Determination of Morpho-Physiological Traits for Assessing Drought Tolerance in Sugarcane.

Drought is a significant constraint to sugarcane productivity. Therefore, understanding how different varieties of sugarcane respond to drought stress can facilitate breeding programs and set up criteria for selecting drought-tolerant varieties. In the present study, we examined eight morpho-physiological traits to distinguish 40 sugarcane genotypes categorized into four groups based on significant differences in cane yield under non-stressed conditions and reduction of cane yield under drought-stressed conditions. The study was conducted during the formative stage in a greenhouse, encompassing both control and drought conditions. Drought treatments resulted in significant changes and differences in the mean values of various morpho-physiological traits. The hierarchical clustering analysis, utilizing stay-green traits such as higher chlorophyll fluorescence ratio (Fv/Fm), leaf chlorophyll content (SPAD), leaf relative water content (RWC), and lower leaf rolling score (LR), leaf drying score (LD), and drought recovery score (DR), successfully grouped 40 sugarcane genotypes into four major clusters, similar to the previously categorized groups. Correlation analysis showed significant relationships among cane yield, reduction of cane yield under drought conditions, and the stay-green traits. Our results demonstrated that morpho-physiological traits contributing to the "stay-green" phenotypes could be useful as selection criteria for drought tolerance in sugarcane.

A SNP variation in the Sucrose synthase (SoSUS) gene associated with sugar-related traits in sugarcane.

Background: Sugarcane (Saccharum spp.) is an economically significant crop for both the sugar and biofuel industries. Breeding sugarcane cultivars with high-performance agronomic traits is the most effective approach for meeting the rising demand for sugar and biofuels. Molecular markers associated with relevant agronomic traits could drastically reduce the time and resources required to develop new sugarcane varieties. Previous sugarcane candidate gene association analyses have found single nucleotide polymorphism (SNP) markers associated with sugar-related traits. This study aims to validate these associated SNP markers of six genes, including Lesion simulating disease 1 (LSD), Calreticulin (CALR), Sucrose synthase 1 (SUS1), DEAD-box ATP-dependent RNA helicase (RH), KANADI1 (KAN1), and Sodium/hydrogen exchanger 7 (NHX7), in a diverse population in 2-year and two-location evaluations. Methods: After genotyping of seven targeted SNP markers was performed by PCR Allelic Competitive Extension (PACE) SNP genotyping, the association with sugar-related traits and important cane yield component traits was determined on a set of 159 sugarcane genotypes. The marker-trait relationships were validated and identified by both t-test analysis and an association analysis based on the general linear model. Results: The mSoSUS1_SNPCh10.T/C and mSoKAN1_SNPCh7.T/C markers that were designed from the SUS1 and KAN1 genes, respectively, showed significant associations with different amounts of sugar-related traits and yield components. The mSoSUS1_SNPCh10.T/C marker was found to have more significant association with sugar-related traits, including pol, CCS, brix, fiber and sugar yield, with p values of 6.08 × 10-6 to 4.35 × 10-2, as well as some cane yield component traits with p values of 1.61 × 10-4 to 3.35 × 10-2. The significant association is consistent across four environments. Conclusion: Sucrose synthase (SUS) is considered a crucial enzyme involved in sucrose metabolism. This marker is a high potential functional marker that may be used in sugarcane breeding programs to select superior sugarcane with good fiber and high sugar contents.

A Large-Scale Candidate-Gene Association Mapping for Drought Tolerance and Agronomic Traits in Sugarcane.

Dissection of the genetic loci controlling drought tolerance traits with a complex genetic inheritance is important for drought-tolerant sugarcane improvement. In this study, we conducted a large-scale candidate gene association study of 649 candidate genes in a sugarcane diversity panel to identify genetic variants underlying agronomic traits and drought tolerance indices evaluated in plant cane and ratoon cane under water-stressed (WS) and non-stressed (NS) environments. We identified 197 significant marker-trait associations (MTAs) in 141 candidate genes associated with 18 evaluated traits with the Bonferroni correction threshold (α = 0.05). Out of the total, 95 MTAs in 78 candidate genes and 62 MTAs in 58 candidate genes were detected under NS and WS conditions, respectively. Most MTAs were found only in specific water regimes and crop seasons. These MTAs explained 7.93-30.52% of phenotypic variation. Association mapping results revealed that 34, 59, and 104 MTAs involved physiological and molecular adaptation, phytohormone metabolism, and drought-inducible genes. They identified 19 pleiotropic genes associated with more than one trait and many genes related to drought tolerance indices. The genetic and genomic resources identified in this study will enable the combining of yield-related traits and sugar-related traits with agronomic value to optimize the yield of sugarcane cultivars grown under drought-stressed and non-stressed environments.

A draft chromosome-scale genome assembly of a commercial sugarcane.

Sugarcane accounts for a large portion of the worlds sugar production. Modern commercial cultivars are complex hybrids of S. officinarum, S. spontaneum, and several other Saccharum species, resulting in an auto-allopolyploid with 8-12 copies of each chromosome. The current genome assembly gold standard is to generate a long read assembly followed by chromatin conformation capture sequencing to scaffold. We used the PacBio RSII and chromatin conformation capture sequencing to sequence and assemble the genome of a South East Asian commercial sugarcane cultivar, known as Khon Kaen 3. The Khon Kaen 3 genome assembled into 104,477 contigs totalling 7 Gb, which scaffolded into 56 pseudochromosomes containing 5.2 Gb of sequence. Genome annotation produced 242,406 genes from 30,927 orthogroups. Aligning the Khon Kaen 3 genome sequence to S. officinarum and S. spontaneum revealed a high level of apparent recombination, indicating a chimeric assembly. This assembly error is explained by high nucleotide identity between S. officinarum and S. spontaneum, where 91.8% of S. spontaneum aligns to S. officinarum at 94% identity. Thus, the subgenomes of commercial sugarcane are so similar that using short reads to correct long PacBio reads produced chimeric long reads. Future attempts to sequence sugarcane must take this information into account.

Identification of Candidate Gene-Based Markers for Girth Growth in Rubber Trees.

Girth growth is an important factor in both latex and timber production of the rubber tree. In this study, we performed candidate gene association mapping for girth growth in rubber trees using intron length polymorphism markers (ILP) in identifying the candidate genes responsible for girth growth. The COBL064_1 marker developed from the candidate gene (COBL4) regulating cellulose deposition and oriented cell expansion in the plant cell wall showed the strongest association with girth growth across two seasons in the Amazonian population and was validated in the breeding lines. We then applied single molecule real-time (SMRT) circular consensus sequencing (CCS) to analyze a wider gene region of the COBL4 to pinpoint the single nucleotide polymorphism (SNP) that best explains the association with the traits. A SNP in the 3' UTR showing linkage disequilibrium with the COBL064_1 most associated with girth growth. This study showed that the cost-effective method of ILP gene-based markers can assist in identification of SNPs in the candidate gene associated with girth growth. The SNP markers identified in this study added useful markers for the improvement of girth growth in rubber tree breeding programs.

Candidate gene association of gene expression data in sugarcane contrasting for sucrose content.

Association mapping of gene expression data, generated from transcriptome and proteome studies, provides a means of understanding the functional significance and trait association potential of candidate genes. In this study, we applied candidate gene association mapping to validate sugarcane genes, using data from the starch and sucrose metabolism pathway, transcriptome, and proteome. We performed multiplex PCR targeted amplicon sequencing of 109 candidate genes, using NGS technology. A range of statistical models, both single-locus and multi-locus, were compared for minimization of false positives in association mapping of four sugar-related traits with different heritability. The Fixed and random model Circulating Probability Unification model effectively suppressed false positives for both low- and high-heritability traits. We identified favorable alleles of the candidate genes involved in signalling and transcriptional regulation. The results will support genetic improvement of sugarcane and may help clarify the genetic architecture of sugar-related traits.

Differential expression between drought-tolerant and drought-sensitive sugarcane under mild and moderate water stress as revealed by a comparative analysis of leaf transcriptome.

Sugarcane contributes 80% of global sugar production and to bioethanol generation for the bioenergy industry. Its productivity is threatened by drought that can cause up to 60% yield loss. This study used RNA-Seq to gain a better understanding of the underlying mechanism by which drought-tolerant sugarcane copes with water stress. We compared gene expression in KPS01-12 (drought-tolerant genotype) and UT12 (drought-sensitive genotype) that have significantly different yield loss rates under drought conditions. We treated KPS01-12 and UT12 with mild and moderate water stress and found differentially expressed genes in various biological processes. KPS01-12 had higher expression of genes that were involved in water retention, antioxidant secondary metabolite biosynthesis, and oxidative and osmotic stress response than UT12. In contrast, the sensitive genotype had more down-regulated genes that were involved in photosynthesis, carbon fixation and Calvin cycle than the tolerant genotype. Our obtained expression profiles suggest that the tolerant sugarcane has a more effective genetic response than the sensitive genotype at the initiation of drought stress. The knowledge gained from this study may be applied in breeding programs to improve sugarcane production in drought conditions.

Detection and validation of EST-SSR markers associated with sugar-related traits in sugarcane using linkage and association mapping.

Sugar-related traits are of great importance in sugarcane breeding. In the present study, quantitative trait loci (QTL) mapping validated with association mapping was used to identify expressed sequence tag-simple sequence repeats (EST-SSRs) associated with sugar-related traits. For linkage mapping, 524 EST-SSRs, 241 Amplified Fragment Length Polymorphisms, and 10 genomic SSR markers were mapped using 283 F1 progenies derived from an interspecific cross. Six regions were identified using Multiple QTL Mapping, and 14 unlinked markers using single marker analysis. Association analysis was performed on a set of 200 accessions, based on the mixed linear model. Validation of the EST-SSR markers using association mapping within the target QTL genomic regions identified two EST-SSR markers showing a putative relationship with uridine diphosphate (UDP) glycosyltransferase, and beta-amylase, which are associated with pol and sugar yield. These functional markers can be used for marker-assisted selection of sugarcane.

Genome-wide association mapping of latex yield and girth in Amazonian accessions of Hevea brasiliensis grown in a suboptimal climate zone.

Latex yield and growth are the key complex traits in commercial rubber production. The present study is the first to report genome-wide association mapping of latex yield and girth, for 170 Amazonian accessions grown in a suboptimal area characterized by limited rainfall and a lengthy dry season. Targeted sequence enrichment to capture gene transcripts generated 14,155 high quality filtered single nucleotide polymorphisms (SNPs) of which 94.3% resided in coding regions. The rapid decay of linkage disequilibrium over physical and genetic distance found in the accessions was comparable to those previously reported for several outcrossing species. A mixed linear model detected three significant SNPs in three candidate genes involved in plant adaptation to drought stress, individually explaining 12.7-15.7% of the phenotypic variance. The SNPs identified in the study will help to extend understanding, and to support genetic improvement of rubber trees grown in drought-affected regions.

ACC oxidase and miRNA 159a, and their involvement in fresh fruit bunch yield (FFB) via sex ratio determination in oil palm.

Oil palm (Elaeis guineesis Jacq.) is the most productive oil-bearing crop, yielding more oil per area than any other oil-bearing crops. However, there are still efforts to improve oil palm yield, in order to serve consumer and manufacturer demand. Oil palm produces female and male inflorescences in an alternating cycle. So, high sex ratio (SR), the ratio of female inflorescences to the total inflorescences, is a favorable trait in term of increasing yields in oil palm. This study aims to understand the genetic control for SR related traits, such as fresh fruit bunch yield (FFB), by characterizing genes at FFB quantitative trait loci (QTLs) on linkage 10 (chromosome 6) and linkage 15 (chromosome 10). Published oil palm sequences at the FFB QTLs were used to develop gene-based and simple sequence repeat (SSR) markers. We used the multiple QTL analysis model (MQM) to characterize the relationship of new markers with the SR traits in the oil palm population. The RNA expression of the most linked QTL genes was also evaluated in various tissues of oil palm. We identified EgACCO1 (encoding aminocyclopropane carboxylate (ACC) oxidase) at chromosome 10 and EgmiR159a (microRNA 159a) at chromosome 6 to be the most linked QTL genes or determinants for FFB yield and/or female inflorescence number with a phenotype variance explained (PVE) from 10.4 to 15 % and suggest that these play the important roles in sex determination and differentiation in oil palm. The strongest expression of EgACCO1 and the predicted precursor of EgmiR159a was found in ovaries and, to a lesser extent, fruit development. In addition, highly normalized expression of EgmiR159a was found in female flowers. In summary, the QTL analysis and the RNA expression reveal that EgACCO1 and EgmiR159a are the potential genetic factors involved in female flower determination and hence would affect yield in oil palm. However, to clarify how these genetic factors regulate female flower determination, more investigation of their down regulation or target may be essential. Additionally, if more sex determination genes controlled by plant hormones are identified, it may possible to reveal a crosstalk of sex determination genes with hormones and environment factors.

The AKR gene family and modifying sex ratios in palms through abiotic stress responsiveness.

Sex ratio (SR), the ratio of female inflorescences to total inflorescences, is one of the main yield components of oil palm (Elaeis guineensis Jacq). The SR quantitative trait locus (QTL) was recently identified on linkage (LG) 8 with a phenotype variance explained (PVE) of 11.3 %. The use of both genetic and physical mapping is one strategy for uncovering the genetic basis of the traits. Here, we report the construction of bacterial artificial chromosome (BAC) and fosmid libraries, and their use for physical mapping in oil palm. Combined, the libraries consist of more than 200,000 clones, representing 6.35 genome equivalents. Physical mapping at the SR locus was implemented by incorporating the published oil palm genome sequence and positive BAC/fosmid clones as identified by colony PCR screening. Based on the previously published sequences, the interval (about 184 kb) was comprised of 19 contigs of the known sequences (~117 kb, 64 %). After, combining the 454 pyrosequences of 15 positive clones and the previously published sequences, the known sequences were revealed to cover about 82 % of the interval (~150 kb), and were used for identifying the new markers by designing 35 gene-based and 23 simple sequence repeat (SSR)-amplified primers. As a result, a putative aldo-keto reductase gene (named EgAKR1) was revealed to be a promising candidate for sex ratio determination, via controlling female inflorescence number (11 % of PVE). This was predicted from the two newly identified polymorphic marker loci (mEgSSRsr8-21LB and mEgAKR1-9) designing from EgAKR1. The functions of AKR gene families in other plant species and our promoter analysis suggested that EgAKR1 may contribute to the sex ratio through abiotic stress responsiveness.

Genetic structure of Thai rice and rice accessions obtained from the International Rice Research Institute.

BACKGROUND: Although the genetic structure of rice germplasm has been characterized worldwide, few studies investigated germplasm from Thailand, the world's largest exporter of rice. Thailand and the International Rice Research Institute (IRRI) have diverse collections of rice germplasm, which could be used to develop breeding lines with desirable traits. This study aimed to investigate the level of genetic diversity and structures of Thai and selected IRRI germplasm. Understanding the genetic structure and relationships among these germplasm will be useful for parent selection used in rice breeding programs. RESULTS: From the 98 InDel markers tested for single copy and polymorphism, 19 markers were used to evaluate 43 Thai and 57 IRRI germplasm, including improved cultivars, breeding lines, landraces, and 5 other Oryza species. The Thai accessions were selected from all rice ecologies such as irrigated, deep water, upland, and rainfed lowland ecosystems. The IRRI accessions were groups of germplasm having agronomic desirable traits, including temperature-sensitive genetic male sterility (TGMS), new plant type, early flowering, and biotic and abiotic stress resistances. Most of the InDel markers were genes with diverse functions. These markers produced the total of 127 alleles for all loci, with a mean of 6.68 alleles per locus, and a mean Polymorphic Information Content (PIC) of 0.440. Genetic diversity of Thai rice were 0.3665, 0.4479 and 0.3972 for improved cultivars, breeding lines, and landraces, respectively, while genetic diversity of IRRI improved and breeding lines were 0.3272 and 0.2970, respectively. Cluster, structure, and differentiation analyses showed six distinct groups: japonica, TGMS, deep-water, IRRI germplasm, Thai landraces and breeding lines, and other Oryza species. CONCLUSIONS: Thai and IRRI germplasm were significantly different. Thus, they can be used to broaden the genetic base and trait improvements. Cluster, structure, and differentiation analyses showed concordant results having six distinct groups, in agreement with their development, and ecologies.