Lijuan Wei

Identification of Rapeseed MicroRNAs Involved in Early Stage Seed Germination under Salt and Drought Stresses

Drought and salinity are severe and wide-ranging abiotic stresses that substantially affect crop germination, development and productivity, and seed germination is the first critical step in plant growth and development. To comprehensively investigate small-RNA targets and improve our understanding of miRNA-mediated post-transcriptional regulation networks during Brassica napus seed imbibition under drought and salt stresses, we constructed three small-RNA libraries from B. napus variety ZS11 embryos exposed to salt (200 mM NaCl, denoted “S”), drought (200 g L−1 PEG-6000, denoted “D”), and distilled water (denoted “CK”) during imbibition and sequenced them using an Illumina Genome Analyzer. A total of 11,528,557, 12,080,081, and 12,315,608 raw reads were obtained from the CK, D, and S libraries, respectively. Further analysis identified 85 known miRNAs belonging to 31 miRNA families and 882 novel miRNAs among the three libraries. Comparison of the D and CK libraries revealed significant down-regulation of six miRNA families, miR156, miR169, miR860, miR399, miR171, and miR395, whereas only miR172 was significantly up-regulated. In contrast, comparison of the S library with the CK library showed significant down-regulation of only two miRNA families: miRNA393 and miRNA399. Putative targets for 336, 376, and 340 novel miRNAs were successfully predicted in the CK, D, and S libraries, respectively, and 271 miRNA families and 20 target gene families [including disease resistance protein (DIRP), drought-responsive family protein (DRRP), early responsive to dehydration stress protein (ERD), stress-responsive alpha-beta barrel domain protein (SRAP), and salt tolerance homolog2 (STH2)] were confirmed as being core miRNAs and genes involved in the seed imbibition response to salt and drought stresses. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs as well as the predicted target genes. Our data suggest that diverse and complex miRNAs are involved in seed imbibition, indicating that miRNAs are involved in plant hormone regulation, and may play important roles during seed germination under salt- or drought-stress conditions.

New Insights into Nested Long Terminal Repeat Retrotransposons in Brassica Species

Long terminal repeat (LTR) retrotransposons, one of the foremost types of transposons, continually change or modify gene function and reorganize the genome through bursts of dramatic proliferation. Many LTR-TEs preferentially insert within other LTR-TEs, but the cause and evolutionary significance of these nested LTR-TEs are not well understood. In this study, a total of 1.52Gb of Brassica sequence containing 2020 bacterial artificial chromosomes (BACs) was scanned, and six bacterial artificial chromosome (BAC) clones with extremely nested LTR-TEs (LTR-TEs density: 7.24/kb) were selected for further analysis. The majority of the LTR-TEs in four of the six BACs were found to be derived from the rapid proliferation of retrotransposons originating within the BAC regions, with only a few LTR-TEs originating from the proliferation and insertion of retrotransposons from outside the BAC regions approximately 5-23Mya. LTR-TEs also preferably inserted into TA-rich repeat regions. Gene prediction by Genescan identified 207 genes in the 0.84Mb of total BAC sequences. Only a few genes (3/207) could be matched to the Brassica expressed sequence tag (EST) database, indicating that most genes were inactive after retrotransposon insertion. Five of the six BACs were putatively centromeric. Hence, nested LTR-TEs in centromere regions are rapidly duplicated, repeatedly inserted, and act to suppress activity of genes and to reshuffle the structure of the centromeric sequences. Our results suggest that LTR-TEs burst and proliferate on a local scale to create nested LTR-TE regions, and that these nested LTR-TEs play a role in the formation of centromeres.

Identification of microRNAs Actively Involved in Fatty Acid Biosynthesis in Developing Brassica napus Seeds Using High-Throughput Sequencing

Seed development has a critical role during the spermatophyte life cycle. In Brassica napus, a major oil crop, fatty acids are synthesized and stored in specific tissues during embryogenesis, and understanding the molecular mechanism underlying fatty acid biosynthesis during seed development is an important research goal. In this study, we constructed three small RNA libraries from early seeds at 14, 21, and 28 days after flowering (DAF) and used high-throughput sequencing to examine microRNA (miRNA) expression. A total of 85 known miRNAs from 30 families and 1160 novel miRNAs were identified, of which 24, including 5 known and 19 novel miRNAs, were found to be involved in fatty acid biosynthesis.bna-miR156b, bna-miR156c, bna-miR156g, novel_mir_1706, novel_mir_1407, novel_mir_173, and novel_mir_104 were significantly down-regulated at 21 DAF and 28 DAF, whereas bna-miR159, novel_mir_1081, novel_mir_19 and novel_mir_555 were significantly up-regulated. In addition, we found that some miRNAs regulate functional genes that are directly involved in fatty acid biosynthesis and that other miRNAs regulate the process of fatty acid biosynthesis by acting on a large number of transcription factors. The miRNAs and their corresponding predicted targets were partially validated by quantitative RT-PCR. Our data suggest that diverse and complex miRNAs are involved in the seed development process and that miRNAs play important roles in fatty acid biosynthesis during seed development.

