Limited allele-specific gene expression in highly polyploid sugarcane.

Polyploidy is widespread in plants, allowing the different copies of genes to be expressed differently in a tissue-specific or developmentally specific way. This allele-specific expression (ASE) has been widely reported, but the proportion and nature of genes showing this characteristic have not been well defined. We now report an analysis of the frequency and patterns of ASE at the whole-genome level in the highly polyploid sugarcane genome. Very high depth whole-genome sequencing and RNA sequencing revealed strong correlations between allelic proportions in the genome and in expressed sequences. This level of sequencing allowed discrimination of each of the possible allele doses in this 12-ploid genome. Most genes were expressed in direct proportion to the frequency of the allele in the genome with examples of polymorphisms being found with every possible discrete level of dose from 1:11 for single-copy alleles to 12:0 for monomorphic sites. The rarer cases of ASE were more frequent in the expression of defense-response genes, as well as in some processes related to the biosynthesis of cell walls. ASE was more common in genes with variants that resulted in significant disruption of function. The low level of ASE may reflect the recent origin of polyploid hybrid sugarcane. Much of the ASE present can be attributed to strong selection for resistance to diseases in both nature and domestication.

The genome of Citrus australasica reveals disease resistance and other species specific genes.

BACKGROUND: The finger lime (Citrus australasica), one of six Australian endemic citrus species shows a high natural phenotypic diversity and novel characteristics. The wide variation and unique horticultural features have made this lime an attractive candidate for domestication. Currently no haplotype resolved genome is available for this species. Here we present a high quality, haplotype-resolved reference genome for this species using PacBio HiFi and Hi-C sequencing. RESULTS: Hifiasm assembly and SALSA scaffolding resulted in a collapsed genome size of 344.2 Mb and 321.1 Mb and 323.2 Mb size for the two haplotypes. The nine pseudochromosomes of the collapsed genome had an N50 of 35.2 Mb, 99.1% genome assembly completeness and 98.9% gene annotation completeness (BUSCO). A total of 41,304 genes were predicted in the nuclear genome. Comparison with C. australis revealed that 13,661 genes in pseudochromosomes were unique in C. australasica. These were mainly involved in plant-pathogen interactions, stress response, cellular metabolic and developmental processes, and signal transduction. The two genomes showed a syntenic arrangement at the chromosome level with large structural rearrangements in some chromosomes. Genetic variation among five C. australasica cultivars was analysed. Genes related to defense, synthesis of volatile compounds and red/yellow coloration were identified in the genome. A major expansion of genes encoding thylakoid curvature proteins was found in the C. australasica genome. CONCLUSIONS: The genome of C. australasica present in this study is of high quality and contiguity. This genome helps deepen our understanding of citrus evolution and reveals disease resistance and quality related genes with potential to accelerate the genetic improvement of citrus.

De novo chromosome level assembly of a plant genome from long read sequence data.

Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. Chromosome level genome assemblies using sequence contigs generated from long read sequencing have involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (HiFi; PacBio) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii, a genome that has been used as a model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig (six with telomere repeats at both ends) and the other six assembled from two to four main contigs. The small number of chromosome breaks appears to be the result of highly repetitive regions including ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now appears possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.

Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids).

BACKGROUND: The importance of uridine 5'-diphosphate glucose (UDP-G) synthesis and degradation on carbon (C) partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of involved enzymes had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools. In this study, the expression of 136 genes belonging to 32 gene families related to UDP-G metabolism was studied in 3 major sugarcane organs (including leaf, internode and root) at 6 different developmental stages in 2 commercial genotypes. RESULTS: Analysis of the genes associated with UDP-G metabolism in leaves indicated low expression of sucrose synthase, but relatively high expression of invertase genes, specifically cell-wall invertase 4 and neutral acid invertase 1-1 and 3 genes. Further, organs that are primarily responsible for sucrose synthesis or bioaccumulation, i.e., in source organs (mature leaves) and storage sink organs (mature internodes), had very low expression of sucrose, cellulose and hemicellulose synthesis genes, specifically sucrose synthase 1 and 2, UDP-G dehydrogenase 5 and several cellulose synthase subunit genes. Gene expression was mostly very low in both leaf and mature internode samples; however, leaves did have a comparatively heightened invertase and sucrose phosphate synthase expression. Major differences were observed in the transcription of several genes between immature sink organs (roots and immature internodes). Gene transcription favoured utilisation of UDP-G toward insoluble and respiratory pools in roots. Whereas, there was comparatively higher expression of sucrose synthetic genes, sucrose phosphate synthase 1 and 4, and comparatively lower expression of many genes associated with C flow to insoluble and respiratory pools including myo-Inositol oxygenase, UDP-G dehydrogenase 4, vacuolar invertase 1, and several cell-wall invertases in immature internodes. CONCLUSION: This study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in sugarcane. Transcriptional analysis displayed the likelihood that C partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism. The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

Highly sex specific gene expression in Jojoba.

