Genome-wide identification and expression analysis of VQ gene family under abiotic stress in Coix lacryma-jobi L.

BACKGROUND: Valine-glutamine (VQ) proteins are non-specific plant proteins that have a highly conserved motif: FxxhVQxhTG. These proteins are involved in the development of various plant organs such as seeds, hypocotyls, flowers, leaves and also play a role in response to salt, drought and cold stresses. Despite their importance, there is limited information available on the evolutionary and structural characteristics of VQ family genes in Coix lacryma-jobi. RESULTS: In this study, a total of 31 VQ genes were identified from the coix genome and classified into seven subgroups (I-VII) based on phylogenetic analysis. These genes were found to be unevenly distributed on 10 chromosomes. Gene structure analysis revealed that these genes had a similar type of structure within each subfamily. Moreover, 27 of ClVQ genes were found to have no introns. Conserved domain and multiple sequence alignment analysis revealed the presence of a highly conserved sequences in the ClVQ protein. This research utilized quantitative real-time PCR (qRT-PCR) and promoter analysis to investigate the expression of ClVQ genes under different stress conditions. Results showed that most ClVQ genes responded to polyethylene glycol, heat treatment, salt, abscisic acid and methyl jasmonate treatment with varying degrees of expression. Furthermore, some ClVQ genes exhibited significant correlation in expression changes under abiotic stress, indicating that these genes may act synergistically in response to adversarial stress. Additionally, yeast dihybrid verification revealed an interaction between ClVQ4, ClVQ12, and ClVQ26. CONCLUSIONS: This study conducted a genome-wide analysis of the VQ gene family in coix, including an examination of phylogenetic relationships, conserved domains, cis-elements and expression patterns. The goal of the study was to identify potential drought resistance candidate genes, providing a theoretical foundation for molecular resistance breeding.

Differential genome evolution and speciation of Coix lacryma-jobi L. and Coix aquatica Roxb. hybrid guangxi revealed by repetitive sequence analysis and fine karyotyping.

BACKGROUND: Coix, Sorghum and Zea are closely related plant genera in the subtribe Maydeae. Coix comprises 9-11 species with different ploidy levels (2n = 10, 20, 30, and 40). The exclusively cultivated C. lacryma-jobi L. (2n = 20) is widely used in East and Southeast Asia for food and medicinal applications. Three fertile cytotypes (2n = 10, 20, and 40) have been reported for C. aquatica Roxb. One sterile cytotype (2n = 30) closely related to C. aquatica has been recently found in Guangxi of China. This putative hybrid has been named C. aquatica HG (Hybrid Guangxi). The genome composition and the evolutionary history of C. lacryma-jobi and C. aquatica HG are largely unclear. RESULTS: About 76% of the genome of C. lacryma-jobi and 73% of the genome of C. aquatica HG are repetitive DNA sequences as shown by low coverage genome sequencing followed by similarity-based cluster analysis. In addition, long terminal repeat (LTR) retrotransposable elements are dominant repetitive sequences in these two genomes, and the proportions of many repetitive sequences in whole genome varied greatly between the two species, indicating evolutionary divergence of them. We also found that a novel 102 bp variant of centromeric satellite repeat CentX and two other satellites only appeared in C. aquatica HG. The results from FISH analysis with repeat probe cocktails and the data from chromosomes pairing in meiosis metaphase showed that C. lacryma-jobi is likely a diploidized paleotetraploid species and C. aquatica HG is possibly a recently formed hybrid. Furthermore, C. lacryma-jobi and C. aquatica HG shared more co-existing repeat families and higher sequence similarity with Sorghum than with Zea. CONCLUSIONS: The composition and abundance of repetitive sequences are divergent between the genomes of C. lacryma-jobi and C. aquatica HG. The results from fine karyotyping analysis and chromosome pairing suggested diploidization of C. lacryma-jobi during evolution and C. aquatica HG is a recently formed hybrid. The genome-wide comparison of repetitive sequences indicated that the repeats in Coix were more similar to those in Sorghum than to those in Zea, which is consistent with the phylogenetic relationship reported by previous work.