Transposon variation by order during allopolyploidisation between Brassica oleracea and Brassica rapa

Although many studies have shown that transposable element (TE) activation is induced by hybridisation and polyploidisation in plants, much less is known on how different types of TE respond to hybridisation, and the impact of TE-associated sequences on gene function. We investigated the frequency and regularity of putative transposon activation for different types of TE, and determined the impact of TE-associated sequence variation on the genome during allopolyploidisation. We designed different types of TE primers and adopted the Inter-Retrotransposon Amplified Polymorphism (IRAP) method to detect variation in TE-associated sequences during the process of allopolyploidisation between Brassica rapa (AA) and Brassica oleracea (CC), and in successive generations of self-pollinated progeny. In addition, fragments with TE insertions were used to perform Blast2GO analysis to characterise the putative functions of the fragments with TE insertions. Ninety-two primers amplifying 548 loci were used to detect variation in sequences associated with four different orders of TE sequences. TEs could be classed in ascending frequency into LTR-REs, TIRs, LINEs, SINEs and unknown TEs. The frequency of novel variation (putative activation) detected for the four orders of TEs was highest from the F1 to F2 generations, and lowest from the F2 to F3 generations. Functional annotation of sequences with TE insertions showed that genes with TE insertions were mainly involved in metabolic processes and binding, and preferentially functioned in organelles. TE variation in our study severely disturbed the genetic compositions of the different generations, resulting in inconsistencies in genetic clustering. Different types of TE showed different patterns of variation during the process of allopolyploidisation.

New insights into helitron transposable elements in the mesopolyploid species Brassica rapa

Helitrons are DNA transposable elements that are widely present in the genomes of diverse eukaryotic taxa. Helitrons are distinct from other transposons in their ability to capture gene fragments and their rolling-replication mechanism. Brassica rapa is a mesopolyploid species and one of the most important vegetable and oil crops globally. A total of 787 helitrons were identified in the B. rapa genome and were assigned to 662 families and 700 subfamilies. More than 21,806 repetitive sequences were found within the helitrons, whose G+C content correlated negatively to that of the host helitron. Each helitron contained an average of 2.9 gene fragments and 1.9 intact genes, of which the majority were annotated with binding functions in metabolic processes. In addition, a set of 114 nonredundant microRNAs were detected within 174 helitrons and predicted to regulate a set of 787 nonredundant target genes. These results suggest that helitrons contribute to genomic structural and transcriptional variation by capturing gene fragments and generating microRNAs.

Genetic and transcriptomic analyses of lignin- and lodging-related traits in Brassica napus

Key messageCandidate genes associated with lignin and lodging traits were identified by combining phenotypic, genotypic, and gene expression data in B. napus.AbstractBrassica napus is one of the world’s most important oilseed crops, but its yield can be dramatically reduced by lodging, bending, and falling of its vertical stems. Lignin has been shown to contribute to stem mechanical strength. In this study, we found that the syringyl/guaiacyl (S/G) monolignol ratio exhibits a significant negative correlation with disease and lodging resistance. A total of 92 and 50 SNP and SSR loci, respectively, were found to be significantly associated with five traits, breaking force, breaking strength, lodging coefficient, acid detergent lignin content, and the S/G monolignol ratio using GWAS. To identify novel genes involved in lignin biosynthesis, transcriptome sequencing of high- (H) and low (L)-ADL content accessions was performed. The up-regulated genes were mainly involved in glycoside catabolic processes (especially glucosinolate catabolism) and cell wall biogenesis, while down-regulated genes were involved in glucosinolate biosynthesis, indicating that crosstalk exists between glucosinolate metabolic processes and lignin biosynthesis. Integrating this differential expression with the GWAS analysis, we identified four candidate genes regulating lignin, including glycosyl hydrolase (BnaA01g00480D), CYT1 (BnaA04g22820D), and two encoding transcription factors, SHINE1 (ERF family) and DAR6 (LIM family). This study provides insight into the genetic control of lodging and lignin in B. napus.

Genome-Wide Analysis of Seed Acid Detergent Lignin (ADL) and Hull Content in Rapeseed (Brassica napus L.)

A stable yellow-seeded variety is the breeding goal for obtaining the ideal rapeseed (Brassica napus L.) plant, and the amount of acid detergent lignin (ADL) in the seeds and the hull content (HC) are often used as yellow-seeded rapeseed screening indices. In this study, a genome-wide association analysis of 520 accessions was performed using the Q + K model with a total of 31,839 single-nucleotide polymorphism (SNP) sites. As a result, three significant associations on the B. napus chromosomes A05, A09, and C05 were detected for seed ADL content. The peak SNPs were within 9.27, 14.22, and 20.86 kb of the key genes BnaA.PAL4, BnaA.CAD2/BnaA.CAD3, and BnaC.CCR1, respectively. Further analyses were performed on the major locus of A05, which was also detected in the seed HC examination. A comparison of our genome-wide association study (GWAS) results and previous linkage mappings revealed a common chromosomal region on A09, which indicates that GWAS can be used as a powerful complementary strategy for dissecting complex traits in B. napus. Genomic selection (GS) utilizing the significant SNP markers based on the GWAS results exhibited increased predictive ability, indicating that the predictive ability of a given model can be substantially improved by using GWAS and GS.

Expression Characteristics of Key Genes in Lignin Pathway among Different Lodging Resistance Lines of Brassica napus L.

Lignin is related to not only plant disease resistance but also lodging resistance.The lignin content and six key genes(PAL,4CL,C4H,CCR1,CCR2,and CAD) expression characteristics of the middle stem were analysed at early flowering stage and podding stage of 15 Brassica napus L.varieties with different lodging resistance level.The results indicated that the lignin content generally increased by 28% from early flowering stage to podding stage,especially by 33.5% in the varieties with stronger lodging resistance.Lignin content was significantly different between the varieties with different lodging resistance levels.The expression of key enzyme genes was remarkably different between two stages,and the expression of genes PAL,4CL,and CCR1 was remarkably different between the varieties with different lodging resistance levels.The expression of gene PAL was significantly correlated with lignin content at both stages,and the expression of gene 4CL was significantly correlated with that of most other genes.Our results suggested that all the genes studied in the paper could control the lignin synthesis,and PAL and 4CL were the most important genes in lignin pathway.

Extensive tRNA gene changes in synthetic Brassica napus

Allopolyploidization, where two species come together to form a new species, plays a major role in speciation and genome evolution. Transfer RNAs (abbreviated tRNA) are typically 73–94 nucleotides in length, and are indispensable in protein synthesis, transferring amino acids to the cell protein synthesis machinery (ribosome). To date, the regularity and function of tRNA gene sequence variation during the process of allopolyploidization have not been well understood. In this study, the inter-tRNA gene corresponding to tRNA amplification polymorphism method was used to detect changes in tRNA gene sequences in the progeny of interspecific hybrids between Brassica rapa and B. oleracea, mimicking the original B. napus (canola) species formation event. Cluster analysis showed that tRNA gene variation during allopolyploidization did not appear to have a genotypic basis. Significant variation occurred in the early generations of synthetic B. napus (F1 and F2 generations), but fewer alterations were observed in the later generation (F3). The variation-prone tRNA genes tended to be located in AT-rich regions. BlastN analysis of novel tRNA gene variants against a Brassica genome sequence database showed that the variation of these tRNA-gene-associated sequences in allopolyploidization might result in variation of gene structure and function, e.g., metabolic process and transport.

Characterization and evolutionary analysis of Brassica species-diverged sequences containing simple repeat units

Brassica species, B. napus (canola), B. rapa and B. oleracea, are important sources of nutritionally valuable vegetable oil and protein-rich meal for animals and humans. Sequencing of the model plant Arabidopsis thaliana, has opened the way for investigations into the complex structure of the Brassica genomes, offering important insight into their evolution and composition. We use this sequence information for the characterization and functional analysis of SSR sequences that have diverged between the Brassica species. A total of 56 species-diverged sequences containing simple repeat units (SDS-SSR) of B. napus and its diploid progenitor species B. rapa and B. oleracea were isolated and characterized. Of these, 40 sequences showed homology with other Brassica sequences. Using the SSR Locator software, only 23 sequences were found to have SSRs, possibly due to the loss of SSR units in the process of species divergence. Sequence alignments with A. thaliana revealed that these species-diverged SSR sequences were responsible for Brassica divergence for differences between Brassica species in several genomic regions. Six active genes related to transferase, protein, transcription factor and retroelements were found in the SDS-SSRs. These results will further improve our understanding of the characteristics of species-diverged SSR fragments and their contribution to genome differentiation.