The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L.) response to repeated drought stresses.

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L) response to repeated drought stresses

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

Comparative Metabolomics Analysis of Different Perilla Varieties Provides Insights into Variation in Seed Metabolite Profiles and Antioxidant Activities.

Perilla seeds are essential functional foods and key ingredients in traditional medicine. Herein, we investigated the variation in phytochemical profiles and antioxidant activities of twelve different perilla seeds. The seeds showed significant variations in total phenolic and flavonoid contents ranging from 16.92 to 37.23 mg GAE/g (GAE, gallic acid equivalent) and 11.6 to 19.52 mg CAE/g (CAE, catechin equivalent), respectively. LC-QqQ-MS (liquid chromatography triple quadrupole tandem mass spectrometry)-based widely targeted metabolic profiling identified a total of 975 metabolites, including 68-269 differentially accumulated metabolites (DAMs). Multivariate analyses categorized the seeds into four groups based on the seed coat and leaf colors. Most key bioactive DAMs, including flavonoids (quercetin-3'-O-glucoside, prunin, naringenin, naringenin chalcone, butin, genistin, kaempferol-3-O-rutinoside, etc.), amino acids (valine, lysine, histidine, glutamine, threonine, etc.), and vitamins (B1, B3, B6, U, etc.) exhibited the highest relative content in PL3 (brown seed, purple leaf), PL1 (white seed, green-purple leaf), and PL4 (white seed, green leaf) groups, respectively. Meanwhile, key differentially accumulated phenolic acids showed a higher relative content in PL1 and PL4 than in other groups. Both seeds exhibited high antioxidant activities, although those of PL2 (brown seed, green leaf) group seeds were the lowest. Our results may facilitate the comprehensive use of perilla seeds in food and pharmaceutical industries.

Illustration of the variation in the content of flavanone rutinosides in various citrus germplasms from genetic and enzymatic perspectives.

In citrus, 1,6-rhamnosytransferase (1,6RhaT) and 1,2-rhamnosytransferase (1,2RhaT) catalyze flavanone-7-O-glucosides to form nonbitter flavanone rutinosides (FRs) and bitter flavanone neohesperidosides (FNs), respectively. As revealed in this study of fruit peels from 36 citrus accessions, FRs varied from undetectable levels in pummelo and kumquat to being the dominant flavonoids in sweet orange and loose-skin mandarins. Furthermore, a previously annotated full-length 1,6RhaT-like gene was identified as another 1,6RhaT-encoding gene by in vitro experiments. In total, 28 alleles of full-length 1,6RhaTs were isolated and classified into A, B and C types with only type A alleles encoding a functional protein. Coincidently, only the accessions that contained FRs harbored type A alleles, as was further verified in two F1 hybrid populations. Moreover, the inferior substrate conversion efficiency of 1,6RhaTs in comparison with that of 1,2RhaT in vitro might partly explain the lower proportions of FRs to total flavanone disaccharides in citrus hybrids harboring both functional rhamnosyltransferases. Our findings provide a better understanding of FR content variations among citrus and are meaningful for a mechanistic illustration of citrus flavonoid metabolism and fruit quality improvement practices.

Transcriptome analysis and molecular mechanism of linseed (Linum usitatissimum L.) drought tolerance under repeated drought using single-molecule long-read sequencing.

BACKGROUND: Oil flax (linseed, Linum usitatissimum L.) is one of the most important oil crops., However, the increases in drought resulting from climate change have dramatically reduces linseed yield and quality, but very little is known about how linseed coordinates the expression of drought resistance gene in response to different level of drought stress (DS) on the genome-wide level. RESULTS: To explore the linseed transcriptional response of DS and repeated drought (RD) stress, we determined the drought tolerance of different linseed varieties. Then we performed full-length transcriptome sequencing of drought-resistant variety (Z141) and drought-sensitive variety (NY-17) under DS and RD stress at the seedling stage using single-molecule real-time sequencing and RNA-sequencing. Gene Ontology (GO) and reduce and visualize GO (REVIGO) enrichment analysis showed that upregulated genes of Z141 were enriched in more functional pathways related to plant drought tolerance than those of NY-17 were under DS. In addition, 4436 linseed transcription factors were identified, and 1190 were responsive to stress treatments. Moreover, protein-protein interaction (PPI) network analysis showed that the proline biosynthesis pathway interacts with stress response genes through RAD50 (DNA repair protein 50) interacting protein 1 (RIN-1). Finally, proline biosynthesis and DNA repair structural gene expression patterns were verified by RT- PCR. CONCLUSIONS: The drought tolerance of Z141 may be related to its upregulation of drought tolerance genes under DS. Proline may play an important role in linseed drought tolerance by maintaining cell osmotic and protecting DNA from ROS damage. In summary, this study provides a new perspective to understand the drought adaptability of linseed.