Genome-wide investigation of ARF transcription factor gene family and its responses to abiotic stress in Coix (Coix lacryma-jobi L.).

Auxin response factor (ARF) is an important transcription factor that regulates the expression of auxin-responsive genes by direct binding to their promoters, which play a central role in plant growth, development, and response to abiotic stresses. The availability of the entire Coix (Coix lacryma-jobi L.) genome sequence provides an opportunity to investigate the characteristics and evolutionary history of the ARF gene family in this medicine and food homology plant for the first time. In this study, a total of 27 ClARF genes were identified based on the genome-wide sequence of Coix. Twenty-four of the 27 ClARF genes were unevenly distributed on 8 chromosomes except Chr 4 and 10, and the remaining three genes (ClARF25-27) were not assigned to any chromosome. Most of the ClARF proteins were predicted to be localized to the nucleus, except ClARF24, which was localized to both the plasma membrane and nucleus. Twenty-seven ClARFs were clustered into six subgroups based on the phylogenetic analysis. Duplication analysis showed that segmental duplication, rather than tandem duplications promoting the expansion of the ClARF gene family. Synteny analysis showed that purifying selection might have been a primary driving force in the development of the ARF gene family in Coix and other investigated cereal plants. The prediction of the cis element of the promoter showed that 27 ClARF genes contain several stress response elements, suggesting that ClARFs might be involved in the abiotic stress response. Expression profile analysis shows that 27 ClARF genes were all expressed in the root, shoot, leaf, kernel, glume, and male flower of Coix with varying expression levels. Furthermore, qRT-PCR analyses revealed that the majority of ClARFs members were upregulated or downregulated in response to hormone treatment and abiotic stress. The current study expands our understanding of the functional roles of ClARFs in stress responses and provides basic information for the ClARF genes.

Divergence in centromere structure distinguishes related genomes in Coix lacryma-jobi and its wild relative.

Knowledge about the composition and structure of centromeres is critical for understanding how centromeres perform their functional roles. Here, we report the sequences of one centromere-associated bacterial artificial chromosome clone from a Coix lacryma-jobi library. Two Ty3/gypsy-class retrotransposons, centromeric retrotransposon of C. lacryma-jobi (CRC) and peri-centromeric retrotransposon of C. lacryma-jobi, and a (peri)centromere-specific tandem repeat with a unit length of 153 bp were identified. The CRC is highly homologous to centromere-specific retrotransposons reported in grass species. An 80-bp DNA region in the 153-bp satellite repeat was found to be conserved to centromeric satellite repeats from maize, rice, and pearl millet. Fluorescence in situ hybridization showed that the three repetitive sequences were located in (peri-)centromeric regions of both C. lacryma-jobi and Coix aquatica. However, the 153-bp satellite repeat was only detected on 20 out of the 30 chromosomes in C. aquatica. Immunostaining with an antibody against rice CENH3 indicates that the 153-bp satellite repeat and CRC might be both the major components for functional centromeres, but not all the 153-bp satellite repeats or CRC sequences are associated with CENH3. The evolution of centromeric repeats of C. lacryma-jobi during the polyploidization was discussed.

Transcriptome characterization and expression profile of Coix lacryma-jobi L. in response to drought.

Coix lacryma-jobi L. is a very important economic crop widely cultivated in Southeast Asia. Drought affects more than four million square kilometers every year, and is a significant factor limiting agricultural productivity. However, relatively little is known about how Coix lacryma-jobi L. responds to drought treatments. To obtain a detailed and comprehensive understanding of the mechanisms regulating the transcriptional responses of Coix lacryma-jobi L. to drought treatment, we employed high throughput short-read sequencing of cDNA prepared from polyadenylated RNA to explore global gene expression after a seven-day drought treatment. We generated a de novo assembled transcriptome comprising 65,480 unique sequences. Differential expression analysis based on RSEM-estimated transcript abundances identified 5,315 differentially expressed genes (DEGs) when comparing samples from plants following drought-treatment and from the appropriate controls. Among these, the transcripts for 3,460 genes were increased in abundance, whereas 1,855 were decreased. Real-time quantitative PCR for 5 transcripts confirmed the changes identified by RNA-Seq. The results provide a transcriptional overview of the changes in Coix lacryma-jobi L. in response to drought, and will be very useful for studying the function of associated genes and selection of molecular marker of Coix lacryma-jobi L in the future.

