Guixin Yan

Genome-Wide Association Study Dissects the Genetic Architecture of Seed Weight and Seed Quality in Rapeseed (Brassica napus L.)

Association mapping can quickly and efficiently dissect complex agronomic traits. Rapeseed is one of the most economically important polyploid oil crops, although its genome sequence is not yet published. In this study, a recently developed 60K Brassica Infinium® SNP array was used to analyse an association panel with 472 accessions. The single-nucleotide polymorphisms (SNPs) of the array were in silico mapped using ‘pseudomolecules’ representative of the genome of rapeseed to establish their hypothetical order and to perform association mapping of seed weight and seed quality. As a result, two significant associations on A8 and C3 of Brassica napus were detected for erucic acid content, and the peak SNPs were found to be only 233 and 128 kb away from the key genes BnaA.FAE1 and BnaC.FAE1. BnaA.FAE1 was also identified to be significantly associated with the oil content. Orthologues of Arabidopsis thaliana HAG1 were identified close to four clusters of SNPs associated with glucosinolate content on A9, C2, C7 and C9. For seed weight, we detected two association signals on A7 and A9, which were consistent with previous studies of quantitative trait loci mapping. The results indicate that our association mapping approach is suitable for fine mapping of the complex traits in rapeseed.

A genome-wide association study of plant height and primary branch number in rapeseed (Brassica napus)

Crop plant architecture plays a highly important role in its agronomic performance. Plant height (PH) and primary branch number (PB) are two major factors that affect the plant architecture of rapeseed (Brassica napus). Previous studies have shown that these two traits are controlled by multiple quantitative trait loci (QTL); however, QTLs have not been delimited to regions less than 10cM. Genome-wide association study (GWAS) is a highly efficient approach for identifying genetic loci controlling traits at relatively high resolution. In this study, variations in PH and PB of a panel of 472 rapeseed accessions that had previously been analyzed by a 60k SNP array were investigated for three consecutive years and studied by GWAS. Eight QTLs on chromosome A03, A05, A07 and C07 were identified for PH, and five QTLs on A01, A03, A07 and C07 were identified for PB. Although most QTLs have been detected in previous studies based on linkage analyses, the two QTLs of PH on A05 and the QTL of PB on C07 were novel. In the genomic regions close to the GWAS peaks, orthologs of the genes involved in flower development, phytohormone biosynthesis, metabolism and signaling in Arabidopsis were identified.

Association Mapping of Flowering Time QTLs and Insight into Their Contributions to Rapeseed Growth Habits

Plants have developed sophisticated systems to adapt to local conditions during evolution, domestication and natural or artificial selection. The selective pressures of these different growing conditions have caused significant genomic divergence within species. The flowering time trait is the most crucial factor because it helps plants to maintain sustainable development. Controlling flowering at appropriate times can also prevent plants from suffering from adverse growth conditions, such as drought, winter hardness, and disease. Hence, discovering the genome-wide genetic mechanisms that influence flowering time variations and understanding their contributions to adaptation should be a central goal of plant genetics and genomics. A global core collection panel with 448 inbred rapeseed lines was first planted in four independent environments, and their flowering time traits were evaluated. We then performed a genome-wide association mapping of flowering times with a 60 K SNP array for this core collection. With quality control and filtration, 20,342 SNP markers were ultimately used for further analyses. In total, 312 SNPs showed marker-trait associations in all four environments, and they were based on a threshold p-value of 4.06 × 10−4; the 40 QTLs showed significant association with flowering time variations. To explore flowering time QTLs and genes related to growth habits in rapeseed, selection signals related to divergent habits were screened at the genome-wide level and 117 genomic regions were found. Comparing locations of flowering time QTLs and genes with these selection regions revealed that 20 flowering time QTLs and 224 flowering time genes overlapped with 24 and 81 selected regions, respectively. Based on this study, a number of marker-trait associations and candidate genes for flowering time variations in rapeseed were revealed. Moreover, we also showed that both flowering time QTLs and genes play important roles in rapeseed growth habits. These results will be applied to rapeseed breeding programs, and they will aid in our understanding of the relation between flowering time variations and growth habits in plants.

Genome-Wide Survey and Expression Analysis of the Putative Non-Specific Lipid Transfer Proteins in Brassica rapa L

Background Plant non-specific lipid transfer proteins (nsLtps) are small, basic proteins encoded by multigene families and have reported functions in many physiological processes such as mediating phospholipid transfer, defense reactions against phytopathogens, the adaptation of plants to various environmental conditions, and sexual reproduction. To date, no genome-wide overview of the Brassica rapa nsLtp (BrnsLtp) gene family has been performed. Therefore, as the first step and as a helpful strategy to elucidate the functions of BrnsLtps, a genome-wide study for this gene family is necessary. Methodology/Principal Finding In this study, a total of 63 putative BrnsLtp genes were identified through a comprehensive in silico analysis of the whole genome of B. rapa. Based on the sequence similarities, these BrnsLtps was grouped into nine types (I, II, III, IV, V, VI, VIII, IX, and XI). There is no type VII nsLtps in B. rapa, and a new type, XI nsLtps, was identified in B. rapa. Furthermore, nine type II AtLtps have no homologous genes in B. rapa. Gene duplication analysis demonstrated that the conserved collinear block of each BrnsLtp is highly identical to those in Arabidopsis and that both segmental duplications and tandem duplications seem to play equal roles in the diversification of this gene family. Expression analysis indicated that 29 out of the 63 BrnsLtps showed specific expression patterns. After careful comparison and analysis, we hypothesize that some of the type I BrnsLtps may function like Arabidopsis pathogenesis-related-14 (PR-14) proteins to protect the plant from phytopathogen attack. Eleven BrnsLtps with inflorescence-specific expression may play important roles in sexual reproduction. Additionally, BrnsLtpI.3 may have functions similar to Arabidopsis LTP1. Conclusions/Significance The genome-wide identification, bioinformatic analysis and expression analysis of BrnsLtp genes should facilitate research of this gene family and polyploidy evolution and provide new insight towards elucidating their biological functions in plants.

