Jiana Li

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

Enhanced Seed Oil Production in Canola by Conditional Expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in Developing Seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.

A High-Density SNP Map for Accurate Mapping of Seed Fibre QTL in Brassica napus L

A high density genetic linkage map for the complex allotetraploid crop species Brassica napus (oilseed rape) was constructed in a late-generation recombinant inbred line (RIL) population, using genome-wide single nucleotide polymorphism (SNP) markers assayed by the Brassica 60 K Infinium BeadChip Array. The linkage map contains 9164 SNP markers covering 1832.9 cM. 1232 bins account for 7648 of the markers. A subset of 2795 SNP markers, with an average distance of 0.66 cM between adjacent markers, was applied for QTL mapping of seed colour and the cell wall fiber components acid detergent lignin (ADL), cellulose and hemicellulose. After phenotypic analyses across four different environments a total of 11 QTL were detected for seed colour and fiber traits. The high-density map considerably improved QTL resolution compared to the previous low-density maps. A previously identified major QTL with very high effects on seed colour and ADL was pinpointed to a narrow genome interval on chromosome A09, while a minor QTL explaining 8.1% to 14.1% of variation for ADL was detected on chromosome C05. Five and three QTL accounting for 4.7% to 21.9% and 7.3% to 16.9% of the phenotypic variation for cellulose and hemicellulose, respectively, were also detected. To our knowledge this is the first description of QTL for seed cellulose and hemicellulose in B. napus, representing interesting new targets for improving oil content. The high density SNP genetic map enables navigation from interesting B. napus QTL to Brassica genome sequences, giving useful new information for understanding the genetics of key seed quality traits in rapeseed.

Genome-wide association study reveals the genetic architecture of flowering time in rapeseed (Brassica napus L.)

Flowering time adaptation is a major breeding goal in the allopolyploid species Brassica napus. To investigate the genetic architecture of flowering time, a genome-wide association study (GWAS) of flowering time was conducted with a diversity panel comprising 523 B. napus cultivars and inbred lines grown in eight different environments. Genotyping was performed with a Brassica 60K Illumina Infinium SNP array. A total of 41 single-nucleotide polymorphisms (SNPs) distributed on 14 chromosomes were found to be associated with flowering time, and 12 SNPs located in the confidence intervals of quantitative trait loci (QTL) identified in previous researches based on linkage analyses. Twenty-five candidate genes were orthologous to Arabidopsis thaliana flowering genes. To further our understanding of the genetic factors influencing flowering time in different environments, GWAS was performed on two derived traits, environment sensitivity and temperature sensitivity. The most significant SNPs were found near Bn-scaff_16362_1-p380982, just 13 kb away from BnaC09g41990D, which is orthologous to A. thaliana CONSTANS (CO), an important gene in the photoperiod flowering pathway. These results provide new insights into the genetic control of flowering time in B. napus and indicate that GWAS is an effective method by which to reveal natural variations of complex traits in B. napus.

Identification of resistant sources against Sclerotinia sclerotiorum in Brassica species with emphasis on B. oleracea

Stem rot caused by Sclerotinia sclerotiorum is one of the most devastating diseases of rapeseed (Brassica napus L.) which causes huge loss in rapeseed production. Genetic sources with high level of resistance has not been found in rapeseed. In this study, 68 accessions in six Brassica species, including 47 accessions of B. oleracea, were evaluated for leaf and stem resistance to S. sclerotiorum. Large variation of resistance was found in Brassica, with maximum differences of 5- and 57-folds in leaf and stem resistance respectively. B. oleracea, especially its wild types such as B. rupestris, B. incana, B. insularis, and B. villosa showed high level of resistance. Our data suggest that wild types of B. oleracea possess tremendous potential for improving S. sclerotiorum resistance of rapeseed.

Horizontal gene transfer in plants

Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries. HGT often occurs in microbic and eukaryotic genomes. However, the pathways by which HGTs occur in multicellular eukaryotes, especially in plants, are not well understood. We systematically summarized more than ten possible pathways for HGT. The intimate contact which frequently occurs in parasitism, symbiosis, pathogen, epiphyte, entophyte, and grafting interactions could promote HGTs between two species. Besides these direct transfer methods, genes can be exchanged with a vector as a bridge: possible vectors include pollen, fungi, bacteria, viruses, viroids, plasmids, transposons, and insects. HGT, especially when involving horizontal transfer of transposable elements, is recognized as a significant force propelling genomic variation and biological innovation, playing an important functional and evolutionary role in both eukaryotic and prokaryotic genomes. We proposed possible mechanisms by which HGTs can occur, which is useful in understanding the genetic information exchange among distant species or distant cellular components.

Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

BackgroundSingle nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.ResultsA total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.ConclusionsDespite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.

Differential accumulation of phenolic compounds and expression of related genes in black- and yellow-seeded Brassica napus

Developing yellow-seeded Brassica napus (rapeseed) with improved qualities is a major breeding goal. The intermediate and final metabolites of the phenylpropanoid and flavonoid pathways affect not only oil quality but also seed coat colour of B. napus. Here, the accumulation of phenolic compounds was analysed in the seed coats of black-seeded (ZY821) and yellow-seeded (GH06) B. napus. Using toluidine blue O staining and liquid chromatography–mass spectrometry, histochemical and biochemical differences were identified in the accumulation of phenolic compounds between ZY821 and GH06. Two and 13 unique flavonol derivatives were detected in ZY821 and GH06, respectively. Quantitative real-time PCR analysis revealed significant differences between ZY821 and GH06 in the expression of common phenylpropanoid biosynthetic genes (BnPAL and BnC4H), common flavonoid biosynthetic genes (BnTT4 and BnTT6), anthocyanin- and proanthocyandin-specific genes (BnTT3 and BnTT18), proanthocyandin-specific genes (BnTT12, BnTT10, and BnUGT2) and three transcription factor genes (BnTTG1, BnTTG2, and BnTT8) that function in the flavonoid biosynthetic pathway. These data provide insight into pigment accumulation in B. napus, and serve as a useful resource for researchers analysing the formation of seed coat colour and the underlying regulatory mechanisms in B. napus.

Gene silencing of BnTT10 family genes causes retarded pigmentation and lignin reduction in the seed coat of Brassica napus.

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.

The Evolutionary History of R2R3-MYB Proteins Across 50 Eukaryotes: New Insights Into Subfamily Classification and Expansion.

R2R3-MYB proteins (2R-MYBs) are one of the main transcription factor families in higher plants. Since the evolutionary history of this gene family across the eukaryotic kingdom remains unknown, we performed a comparative analysis of 2R-MYBs from 50 major eukaryotic lineages, with particular emphasis on land plants. A total of 1548 candidates were identified among diverse taxonomic groups, which allowed for an updated classification of 73 highly conserved subfamilies, including many newly identified subfamilies. Our results revealed that the protein architectures, intron patterns, and sequence characteristics were remarkably conserved in each subfamily. At least four subfamilies were derived from early land plants, 10 evolved from spermatophytes, and 19 from angiosperms, demonstrating the diversity and preferential expansion of this gene family in land plants. Moreover, we determined that their remarkable expansion was mainly attributed to whole genome and segmental duplication, where duplicates were preferentially retained within certain subfamilies that shared three homologous intron patterns (a, b, and c) even though up to 12 types of patterns existed. Through our integrated distributions, sequence characteristics, and phylogenetic tree analyses, we confirm that 2R-MYBs are old and postulate that 3R-MYBs may be evolutionarily derived from 2R-MYBs via intragenic domain duplication.