Genomic Comparison and Population Diversity Analysis Provide Insights into the Domestication and Improvement of Flax.

Flax has been cultivated for its oil and fiber for thousands of years. However, it remains unclear how the modifications of agronomic traits occurred on the genetic level during flax cultivation. In this study, we conducted genome-wide variation analyses on multiple accessions of oil-use, fiber-use, landraces, and pale flax to identify the genomic variations during flax cultivation. Our findings indicate that, during flax domestication, genes relevant to flowering, dehiscence, oil production, and plant architecture were preferentially selected. Furthermore, regardless of origins, the improvement of the modern oil-use flax preceded that of the fiber-use flax, although the dual selection on oil-use and fiber-use characteristics might have occurred in the early flax domestication. We also found that the expansion of MYB46/MYB83 genes may have contributed to the unique secondary cell wall biosynthesis in flax and the directional selections on MYB46/MYB83 may have shaped the morphological profile of the current oil-use and fiber-use flax.

Sequencing of Chinese castor lines reveals genetic signatures of selection and yield-associated loci.

Oil produced by castor (Ricinus communis) has broad industrial applications. However, knowledge on the genetic diversity, especially genetic alterations that occurred during domestication and subsequent traits selection, of this oil crop is limited. Here, our population genomics analyses show that the Chinese castors have developed a geographic pattern, classified into the southern-, the middle-, and the northern-China groups. We detect a number of candidate genomic loci that are associated with the selection signals during the geographical differentiation and domestication. Using genome-wide association analysis, we identify candidate genes associated with nine agronomically important traits. One of the candidate genes encoding a glycosyltransferase related to cellulose and lignin biosynthesis is associated with both capsule dehiscence and endocarp thickness. We hypothesize that the abundance of cellulose or lignin in endocarp is an important factor for capsule dehiscence. Our results provide foundation for castor breeding and genetic study.

Introgression lines of Solanum sitiens, a wild nightshade of the Atacama Desert, in the genome of cultivated tomato.

The wild tomato relative Solanum sitiens is a xerophyte endemic to the Atacama Desert of Chile and a potential source of genes for tolerance to drought, salinity and low-temperature stresses. However, until recently, strong breeding barriers prevented its hybridization and introgression with cultivated tomato, Solanum lycopersicum L. We overcame these barriers using embryo rescue, bridging lines and allopolyploid hybrids, and synthesized a library of introgression lines (ILs) that captures the genome of S. sitiens in the background of cultivated tomato. The IL library consists of 56 overlapping introgressions that together represent about 93% of the S. sitiens genome: 65% in homozygous and 28% in heterozygous (segregating) ILs. The breakpoints of each segment and the gaps in genome coverage were mapped by single nucleotide polymorphism (SNP) genotyping using the SolCAP SNP array. Marker-assisted selection was used to backcross selected introgressions into tomato, to recover a uniform genetic background, to isolate recombinant sub-lines with shorter introgressions and to select homozygous genotypes. Each IL contains a single S. sitiens chromosome segment, defined by markers, in the genetic background of cv. NC 84173, a fresh market inbred line. Large differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs contain highly divergent allelic variation. Several loci contributing to unilateral incompatibility or hybrid necrosis were mapped with the lines. This IL population will facilitate studies of the S. sitiens genome and expands the range of genetic variation available for tomato breeding and research.

Natural Variation in CCD4 Promoter Underpins Species-Specific Evolution of Red Coloration in Citrus Peel.

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference. Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments. In this study, using an integrated genetic approach we revealed that a 5' cis-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C30 apocarotenoids responsible for red coloration of citrus peel. Functional analyses demonstrated that in addition the known role in synthesizing ß-citraurin, CCD4b is also responsible for the production of another important C30 apocarotenoid pigment, ß-citraurinene. Furthermore, analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5' cis-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C30 apocarotenoid-rich red-peeled accessions. Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids. Taken together, our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants, and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.