The amplification and evolution of orthologous 22-kDa α-prolamin tandemly arrayed genes in coix, sorghum and maize genomes.

Tandemly arrayed genes (TAGs) account for about one-third of the duplicated genes in eukaryotic genomes. They provide raw genetic material for biological evolution, and play important roles in genome evolution. The 22-kDa prolamin genes in cereal genomes represent typical TAG organization, and provide the good material to investigate gene amplification of TAGs in closely related grass genomes. Here, we isolated and sequenced the Coix 22-kDa prolamin (coixin) gene cluster (283 kb), and carried out a comparative analysis with orthologous 22-kDa prolamin gene clusters from maize and sorghum. The 22-kDa prolamin gene clusters descended from orthologous ancestor genes, but underwent independent gene amplification paths after the separation of these species, therefore varied dramatically in sequence and organization. Our analysis indicated that the gene amplification model of 22-kDa prolamin gene clusters can be divided into three major stages. In the first stage, rare gene duplications occurred from the ancestor gene copy accidentally. In the second stage, rounds of gene amplification occurred by unequal crossing over to form tandem gene array(s). In the third stage, gene array was further diverged by other genomic activities, such as transposon insertions, segmental rearrangements, etc. Unlike their highly conserved sequences, the amplified 22-kDa prolamin genes diverged rapidly at their expression capacities and expression levels. Such processes had no apparent correlation to age or order of amplified genes within TAG cluster, suggesting a fast evolving nature of TAGs after gene amplification. These results provided insights into the amplification and evolution of TAG families in grasses.

Construction of a Coix BAC library and isolation of the 22 kDa α-coixin gene cluster.

Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.

[Characteristics of germplasm resources in Coix from xishuangbanna].

OBJECTIVE: To evaluate the genetic diversity among wild germplasm resources of Coix that distributed at altitude of 550-1550 m in Xishuangbanna, Yunnan province by comparing the morphological variation, biological and economical factors. METHOD: The field plot experiments were conducted to observation the germination rate, growth period. While the milled rice rate, content of protein, crude fat and ethanol extract of 9 wild Coix germplasm resources were measured. RESULT: The germination rate of 9 germplasm was from 22% to 81%, and the growth period was among 139-156 d, which belongs to the medium-late mature type. The germination rate, growth stage, plant height, leaf length and width, tiller number, ramification number, seed number per stem of 9 Coix germplasm were significantly different (P < 0.05), respectively. The range of seed total protein contents were from 15.63% 25.74%, crude fat contents were from 5.05%-7.14%, and the contents of alcohol extract, which showed antitumor activity, were from 5.85%-7.27%. CONCLUSION: The fact of quite different in plant morphological, biological and quality characters of 9 germplasm suggested that the genetic diversity in the wild populations of Coix distributed throughout the regions of Xishuangbanna is relatively abundant.

Evolution and Domestication Footprints Uncovered from the Genomes of Coix.

Coix lacryma-jobi, a plant species closely related to Zea and Sorghum, is an important food and medicinal crop in Asia. However, no reference genome of this species has been reported, and its exact phylogeny within the Andropogoneae remains unresolved. Here, we generated a high-quality genome assembly of coix comprising ∼1.73 Gb with 44 485 predicted protein-coding genes. We found coix to be a typical diploid plant with an overall 1-to-1 syntenic relationship with the Sorghum genome, despite its drastic genome expansion (∼2.3-fold) due mainly to the activity of transposable elements. Phylogenetic analysis revealed that coix diverged with sorghum ∼10.41 million years ago, which was ∼1.49 million years later than the divergence between sorghum and maize. Resequencing of 27 additional coix accessions revealed that they could be unambiguously separated into wild relatives and cultivars, and suggested that coix experienced a strong genetic bottleneck, resulting in the loss of about half of the genetic diversity during domestication, even though many traits have remained undomesticated. Our data not only provide novel comparative genomic and evolutionary insights into the Andropogoneae lineage, but also an important resource that will greatly benefit molecular breeding of this important crop.