BACKGROUND: Dioecious plants have male and female flowers on separate plants. Jojoba is a dioecious plant that is drought-tolerant and native to arid areas. The genome sequence of male and female plants was recently reported and revealed an X and Y chromosome system, with two large male-specific insertions in the Y chromosome. RESULTS: A total of 16,923 differentially expressed genes (DEG) were identified between the flowers of the male and female jojoba plants. This represented 40% of the annotated genes in the genome. Many genes, including those responsible for plant environmental responses and those encoding transcription factors (TFs), were specific to male or female reproductive organs. Genes involved in plant hormone metabolism were also found to be associated with flower and pollen development. A total of 8938 up-regulated and 7985 down-regulated genes were identified in comparison between male and female flowers, including many novel genes specific to the jojoba plant. The most differentially expressed genes were associated with reproductive organ development. The highest number of DEG were linked with the Y chromosome in male plants. The male specific parts of the Y chromosome encoded 12 very highly expressed genes including 9 novel genes and 3 known genes associated with TFs and a plant hormone which may play an important role in flower development. CONCLUSION: Many genes, largely with unknown functions, may explain the sexual dimorphisms in jojoba plants and the differentiation of male and female flowers.

The jojoba genome reveals wide divergence of the sex chromosomes in a dioecious plant.

Most flowering plants are hermaphrodites, but around 6% of species are dioecious, having separate male and female plants. Sex chromosomes and some sex-specific genes have been reported in plants, but the genome sequences have not been compared. We now report the genome sequence of male and female jojoba (Simmondsia chinensis) plants, revealing a very large difference in the sex chromosomes. The male genome assembly was 832 Mb and the female 822 Mb. This was explained by the large size differences in the Y chromosome (37.6 Mb) compared with the X chromosome (26.9 Mb). Relative to the X chromosome, the Y chromosome had two large insertions each of more than 5 Mb containing more than 400 genes. Many of the genes in the chromosome-specific regions were novel. These male-specific regions included many flowering-related and stress response genes. Smaller insertions found only in the X chromosome totalled 877 kb. The wide divergence of the sex chromosomes suggests a long period of adaptation to diverging sex-specific roles. Male and female plants may have evolved to accommodate factors such as differing reproductive resource allocation requirements under the stress of the desert environment in which the plants are found. The sex-determining regions accumulate genes beneficial to each sex. This has required the evolution of many more novel sex-specific genes than has been reported for other organisms. This suggest that dioecious plants provide a novel source of genes for manipulation of reproductive performance and environmental adaptation in crops.

Transcript profiles of wild and domesticated sorghum under water-stressed conditions and the differential impact on dhurrin metabolism.

MAIN CONCLUSION: Australian native species of sorghum contain negligible amounts of dhurrin in their leaves and the cyanogenesis process is regulated differently under water-stress in comparison to domesticated sorghum species. Cyanogenesis in forage sorghum is a major concern in agriculture as the leaves of domesticated sorghum are potentially toxic to livestock, especially at times of drought which induces increased production of the cyanogenic glucoside dhurrin. The wild sorghum species endemic to Australia have a negligible content of dhurrin in the above ground tissues and thus represent a potential resource for key agricultural traits like low toxicity. In this study we investigated the differential expression of cyanogenesis related genes in the leaf tissue of the domesticated species Sorghum bicolor and the Australian native wild species Sorghum macrospermum grown in glasshouse-controlled water-stress conditions using RNA-Seq analysis to analyse gene expression. The study identified genes, including those in the cyanogenesis pathway, that were differentially regulated in response to water-stress in domesticated and wild sorghum. In the domesticated sorghum, dhurrin content was significantly higher compared to that in the wild sorghum and increased with stress and decreased with age whereas in wild sorghum the dhurrin content remained negligible. The key genes in dhurrin biosynthesis, CYP79A1, CYP71E1 and UGT85B1, were shown to be highly expressed in S. bicolor. DHR and HNL encoding the dhurrinase and α-hydroxynitrilase catalysing bio-activation of dhurrin were also highly expressed in S. bicolor. Analysis of the differences in expression of cyanogenesis related genes between domesticated and wild sorghum species may allow the use of these genetic resources to produce more acyanogenic varieties in the future.