Comparison of the heat stress induced variations in DNA methylation between heat-tolerant and heat-sensitive rapeseed seedlings

DNA methylation is responsive to various biotic and abiotic stresses. Heat stress is a serious threat to crop growth and development worldwide. Heat stress results in an array of morphological, physiological and biochemical changes in plants. The relationship between DNA methylation and heat stress in crops is relatively unknown. We investigated the differences in methylation levels and changes in the cytosine methylation patterns in seedlings of two rapeseed genotypes (heat-sensitive and heat-tolerant) under heat stress. Our results revealed that the methylation levels were different between a heat-tolerant genotype and a heat-sensitive one under control conditions. Under heat treatment, methylation increased more in the heat-sensitive genotype than in the heat-tolerant genotype. More DNA demethylation events occurred in the heat-tolerant genotype, while more DNA methylation occurred in the heat-sensitive genotype. A large and diverse set of genes were affected by heat stress via cytosine methylation changes, suggesting that these genes likely play important roles in the response and adaption to heat stress in Brassica napus L. This study indicated that the changes in DNA methylation differed between heat-tolerant and heat-sensitive genotypes of B. napus in response to heat stress, which further illuminates the molecular mechanisms of the adaption to heat stress in B. napus.

Assessing and broadening genetic diversity of a rapeseed germplasm collection

Assessing the level of genetic diversity within a germplasm collection contributes to evaluating the potential for its utilization as a gene pool to improve the performance of cultivars. In this study, 45 high-quality simple sequence repeat (SSR) markers were screened and used to estimate the genetic base of a world-wide collection of 248 rapeseed (Brassica napus) inbred lines. For the whole collection, the genetic diversity of A genome was higher than that of C genome. The genetic diversity of C genome for the semi-winter type was the lowest among the different germplasm types. Because B. oleracea is usually used to broaden the genetic diversity of C genome in rapeseed, we evaluated the potential of 25 wild B. oleracea lines. More allelic variations and a higher genetic diversity were observed in B. oleracea than in rapeseed. One B. oleracea line and one oilseed B. rapa line were used to generate a resynthesized Brassica napus line, which was then crossed with six semi-winter rapeseed cultivars to produce 7 F1 hybrids. Not only the allele introgression but also mutations were observed in the hybrids, resulting in significant improvement of the genetic base.

Global DNA methylation variations after short-term heat shock treatment in cultured microspores of Brassica napus cv. Topas.

Heat stress can induce the cultured microspores into embryogenesis. In this study, whole genome bisulphite sequencing was employed to study global DNA methylation variations after short-term heat shock (STHS) treatments in cultured microspores of Brassica napus cv. Topas. Our results indicated that treatment on cultured Topas microspores at 32 °C for 6 h triggered DNA hypomethylation, particularly in the CG and CHG contexts. And the total number of T32 (Topas 32 °C for 6 h) vs. T0 (Topas 0 h) differentially methylated region-related genes (DRGs) was approximately two-fold higher than that of T18 (Topas 18 °C for 6 h) vs. T0 DRGs, which suggested that 32 °C might be a more intense external stimulus than 18 °C resulting in more changes in the DNA methylation status of cultured microspores. Additionally, 32 °C treatment for 6 h led to increased CHG differential methylations of transposons (DMTs), which were mainly constituted by overlaps between the hypomethylated differentially methylated regions (hypo-DMRs) and transposon elements (TEs). Further analysis demonstrated that the DRGs and their paralogs exhibited differential methylated/demethylated patterns. To summarize, the present study is the first methylome analysis of cultured microspores in response to STHS and may provide valuable information on the roles of DNA methylation in heat response.

High-throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus, Brassica rapa and Brassica oleracea.

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole-genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C-genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.

High-Throughput Discovery of Chloroplast and Mitochondrial DNA Polymorphisms in Brassicaceae Species by ORG-EcoTILLING

Background Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used. Methodology/Principal Findings To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results. Conclusions/Significance These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.

Identification and Characterization of a Glyoxalase I Gene in a Rapeseed Cultivar with Seed Thermotolerance.

Glyoxalase I (GLYI) is a ubiquitous enzyme in all organisms that catalyzes the conversion of the potent cytotoxin methylglyoxal to S-D-lactoylglutathione. Although many reports suggest the importance of GLYI in the plant response to stress, its function in seeds requires further study. Here, we identified a heat-induced GLYI from Brassica napus seeds, BnGLYI, using a comparative proteomics approach. Two-dimensional gel analyses revealed that BnGLYI protein expression upon heat treatment was significantly elevated in thermotolerant seeds but was diminished in heat-sensitive seeds. The BnGLYI-2 and BnGLYI-3 genes from the heat-sensitive and thermotolerant cultivars, respectively, were characterized, and analyzed. Only two amino acid residue variations were found between the amino acid sequences of the two genes. Moreover, overexpressing BnGLYI-3 in yeast cells enhanced tolerance to heat and cold stress and significantly increased GLYI activity compared to overexpressing BnGLYI-2. In addition, BnGLYI-3 transformants showed enhanced superoxide dismutase activities under heat and cold treatment, whereas these activities were diminished for BnGLYI-2 transformants. Taken together, these results indicate that overexpression of the BnGLYI-3 gene imparts thermotolerance and cold tolerance in yeast and that the variations in BnGLYI-3 may play an important role in the responses to temperature stresses.