SLAF-Based Construction of a High-Density Genetic Map and Its Application in QTL Mapping of Carotenoids Content in Citrus Fruit.

Carotenoids are important antioxidant components in the human diet. To develop carotenoid-rich agricultural products by genetic intervention, understanding the genetic basis of carotenoids variation is essential. In this study, we constructed a high-density integrated genetic map with 3817 molecular markers using specific locus amplified fragment (SLAF) sequencing from a C. reticulata × P. trifoliata F1 pseudotestcross population. A total of 17 significant quantitative trait loci (QTLs) distributed on Chromosomes (Chr) 2, 3, 5, 6, and 9 were detected to determine the carotenoid variation in the population. In particular, three QTL colocalizations for multiple carotenoid constituents were observed on Chr 2, 3, and 9, one of which was located on Chr2:34,654,608-35430715 accounted for 20.1-25.4% of the variation of luteoxanthin, auroxanthin, lutein, violaxanthin, and total carotenoid content. Overall, this study provides a genetic foundation for marker-assisted selection (MAS) breeding of nutritionally enhanced citrus fruit.

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S-RNase-independent unilateral incompatibility.

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self-incompatible (SI) and the male parent self-compatible (SC) (the 'SI × SC rule'). Additional, as yet unknown, UI mechanisms are independent of self-incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen-side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S-RNase, the pistil determinant of S-specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18-fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F1 hybrid, leading to transmission ratio distortion in the F2 progeny. CRISPR/Cas9-generated knockout mutants (fps2) in S. pennelliiLA0716 are self-sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F2 progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2-based pollen selectivity does not involve S-RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.

Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction.

The emergence of apomixis-the transition from sexual to asexual reproduction-is a prominent feature of modern citrus. Here we de novo sequenced and comprehensively studied the genomes of four representative citrus species. Additionally, we sequenced 100 accessions of primitive, wild and cultivated citrus. Comparative population analysis suggested that genomic regions harboring energy- and reproduction-associated genes are probably under selection in cultivated citrus. We also narrowed the genetic locus responsible for citrus polyembryony, a form of apomixis, to an 80-kb region containing 11 candidate genes. One of these, CitRWP, is expressed at higher levels in ovules of polyembryonic cultivars. We found a miniature inverted-repeat transposable element insertion in the promoter region of CitRWP that cosegregated with polyembryony. This study provides new insights into citrus apomixis and constitutes a promising resource for the mining of agriculturally important genes.

Transcriptomic Analysis for Different Sex Types of Ricinus communis L. during Development from Apical Buds to Inflorescences by Digital Gene Expression Profiling.

The castor plant (Ricinus communis L.) is a versatile industrial oilseed crop with a diversity of sex patterns, its hybrid breeding for improving yield and high purity is still hampered by genetic instability of female and poor knowledge of sex expression mechanisms. To obtain some hints involved in sex expression and provide the basis for further insight into the molecular mechanisms of castor plant sex determination, we performed DGE analysis to investigate differences between the transcriptomes of apices and racemes derived from female (JXBM0705P) and monoecious (JXBM0705M) lines. A total of 18 DGE libraries were constructed from the apices and racemes of a wild monoecious line and its isogenic female derivative at three stages of apex development, in triplicate. Approximately 5.7 million clean tags per library were generated and mapped to the reference castor genome. Transcriptomic analysis showed that identical dynamic changes of gene expression were indicated in monoecious and female apical bud during its development from vegetation to reproduction, with more genes expressed at the raceme formation and infant raceme stages compare to the early leaf bud stage. More than 3000 of differentially expressed genes (DEGs) were detected in Ricinus apices at three developmental stages between two different sex types. A number of DEGs involved in hormone response and biosynthesis, such as auxin response and transport, transcription factors, signal transduction, histone demethylation/methylation, programmed cell death, and pollination, putatively associated with sex expression and reproduction were discovered, and the selected DEGs showed consistent expression between qRT-PCR validation and the DGE patterns. Most of those DEGs were suppressed at the early leaf stage in buds of the mutant, but then activated at the following transition stage (5-7-leaf stage) of buds in the mutant, and ultimately, the number of up-regulated DEGs was equal to that of down-regulation in the small raceme of the mutant. In this study, a large number of DEGs and some suggestions involved in sex expression and reproduction were discovered using DGE analysis, which provides large information and valuable hints for next insights into the molecular mechanism of sex determination. It is useful for other further studies in Ricinus.