Transcriptome changes in the developing sugarcane culm associated with high yield and early-season high sugar content.

Sugarcane, with its exceptional carbon dioxide assimilation, biomass and sugar yield, has a high potential for the production of bio-energy, bio-plastics and high-value products in the food and pharmaceutical industries. A crucial challenge for long-term economic viability and environmental sustainability is also to optimize the production of biomass composition and carbon sequestration. Sugarcane varieties such as KQ228 and Q253 are highly utilized in the industry. These varieties are characterized by a high early-season sugar content associated with high yield. In order to investigate these correlations, 1,440 internodes were collected and combined to generate a set of 120 samples in triplicate across 24 sugarcane cultivars at five different development stages. Weighted gene co-expression network analysis (WGCNA) was used and revealed for the first time two sets of co-expressed genes with a distinct and opposite correlation between fibre and sugar content. Gene identification and metabolism pathways analysis was used to define these two sets of genes. Correlation analysis identified a large number of interconnected metabolic pathways linked to sugar content and fibre content. Unsupervised hierarchical clustering of gene expression revealed a stronger level of segregation associated with the genotypes than the stage of development, suggesting a dominant genetic influence on biomass composition and facilitating breeding selection. Characterization of these two groups of co-expressed key genes can help to improve breeding program for high fibre, high sugar species or plant synthetic biology.

Gene Expression in the Developing Seed of Wild and Domesticated Rice.

The composition and nutritional properties of rice are the product of the expression of genes in the developing seed. RNA-Seq was used to investigate the level of gene expression at different stages of seed development in domesticated rice (Oryza sativa ssp. japonica var. Nipponbare) and two Australian wild taxa from the primary gene pool of rice (Oryza meridionalis and Oryza rufipogon type taxa). Transcriptome profiling of all coding sequences in the genome revealed that genes were significantly differentially expressed at different stages of seed development in both wild and domesticated rice. Differentially expressed genes were associated with metabolism, transcriptional regulation, nucleic acid processing, and signal transduction with the highest number of being linked to protein synthesis and starch/sucrose metabolism. The level of gene expression associated with domestication traits, starch and sucrose metabolism, and seed storage proteins were highest at the early stage (5 days post anthesis (DPA)) to the middle stage (15 DPA) and declined late in seed development in both wild and domesticated rice. However, in contrast, black hull colour (Bh4) gene was significantly expressed throughout seed development. A substantial number of novel transcripts (38) corresponding to domestication genes, starch and sucrose metabolism, and seed storage proteins were identified. The patterns of gene expression revealed in this study define the timing of metabolic processes associated with seed development and may be used to explain differences in rice grain quality and nutritional value.

Fragrance in Pandanus amaryllifoliusRoxb. Despite the Presence of a Betaine Aldehyde Dehydrogenase 2.

Pandanus amaryllifoliusRoxb. accumulates the highest concentration of the major basmati aroma volatile 2-acetyl-1-pyrroline (2AP) in the plant kingdom. The expression of 2AP is correlated with the presence of a nonfunctional betaine aldehyde dehydrogenase 2(BADH2) in aromatic rice and other plant species. In the present study, a full-length BADH2 sequence was reconstructed from the transcriptome data of leaf tissue from P. amaryllifolius seedlings. Based on this sequence, a 1509 bp coding sequence was defined that encoded a 54 kD PaBADH2protein. This revealed the presence of a full-length BADH2 protein in P. amaryllifolius. Moreover, quantitative real-time PCR analysis, combined with BADH2 enzyme activity, confirmed the expression and functionality of the PaBADH2 protein. To understand the apparent structural variation, docking analysis was carried out in which protein showed a good affinity with both betaine aldehyde (BAD) and γ-aminobutyraldehyde (GAB-ald) as substrates. Overall, the analysis showed the presence of a functional BADH2, along with substantial 2AP synthesis (4.38 ppm). Therefore, we conclude that unlike all other plants studied to date, 2AP biosynthesis in P. amaryllifolius is not due to the inactivation of BADH2.

Differential expression in leaves of Saccharum genotypes contrasting in biomass production provides evidence of genes involved in carbon partitioning.

BACKGROUND: The development of biomass crops aims to meet industrial yield demands, in order to optimize profitability and sustainability. Achieving these goals in an energy crop like sugarcane relies on breeding for sucrose accumulation, fiber content and stalk number. To expand the understanding of the biological pathways related to these traits, we evaluated gene expression of two groups of genotypes contrasting in biomass composition. RESULTS: First visible dewlap leaves were collected from 12 genotypes, six per group, to perform RNA-Seq. We found a high number of differentially expressed genes, showing how hybridization in a complex polyploid system caused extensive modifications in genome functioning. We found evidence that differences in transposition and defense related genes may arise due to the complex nature of the polyploid Saccharum genomes. Genotypes within both biomass groups showed substantial variability in genes involved in photosynthesis. However, most genes coding for photosystem components or those coding for phosphoenolpyruvate carboxylases (PEPCs) were upregulated in the high biomass group. Sucrose synthase (SuSy) coding genes were upregulated in the low biomass group, showing that this enzyme class can be involved with sucrose synthesis in leaves, similarly to sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP). Genes in pathways related to biosynthesis of cell wall components and expansins coding genes showed low average expression levels and were mostly upregulated in the high biomass group. CONCLUSIONS: Together, these results show differences in carbohydrate synthesis and carbon partitioning in the source tissue of distinct phenotypic groups. Our data from sugarcane leaves revealed how hybridization in a complex polyploid system resulted in noticeably different transcriptomic profiles between contrasting genotypes.

Two divergent chloroplast genome sequence clades captured in the domesticated rice gene pool may have significance for rice production.

BACKGROUND: The whole chloroplast genomes of 3018 rice genotypes were assembled from available sequence data by alignment with a reference rice chloroplast genome sequence, providing high quality chloroplast genomes for analysis of diversity on a much larger scale than in any previous plant study. RESULTS: Updated annotation of the chloroplast genome identified 13 more tRNA genes and 30 more introns and defined the function of more of the genes. Domesticated rice had chloroplast genomes that were distinct from those in wild relatives. Analysis confirms an Australian chloroplast clade as a sister to the domesticated clade. All domesticated rice genotypes could be assigned to one of two main clades suggesting the domestication of two distinct maternal genome clades that diverged long before domestication. These clades were very distinct having 4 polymorphisms between all 1486 accession in clade A and all 1532 accessions in clade B. These would result in expression of 3 proteins with altered amino acid sequences and a tRNA with an altered sequence and may be associated with adaptive evolution of the two chloroplast types. Diversity within these pools may have been captured during domestication with subclades enriched in specific groups such as basmati, tropical japonica and temperate japonica. However the phylogenies of the chloroplast and nuclear genomes differed possibly due to modern rice breeding and reticulate evolution prior to domestication. Indica and aus genotypes were common in both chloroplast clades while japonica genotypes were more likely to be found in the same clade (cladeB). CONCLUSIONS: The different evolutionary paths of the cytoplasmic and nuclear genomes of rice have resulted in the presence of apparently functional chloroplast genome diversity and the implications for rice crop performance require further investigation.

Slower development of lower canopy beans produces better coffee.

The production of high-quality coffee is being challenged by changing climates in coffee-growing regions. The coffee beans from the upper and lower canopy at different development stages of the same plants were analyzed to investigate the impact of the microenvironment on gene expression and coffee quality. Compared with coffee beans from the upper canopy, lower canopy beans displayed more intense aroma with higher caffeine, trigonelline, and sucrose contents, associated with greater gene expression in the representative metabolic pathways. Global gene expression indicated a longer ripening in the lower canopy, resulting from higher expression of genes relating to growth inhibition and suppression of chlorophyll degradation during early bean ripening. Selection of genotypes or environments that enhance expression of the genes slowing bean development may produce higher quality coffee beans, allowing coffee production in a broader range of available future environments.

Analysis of the diversity and tissue specificity of sucrose synthase genes in the long read transcriptome of sugarcane.

BACKGROUND: Sugarcane accumulates very high levels of sucrose in the culm. Elucidation of the molecular mechanisms that allows such high sucrose synthesis and accumulation (up to 650 mM) is made difficult by the complexity of the highly polyploid genome. Here we report the use of RNA Seq data to characterize the sucrose synthase (SuSy) genes expressed in the transcriptome of the mature sugarcane plant. RESULTS: Four SuSy gene families were identified in the sugarcane Iso-Seq long read transcriptome (SUGIT) through gene annotation of transcripts that mapped to reference SuSy genes from sorghum and maize. In total, 38, 19, 14, and 2 transcripts were identified for the four corresponding SuSy genes 1, 2, 4 and 7, respectively. Comparative studies using available SuSy genes from sorghum (1, 2, 4, 6, 7) and maize (1-7) revealed that the sugarcane SuSy genes were interrupted by multiple introns and that they share a highly conserved gene structure. Spatial expression of the four SuSy genes in sugarcane genotypes and in the progenitor species, Saccharum spontaneum and Saccharum officinarum, was studied in the leaf and root tissues and also in three regions of the culm tissue; top, middle and bottom internodes. Expression profiles indicated that all SuSy transcripts were differentially expressed between the top and bottom tissues, with high expression in the top tissues, lower expression in the bottom and moderate expression in the middle, indicating a gradient of SuSy activity in the sugarcane culm. Further, the root tissue had similar expression levels to that of the top internodes while leaf tissues showed lower expression. In the progenitors, SuSy7 was found to be highly expressed in S. officinarum while the other three SuSy genes had moderate expression in both the progenitors. CONCLUSIONS: The high expression of the SuSy genes in sink tissues, the top internodes and the roots suggests functional roles in sucrose utilization to support growth. The SuSy7 gene has not been previously reported in sugarcane. As sugarcane is unique in storing such high amounts of sucrose, it is possible that there are more SuSy genes/isoforms with specific expression patterns to be discovered in this complex system.

Sequencing of bulks of segregants allows dissection of genetic control of amylose content in rice.

Amylose content (AC) is a key quality trait in rice. A cross between Oryza glaberrima (African rice) and Oryza sativa (Asian rice) segregating for AC was analysed by sequencing bulks of individuals with high and low AC. SNP associated with the granule bound starch synthase (GBSS1) locus on chromosome 6 were polymorphic between the bulks. In particular, a G/A SNP that would result in an Asp to Asn mutation was identified. This amino acid substitution may be responsible for differences in GBSS activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. Other polymorphisms in genomic regions closely surrounding this variation may be the result of linkage drag. In addition to the variant in the starch biosynthesis gene, SNP on chromosomes 1 and 11 linked to AC was also identified. SNP was found in the genes encoding the NAC and CCAAT-HAP5 transcription factors that have previously been linked to starch biosynthesis. This study has demonstrated that the approach of sequencing bulks was able to identify genes on different chromosomes associated with this complex trait.

Use of a draft genome of coffee (Coffea arabica) to identify SNPs associated with caffeine content.

Arabica coffee (Coffea arabica) has a small gene pool limiting genetic improvement. Selection for caffeine content within this gene pool would be assisted by identification of the genes controlling this important trait. Sequencing of DNA bulks from 18 genotypes with extreme high- or low-caffeine content from a population of 232 genotypes was used to identify linked polymorphisms. To obtain a reference genome, a whole genome assembly of arabica coffee (variety K7) was achieved by sequencing using short read (Illumina) and long-read (PacBio) technology. Assembly was performed using a range of assembly tools resulting in 76 409 scaffolds with a scaffold N50 of 54 544 bp and a total scaffold length of 1448 Mb. Validation of the genome assembly using different tools showed high completeness of the genome. More than 99% of transcriptome sequences mapped to the C. arabica draft genome, and 89% of BUSCOs were present. The assembled genome annotated using AUGUSTUS yielded 99 829 gene models. Using the draft arabica genome as reference in mapping and variant calling allowed the detection of 1444 nonsynonymous single nucleotide polymorphisms (SNPs) associated with caffeine content. Based on Kyoto Encyclopaedia of Genes and Genomes pathway-based analysis, 65 caffeine-associated SNPs were discovered, among which 11 SNPs were associated with genes encoding enzymes involved in the conversion of substrates, which participate in the caffeine biosynthesis pathways. This analysis demonstrated the complex genetic control of this key trait in coffee.

Chloroplast phylogeography of AA genome rice species.

