abroad, some collections have a high number of accessions, making it difficult to explore the genetic variability present in a germplasm bank for conservation and breeding purposes. Therefore, the aim of this study was to quantify and analyze the structure of genetic variability among 500 common bean accessions to construct a core collection. A total of 58 SSRs were used for this purpose. The polymorphism information content (PIC) in the 180 common bean accessions selected to compose the core collection ranged from 0.17 to 0.86, and the discriminatory power (DP) ranged from 0.21 to 0.90. The 500 accessions were clustered into 15 distinct groups and the 180 accessions into four distinct groups in the Structure analysis. According to analysis of molecular variance, the most divergent accessions comprised 97.2% of the observed genetic variability present within the base collection, confirming the efficiency of the selection criterion. The 180 selected accessions will be used for association mapping in future studies and could be potentially used by breeders to direct new crosses and generate elite cultivars that meet current and future global market needs.

Duplicate and conquer: multiple homologs of PHOSPHORUS-STARVATION TOLERANCE1 enhance phosphorus acquisition and sorghum performance on low-phosphorus soils.

Low soil phosphorus (P) availability is a major constraint for crop production in tropical regions. The rice (Oryza sativa) protein kinase, PHOSPHORUS-STARVATION TOLERANCE1 (OsPSTOL1), was previously shown to enhance P acquisition and grain yield in rice under P deficiency. We investigated the role of homologs of OsPSTOL1 in sorghum (Sorghum bicolor) performance under low P. Association mapping was undertaken in two sorghum association panels phenotyped for P uptake, root system morphology and architecture in hydroponics and grain yield and biomass accumulation under low-P conditions, in Brazil and/or in Mali. Root length and root surface area were positively correlated with grain yield under low P in the soil, emphasizing the importance of P acquisition efficiency in sorghum adaptation to low-P availability. SbPSTOL1 alleles reducing root diameter were associated with enhanced P uptake under low P in hydroponics, whereas Sb03g006765 and Sb03g0031680 alleles increasing root surface area also increased grain yield in a low-P soil. SbPSTOL1 genes colocalized with quantitative trait loci for traits underlying root morphology and dry weight accumulation under low P via linkage mapping. Consistent allelic effects for enhanced sorghum performance under low P between association panels, including enhanced grain yield under low P in the soil in Brazil, point toward a relatively stable role for Sb03g006765 across genetic backgrounds and environmental conditions. This study indicates that multiple SbPSTOL1 genes have a more general role in the root system, not only enhancing root morphology traits but also changing root system architecture, which leads to grain yield gain under low-P availability in the soil.

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.

QTL mapping of growth-related traits in a full-sib family of rubber tree (Hevea brasiliensis) evaluated in a sub-tropical climate.

The rubber tree (Hevea spp.), cultivated in equatorial and tropical countries, is the primary plant used in natural rubber production. Due to genetic and physiological constraints, inbred lines of this species are not available. Therefore, alternative approaches are required for the characterization of this species, such as the genetic mapping of full-sib crosses derived from outbred parents. In the present study, an integrated genetic map was obtained for a full-sib cross family with simple sequence repeats (SSRs) and expressed sequence tag (EST-SSR) markers, which can display different segregation patterns. To study the genetic architecture of the traits related to growth in two different conditions (winter and summer), quantitative trait loci (QTL) mapping was also performed using the integrated map. Traits evaluated were height and girth growth, and the statistical model was based in an extension of composite interval mapping. The obtained molecular genetic map has 284 markers distributed among 23 linkage groups with a total length of 2688.8 cM. A total of 18 QTLs for growth traits during the summer and winter seasons were detected. A comparison between the different seasons was also conducted. For height, QTLs detected during the summer season were different from the ones detected during winter season. This type of difference was also observed for girth. Integrated maps are important for genetics studies in outbred species because they represent more accurately the polymorphisms observed in the genitors. QTL mapping revealed several interesting findings, such as a dominance effect and unique segregation patterns that each QTL could exhibit, which were independent of the flanking markers. The QTLs identified in this study, especially those related to phenotypic variation associated with winter could help studies of marker-assisted selection that are particularly important when the objective of a breeding program is to obtain phenotypes that are adapted to sub-optimal regions.

A novel linkage map of sugarcane with evidence for clustering of retrotransposon-based markers.

BACKGROUND: The development of sugarcane as a sustainable crop has unlimited applications. The crop is one of the most economically viable for renewable energy production, and CO2 balance. Linkage maps are valuable tools for understanding genetic and genomic organization, particularly in sugarcane due to its complex polyploid genome of multispecific origins. The overall objective of our study was to construct a novel sugarcane linkage map, compiling AFLP and EST-SSR markers, and to generate data on the distribution of markers anchored to sequences of scIvana_1, a complete sugarcane transposable element, and member of the Copia superfamily. RESULTS: The mapping population parents ('IAC66-6' and 'TUC71-7') contributed equally to polymorphisms, independent of marker type, and generated markers that were distributed into nearly the same number of co-segregation groups (or CGs). Bi-parentally inherited alleles provided the integration of 19 CGs. The marker number per CG ranged from two to 39. The total map length was 4,843.19 cM, with a marker density of 8.87 cM. Markers were assembled into 92 CGs that ranged in length from 1.14 to 404.72 cM, with an estimated average length of 52.64 cM. The greatest distance between two adjacent markers was 48.25 cM. The scIvana_1-based markers (56) were positioned on 21 CGs, but were not regularly distributed. Interestingly, the distance between adjacent scIvana_1-based markers was less than 5 cM, and was observed on five CGs, suggesting a clustered organization. CONCLUSIONS: Results indicated the use of a NBS-profiling technique was efficient to develop retrotransposon-based markers in sugarcane. The simultaneous maximum-likelihood estimates of linkage and linkage phase based strategies confirmed the suitability of its approach to estimate linkage, and construct the linkage map. Interestingly, using our genetic data it was possible to calculate the number of retrotransposon scIvana_1 (~60) copies in the sugarcane genome, confirming previously reported molecular results. In addition, this research possibly will have indirect implications in crop economics e.g., productivity enhancement via QTL studies, as the mapping population parents differ in response to an important fungal disease.

Genetic diversity in cultivated carioca common beans based on molecular marker analysis.

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats - SSRs and amplified fragment length polymorphisms - AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger's modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98% and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm.

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats - SSRs and amplified fragment length polymorphisms - AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger's modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98 percent and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm.