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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., (© 2022. The Author(s).)

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 F 1 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs., Methods: We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors., Results: A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5' and 3' untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants., Discussion: The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar., Competing Interests: Warodom Wirojsirasak, Prapat Punpee and Peeraya Klomsa-ard are employed by Mitr Phol Sugarcane Research Center Co., Ltd.

The world-renowned Thai Hom Mali Rice has been the most important aromatic rice originating in Thailand. The aromatic variety was collected from Chachoengsao, a central province, and after pure-line selection, it was officially named as Khao Dawk Mali 105, (KDML105). Because of its superb fragrance and cooking quality, KDML105 has been a model variety for studying genes controlling grain quality and aroma. The aromatic gene was cloned in KDML105, as an amino aldehyde dehydrogenase (AMADH) or better known as BADH2 located on chromosome 8. Later on, all other aromatic rice genes were discovered as allelic to the AMADH. As a selection of local landrace variety found in rainfed areas, the Thai Jasmine rice showed adaptive advantages over improved irrigated rice in less fertile lowland rainfed conditions. Because KDML105 was susceptible to most diseases and insect pests, marker-assisted backcross selection (MABC) was used for the genetic improvement since 2000. After nearly 17 years of MABC for integrating new traits into KDML105, a new generation of KDML105, designated HM84, was developed which maintains the cooking quality and fragrance, and has gained advantages during flash flooding, disease, and insect outbreak.

The oil palm ( Elaeis guineensis ), a monocotyledonous species in the family Arecaceae, has an extraordinarily oil rich fleshy mesocarp, and presents an original model to examine the ripening processes and regulation in this particular monocot fruit. Histochemical analysis and cell parameter measurements revealed cell wall and middle lamella expansion and degradation during ripening and in response to ethylene. Cell wall related transcript profiles suggest a transition from synthesis to degradation is under transcriptional control during ripening, in particular a switch from cellulose, hemicellulose, and pectin synthesis to hydrolysis and degradation. The data provide evidence for the transcriptional activation of expansin, polygalacturonase, mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase proteins in the ripening oil palm mesocarp, suggesting widespread conservation of these activities during ripening for monocotyledonous and eudicotyledonous fruit types. Profiling of the most abundant oil palm polygalacturonase ( EgPG4 ) and 1-aminocyclopropane-1-carboxylic acid oxidase ( ACO ) transcripts during development and in response to ethylene demonstrated both are sensitive markers of ethylene production and inducible gene expression during mesocarp ripening, and provide evidence for a conserved regulatory module between ethylene and cell wall pectin degradation. A comprehensive analysis of NAC transcription factors confirmed at least 10 transcripts from diverse NAC domain clades are expressed in the mesocarp during ripening, four of which are induced by ethylene treatment, with the two most inducible ( EgNAC6 and EgNAC7 ) phylogenetically similar to the tomato NAC-NOR master-ripening regulator. Overall, the results provide evidence that despite the phylogenetic distance of the oil palm within the family Arecaceae from the most extensively studied monocot banana fruit, it appears ripening of divergent monocot and eudicot fruit lineages are regulated by evolutionarily conserved molecular physiological processes.

Sugarcane accounts for a large portion of the worlds sugar production. Modern commercial cultivars are complex hybrids of S. officinarum and several other Saccharum species. Historical records identify New Guinea as the origin of S. officinarum and that a small number of plants originating from there were used to generate all modern commercial cultivars. The mitochondrial genome can be a useful way to identify the maternal origin of commercial cultivars. We have used the PacBio RSII to sequence and assemble the mitochondrial genome of a South East Asian commercial cultivar, known as Khon Kaen 3. The long read length of this sequencing technology allowed for the mitochondrial genome to be assembled into two distinct circular chromosomes with all repeat sequences spanned by individual reads. Comparison of five commercial hybrids, two S. officinarum and one S. spontaneum to our assembly reveals no structural rearrangements between our assembly, the commercial hybrids and an S. officinarum from New Guinea. The S. spontaneum, from India, and one sample of S. officinarum (unknown origin) are substantially rearranged and have a large number of homozygous variants. This supports the record that S. officinarum plants from New Guinea are the maternal source of all modern commercial hybrids.

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 oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function.

Background: Rice contains the lowest grain Fe content among cereals. One biological limiting factor is the tolerance of rice to Fe toxicity. Reverse and forward genetic screenings were used to identify tolerance to Fe toxicity in 4,500 M4 lines irradiated by fast neutrons (FN)., Findings: Fe-tolerant mutants were successfully isolated. In the forward screen, we selected five highly tolerant and four highly intolerant mutants based on the response of seedlings to 300 ppm Fe. Reverse screening based on the polymorphic coding sequence of seven Fe homeostatic genes detected by denaturing high performance liquid chromatography (dHPLC) revealed MuFRO1, a mutant for OsFRO1 (LOC_Os04g36720). The MuFRO1 mutant tolerated Fe toxicity in the vegetative stage and had 21-30% more grain Fe content than its wild type. All five highly Fe-tolerant mutants have the same haplotype as the MuFRO1, confirming the important role of OsFRO1 in Fe homeostasis in rice., Conclusions: FN radiation generated extreme Fe-tolerant mutants capable of tolerating different levels of Fe toxicity in the lowland rice environment. Mutants from both reverse and forward screens suggested a role for OsFRO1 in seedling tolerance to Fe toxicity. The MuFRO1 mutant could facilitate rice production in the high-Fe soil found in Southeast Asia.

Construction of linkage maps is crucial for genetic studies and marker-assisted breeding programs. Recent advances in next generation sequencing technologies allow for the generation of high-density linkage maps, especially in non-model species lacking extensive genomic resources. Here, we constructed a high-density integrated genetic linkage map of rubber tree (Hevea brasiliensis), the sole commercial producer of high-quality natural rubber. We applied a genotyping-by-sequencing (GBS) technique to simultaneously discover and genotype single nucleotide polymorphism (SNP) markers in two rubber tree populations. A total of 21,353 single nucleotide substitutions were identified, 55% of which represented transition events. GBS-based genetic maps of populations P and C comprised 1704 and 1719 markers and encompassed 2041 cM and 1874 cM, respectively. The average marker densities of these two maps were one SNP in 1.23-1.25 cM. A total of 1114 shared SNP markers were used to merge the two component maps. An integrated linkage map consisted of 2321 markers and spanned the cumulative length of 2052 cM. The composite map showed a substantial improvement in marker density, with one SNP marker in every 0.89 cM. To our knowledge, this is the most saturated genetic map in rubber tree to date. This integrated map allowed us to anchor 28,965 contigs, covering 135 Mb or 12% of the published rubber tree genome. We demonstrated that GBS is a robust and cost-effective approach for generating a common set of genome-wide SNP data suitable for constructing integrated linkage maps from multiple populations in a highly heterozygous agricultural species.

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.

Oil palm has become one of the most important oil crops in the world. Marker-assisted selections have played a pivotal role in oil palm breeding programs. Here, we report the use of genotyping-by-sequencing (GBS) approach for a large-scale SNP discovery and genotyping of a mapping population. Reduced representation libraries of 108 F2 progeny were sequenced and a total of 524 million reads were obtained. We detected 21,471 single nucleotide substitutions, most of which (62.6%) represented transition events. Of 3417 fully informative SNP markers, we were able to place 1085 on a linkage map, which spanned 1429.6 cM and had an average of one marker every 1.26 cM. Three QTL affecting trunk height were detected on LG 10, 14 and 15, whereas a single QTL associated with fruit bunch weight was identified on LG 3. The use of GBS approach proved to be rapid, cost-effective and highly reproducible in this species., (Copyright © 2014. Published by Elsevier Inc.)

Hevea brasiliensis, or rubber tree, is an important crop species that accounts for the majority of natural latex production. The rubber tree nuclear genome consists of 18 chromosomes and is roughly 2.15 Gb. The current rubber tree reference genome assembly consists of 1,150,326 scaffolds ranging from 200 to 531,465 bp and totalling 1.1 Gb. Only 143 scaffolds, totalling 7.6 Mb, have been placed into linkage groups. We have performed RNA-seq on 6 varieties of rubber tree to identify SNPs and InDels and used this information to perform target sequence enrichment and high throughput sequencing to genotype a set of SNPs in 149 rubber tree offspring from a cross between RRIM 600 and RRII 105 rubber tree varieties. We used this information to generate a linkage map allowing for the anchoring of 24,424 contigs from 3,009 scaffolds, totalling 115 Mb or 10.4% of the published sequence, into 18 linkage groups. Each linkage group contains between 319 and 1367 SNPs, or 60 to 194 non-redundant marker positions, and ranges from 156 to 336 cM in length. This linkage map includes 20,143 of the 69,300 predicted genes from rubber tree and will be useful for mapping studies and improving the reference genome assembly.

Background: DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained., Results: We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists., Conclusions: This work presents an automated genotyping tool from DNA gel electrophoresis images, called GELect, which was written in Java and made available through the imageJ framework. With a novel automated image processing workflow, the tool can accurately segment lanes from a gel matrix, intelligently extract distorted and even doublet bands that are difficult to identify by existing image processing tools. Consequently, genotyping from DNA gel electrophoresis can be performed automatically allowing users to efficiently conduct large scale DNA fingerprinting via DNA gel electrophoresis. The software is freely available from http://www.biotec.or.th/gi/tools/gelect.

Cassava (Manihot esculenta Crantz) is one of the most important crop species being the main source of dietary energy in several countries. Marker-assisted selection has become an essential tool in plant breeding. Single nucleotide polymorphism (SNP) discovery via transcriptome sequencing is an attractive strategy for genome complexity reduction in organisms with large genomes. We sequenced the transcriptome of 16 cassava accessions using the Illumina HiSeq platform and identified 675,559 EST-derived SNP markers. A subset of those markers was subsequently genotyped by capture-based targeted enrichment sequencing in 100 F1 progeny segregating for starch viscosity phenotypes. A total of 2,110 non-redundant SNP markers were used to construct a genetic map. This map encompasses 1,785 cM and consists of 19 linkage groups. A major quantitative trait locus (QTL) controlling starch pasting properties was identified and shown to coincide with the QTL previously reported for this trait. With a high-density SNP-based linkage map presented here, we also uncovered a novel QTL associated with starch pasting time on LG 10.