Whole chloroplast genome sequence analysis of 58 wild and domesticated rice samples was used to investigate their phylogeny providing more detail on the biogeography of the major groups of wild A genome rices globally. An optimized chloroplast assembly method was developed and applied to extracting high quality whole chloroplast genome sequences from shot gun whole DNA sequencing data. Forty complete high quality chloroplast genome sequences were assembled (including; temperate japonica, tropical japonica and aus). South American, African wild rice relationship were conformed, while the Australian chloroplast type was found to extend north to the Philippines. The remainder could be divided into an African (O. barthii and the domesticated O. glaberrima) clade and the Asian taxa. The Asian taxa was placed in two distinct clades including the domesticated O. sativa ssp. indica and O. sativa ssp. japonica respectively. These two groups of wild rices had substantially overlapping distributions with the O. sativa japonica group extending further west into India. The aromatic rices had japonica chloroplasts as expected. A polyphyletic maternal genome origin of the cultivated aus group of rices was suggested by the identification of aus accessions in both the indica and japonica clades. The current distribution of the chloroplast types appears to differ significantly to that of the nuclear genome diversity suggesting a complex evolutionary history of the rice progenitors leading to the domestication of rice.

The transcriptome of the developing grain: a resource for understanding seed development and the molecular control of the functional and nutritional properties of wheat.

BACKGROUND: Wheat is one of the three major cereals that have been domesticated to feed human populations. The composition of the wheat grain determines the functional properties of wheat including milling efficiency, bread making, and nutritional value. Transcriptome analysis of the developing wheat grain provides key insights into the molecular basis for grain development and quality. RESULTS: The transcriptome of 35 genotypes was analysed by RNA-Seq at two development stages (14 and 30 days-post-anthesis, dpa) corresponding to the mid stage of development (stage Z75) and the almost mature seed (stage Z85). At 14dpa, most of the transcripts were associated with the synthesis of the major seed components including storage proteins and starch. At 30dpa, a diverse range of genes were expressed at low levels with a predominance of genes associated with seed defence and stress tolerance. RNA-Seq analysis of changes in expression between 14dpa and 30dpa stages revealed 26,477 transcripts that were significantly differentially expressed at a FDR corrected p-value cut-off at ≤0.01. Functional annotation and gene ontology mapping was performed and KEGG pathway mapping allowed grouping based upon biochemical linkages. This analysis demonstrated that photosynthesis associated with the pericarp was very active at 14dpa but had ceased by 30dpa. Recently reported genes for flour yield in milling and bread quality were found to influence wheat quality largely due to expression patterns at the earlier seed development stage. CONCLUSIONS: This study serves as a resource providing an overview of gene expression during wheat grain development at the early (14dpa) and late (30dpa) grain filling stages for use in studies of grain quality and nutritional value and in understanding seed biology.

Association of variation in the sugarcane transcriptome with sugar content.

BACKGROUND: Sugarcane is a major crop of the tropics cultivated mainly for its high sucrose content. The crop is genetically less explored due to its complex polyploid genome. Sucrose synthesis and accumulation are complex processes influenced by physiological, biochemical and genetic factors, and the growth environment. The recent focus on the crop for fibre and biofuel has led to a renewed interest on understanding the molecular basis of sucrose and biomass traits. This transcriptome study aimed to identify genes that are associated with and differentially regulated during sucrose synthesis and accumulation in the mature stage of sugarcane. Patterns of gene expression in high and low sugar genotypes as well as mature and immature culm tissues were studied using RNA-Seq of culm transcriptomes. RESULTS: In this study, 28 RNA-Seq libraries from 14 genotypes of sugarcane differing in their sucrose content were used for studying the transcriptional basis of sucrose accumulation. Differential gene expression studies were performed using SoGI (Saccharum officinarum Gene Index, 3.0), SAS (sugarcane assembled sequences) of sugarcane EST database (SUCEST) and SUGIT, a sugarcane Iso-Seq transcriptome database. In total, about 34,476 genes were found to be differentially expressed between high and low sugar genotypes with the SoGI database, 20,487 genes with the SAS database and 18,543 genes with the SUGIT database at FDR < 0.01, using the Baggerley's test. Further, differential gene expression analyses were conducted between immature (top) and mature (bottom) tissues of the culm. The DEGs were functionally annotated using GO classification and the genes consistently associated with sucrose accumulation were identified. CONCLUSIONS: The large number of DEGs may be due to the large number of genes that influence sucrose content or are regulated by sucrose content. These results indicate that apart from being a primary metabolite and storage and transport sugar, sucrose may serve as a signalling molecule that regulates many aspects of growth and development in sugarcane. Further studies are needed to confirm if sucrose regulates the expression of the identified DEGs or vice versa. The DEGs identified in this study may lead to identification of genes/pathways regulating sucrose accumulation and/or regulated by sucrose levels in sugarcane. We propose identifying the master regulators of sucrose if any in the future.

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

BACKGROUND: Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms. RESULTS: The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes. CONCLUSIONS: The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.