The Coix Genome Provides Insights into Panicoideae Evolution and Papery Hull Domestication.

Coix is a grass crop domesticated as early as the Neolithic era. It is still widely cultivated for both highly nutritional food and medicinal use. However, the genetic study and breeding of this crop are hindered by the lack of a sequenced genome. Here, we report de novo sequencing and assembly of the 1619-Mb genome of Coix, and annotation of 75.39% repeats and 39 629 protein-coding genes. Comparative genomics analysis showed that Coix is more closely related to sorghum than maize, but intriguingly only Coix and maize had a recent genome duplication event, which was not detected in sorghum. We further constructed a genetic map and mapped several important traits, especially the strength of hull. Selection of papery hull (thin: easy dehulling) from the stony hull (thick: difficult dehulling) in wild progenitors was a key step in Coix domestication. The papery hull makes seed easier to process and germinate. Anatomic and global transcriptome analysis revealed that the papery hull is a result of inhibition of cell division and wall biogenesis. We also successfully demonstrated that seed hull pressure resistance is controlled by two major quantitative trait loci (QTLs), which are associated with hull thickness and color, respectively. The two QTLs were further fine mapped within intervals of 250 kb and 146 kb, respectively. These resources provide a platform for evolutionary studies and will facilitate molecular breeding of this important crop.

The complete chloroplast genome of Coix lacryma-jobi and a comparative molecular evolutionary analysis of plastomes in cereals.

Graminoid molecular evolution was investigated by chloroplast genome (plastome) scale analyses. A complete plastome from Coix lacryma-jobi (Poaceae) and a draft plastome from Joinvillea plicata (Joinvilleaceae) were sequenced and analyzed. The draft plastome included conserved protein-coding loci routinely analyzed in previous studies plus one additional locus of demonstrated phylogenetic utility. The methodological approach was to directly sequence overlapping amplicons from known plastome regions. Over 100 pairs of amplification and sequencing primers were designed and positioned to flank overlapping 1,200-base pair fragments around the entire plastome. Newly determined sequences were analyzed with published plastomes from representatives of Panicoideae, Ehrhartoideae, and Pooideae. Considerable variation was found for studies within the family and even within Andropogoneae. Readily interpreted mutation patterns were observed, such as small inversions in hairpin-loop regions and indels, which were common in intergenic spacers. Maximum or near-maximum bootstrap support was observed in all analyses resolving relationships between subfamilies. However, the addition of characters from noncoding regions increased the number of parsimony-informative characters and lengthened short internal branches (Andropogoneae), better defining intergeneric relationships. Thus, characters in complete plastomes can be used over a wide scope of phylogenetic studies.

Transcriptome and proteome analyses of the molecular mechanisms associated with coix seed nutritional quality in the process of breeding.

In present study, the content of main nutrients in wild coix seed were significantly higher than cultivated coix seed. Transcriptome and proteome were combined to provide new insight of the molecular mechanisms linked to nutritional quality of wild coix seed and cultivated coix seed by RNA sequencing and isobaric tags for relative and absolute quantification techniques. A total of 20,039 genes and 471 proteins exhibited differential expression level in cultivated coix seed when compared with wild coix seed. These genes and proteins revealed that the pathway of flavonoids biosynthesis, starch and sucrose metabolism, lipid metabolism and amino acid metabolism were linked to nutritional quality of coix seed. Our results provided transcriptomics and proteomics information with respect to the molecular mechanisms of nutritional changes of coix seed, identified key genes and proteins that associated with the metabolism and accumulation of nutrients, and helped clarify the mechanisms of nutrient differences.

De novo transcriptome assembly of the Chinese pearl barley, adlay, by full-length isoform and short-read RNA sequencing.

Adlay (Coix lacryma-jobi) is a tropical grass that has long been used in traditional Chinese medicine and is known for its nutritional benefits. Recent studies have shown that vitamin E compounds in adlay protect against chronic diseases such as cancer and heart disease. However, the molecular basis of adlay's health benefits remains unknown. Here, we generated adlay gene sets by de novo transcriptome assembly using long-read isoform sequencing (Iso-Seq) and short-read RNA-Sequencing (RNA-Seq). The gene sets obtained from Iso-seq and RNA-seq contained 31,177 genes and 57,901 genes, respectively. We confirmed the validity of the assembled gene sets by experimentally analyzing the levels of prolamin and vitamin E biosynthesis-associated proteins in adlay plant tissues and seeds. We compared the screened adlay genes with known gene families from closely related plant species, such as rice, sorghum and maize. We also identified tissue-specific genes from the adlay leaf, root, and young and mature seed, and experimentally validated the differential expression of 12 randomly-selected genes. Our study of the adlay transcriptome will provide a valuable resource for genetic studies that can enhance adlay breeding programs in the future.

[Mutagenic effects of gamma-rays on Coix lacryma-jobi var. ma-yuen].

OBJECTIVE: To study the mutagenic effect of gamma-rays on Coix lacryma-jobi var. ma-yuen. METHOD: Physiological and mutagenic effects of gamma-rays on C. lacryma-jobi var. ma-yuen dormant seeds were studied. The germination percentage, seeding survival, seeding height and root length of M1 plants and the frequency of chlorophyll mutation in M2 generation were selected as criteria. RESULT: The gamma-rays showed obvious inhibitory action to the seedling growth, and a strong ability in inducing the chlorophyll mutation. CONCLUSION: The gamma-rays is one kind of C. lacryma-jobi var. ma-yuen effective mutagen. The appropriate dose of gamma-rays is 450 Gy for C. lacryma-jobi var. ma-yuen dormant seeds.

Assessment of the Genetic Diversity of Different Job's Tears (Coix lacryma-jobi L.) Accessions and the Active Composition and Anticancer Effect of Its Seed Oil.

Job's tears (Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job's tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job's tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job's tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job's tears at the species level (h = 0.21, I = 0.33) and the accession level (h = 0.07, I = 0.10), and strong genetic differentiation (GST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job's tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job's tears.

Quantitative chromosome map of Coix lacryma-jobi L. by imaging methods.

Prometaphase chromosomes of Coix lacryma-jobi L. were quantitatively analyzed based on their distribution patterns of DAPI signals. The DAPI signals showed prominent uneven distribution along the chromosomes. Based on the DAPI signal patterns, a quantitative chromosome map was constructed for the first time in C. lacryma-jobi. The quantitative chromosome map will offer the foundation for genome analysis of C. lacryma-jobi.

Comparative physical localization of maize mir1 gene in Zea mays L. and Coix lacryma-jobi L.

The maize gene mir1 encoded a cystein proteinase which is resistant to fall armyworm. Previously, RFLP map indicated that the mir1 was mapped on chromosome 6. However, physical location of the mir1 gene on chromosome 6 has not been reported. In this study, the mir1 gene was physically located on the short arm of metaphase and pachytene chromosome 6 by dual-color fluorescence in situ hybridization (FISH) with 45S rDNA as a reference marker. The results of Southern blotting suggested that there were sequences homologous to mir1 in Coix lacryma-jobi L. genome. Then, the sequences were mapped on the distal region of long arm of chromosome 7 in C. lacryma-jobi by FISH. The percent distance from the signal site to centromere was 73.33 +/- 0.15.

Ultrastructure and phylogeny of Ustilago coicis.

Ustilago coicis causes serious smut on Coix lacryma-jobi in Dayang Town, Jinyun County, Zhejiang Province of China. In this paper, ultrastructural assessments on fungus-host interactions and teliospore development are presented, and molecular phylogenetic analyses have been done to elucidate the phylogenetic placement of the taxon. Hyphal growth within infected tissues was both intracellular and intercellular and on the surface of fungus-host interaction, and the fungal cell wall and the invaginated host plasma membrane were separated by a sheath comprising two distinct layers between the fungal cell wall and the invaginated host plasma membrane. Ornamentation development of teliospore walls was unique as they appeared to be originated from the exosporium. In addition, internal transcribed spacer (ITS) and large subunit (LSU) sequence data showed that U. coicis is closely related to Ustilago trichophora which infects grass species of the genus Echinochloa (Poaceae).