Jiaqin Mei

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.

Comparative quantitative trait loci for silique length and seed weight in Brassica napus.

Silique length (SL) and seed weight (SW) are important yield-associated traits in rapeseed (Brassica napus). Although many quantitative trait loci (QTL) for SL and SW have been identified in B. napus, comparative analysis for those QTL is seldom performed. In the present study, 20 and 21 QTL for SL and SW were identified in doubled haploid (DH) and DH-derived reconstructed F2 populations in rapeseed, explaining 55.1–74.3% and 24.4–62.9% of the phenotypic variation across three years, respectively. Of which, 17 QTL with partially or completely overlapped confidence interval on chromosome A09, were homologous with two overlapped QTL on chromosome C08 by aligning QTL confidence intervals with the reference genomes of Brassica crops. By high density selective genotyping of DH lines with extreme phenotypes, using a Brassica single-nucleotide polymorphism (SNP) array, the QTL on chromosome A09 was narrowed, and aligned into 1.14-Mb region from 30.84 to 31.98 Mb on chromosome R09 of B. rapa and 1.05-Mb region from 27.21 to 28.26 Mb on chromosome A09 of B. napus. The alignment of QTL with Brassica reference genomes revealed homologous QTL on A09 and C08 for SL. The narrowed QTL region provides clues for gene cloning and breeding cultivars by marker-assisted selection.

Genomic relationships between wild and cultivated Brassica oleracea L. with emphasis on the origination of cultivated crops

Wild taxa in Brassica oleracea L. play an important role to improve cultivated crops, but the genomic relationships between wild and cultivated forms have not been well clarified. An overall survey of genomic relationships among 39 accessions covering 10 wild and 7 cultivared types in B. oleracea was performed using amplified fragment length polymorphism and simple sequence repeat. The cultivated types were clustered together with B. oleracea ssp. oleracea,B. incana, B. bourgeaui, B. montana, B. cretica and B. hilarionis, while 4 wild taxa from Sicily, B. rupestris, B. insularis, B. macrocarpa and B. villosa formed the other group. It implies the low possibility that current B. oleracea crops originated in Sicily.

Screening resistance against Sclerotinia sclerotiorum in Brassica crops with use of detached stem assay under controlled environment

Sclerotinia sclerotiorum is a major pathogen that infects stem tissue, causing yield loss and poor seed quality in rapeseed (Brassica napus). Here we report a reliable detached stem inoculation method for evaluating resistance under controlled environment. Two sets of Brassica materials were employed, including 17 genotypes from five Brassica crops in experiment 1, and 71 F2 lines derived from a cross between susceptible and resistant lines of B. oleracea in experiment 2. High correlations were detected between stem and branch for lesion length in both experiments and between stem and sections of stem in experiment 1. Although the lesion length of detached stem inoculation under controlled environment was positively correlated with that of toothpick inoculation in the field, the variation of lesion length in the detached stem inoculation was lower than that in toothpick inoculation in experiment 2. Moreover, no significant association was detected between lesion length and the diameter of stem or branch. These findings suggest that the detached stem inoculation under controlled conditions is a large-scale, flexible and reliable method of screening for resistance against S. sclerotiorum in Brassica crops. The application of detached stem inoculation is discussed in resistance breeding programs against S. sclerotiorum in Brassica crops.

DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5′-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5′-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola (Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

Genome-wide identification of loci affecting seed glucosinolate contents in Brassica napus L: Ecotype-specific loci for seed glucosinolate content

Glucosinolates are amino acid-derived secondary metabolites that act as chemical defense agents against pests. However, the presence of high levels of glucosinolates severely diminishes the nutritional value of seed meals made from rapeseed (Brassica napus L.). To identify the loci affecting seed glucosinolate content (SGC), we conducted genome-wide resequencing in a population of 307 diverse B. napus accessions from the three B. napus ecotype groups, namely, spring, winter, and semi-winter. These resequencing data were used for a genome-wide association study (GWAS) to identify the loci affecting SGC. In the three ecotype groups, four common and four ecotype-specific haplotype blocks (HBs) were significantly associated with SGC. To identify candidate genes controlling SGC, transcriptome analysis was carried out in 36 accessions showing extreme SGC values. Analyses of haplotypes, genomic variation, and candidate gene expression pointed to five and three candidate genes in the common and spring group-specific HBs, respectively. Our expression analyses demonstrated that additive effects of the three candidate genes in the spring group-specific HB play important roles in the SGC of B. napus.

Identification of quantitative trait loci for Clubroot Resistance in Brassica oleracea with the use of Brassica SNP microarray

Increasing clubroot resistance (CR) of Brassica oleracea by ascertaining the molecular mechanisms has been the key focus in modern B. oleracea breeding. In order to identify the quantitative trait loci (QTLs) associated with CR in B. oleracea, 94 F2 vegetative lines which were developed by tissue culture of selfed seeds from the F1 generation between a clubroot-resistant B. oleracea inbred line and a susceptible line, were identified for disease incidence and six CR-associated traits under a lab inoculation by Plasmodiophora brassicae and were genotyped with the 60K Brassica SNP array. Significant correlations were detected for numbers of fibrous roots and P. brassicae content in roots with disease incidence. Nine linkage groups were constructed from 565 bins which covered around 3,000 SNPs, spanning 1,028 cM of the B. oleracea genome with an average distance of 1.82 cM between adjacent bins. A total of 23 QTLs were identified for disease incidence and the other two correlated traits, individually explaining 6.1–17.8% of the phenotypic variation. Several overlaps were detected among traits, including one three-traits-overlapped locus on linkage group C08 and two important overlapped regions between the two CR-associated traits on C06. The QTLs were compared with known CR loci/genes and the novelty of our QTLs was discussed.

Genetics and epigenetic alterations of hexaploid early generation derived from hybrid between Brassica napus and B. oleracea

Good fertility was observed previously in hexaploid derived from hybrid (ACC) between calona ‘Zhongshuang 9’(Brassica napus, 2n = 38, AACC) and kale ‘SWU01’ (B. oleracea var. acephala, 2n = 18, CC). However, the mechanism to underlying the character is unknown. In the present study, genetic and epigenetic alterations of S0, 6 S1, and 18 of their S2 progenies with hexaploid chromosome conformation (20A + 36C) were selected to compare with ACC and their parental species. 13.08% and 26.45% polymorphism alleles different from two parental species were identified in ACC via 58 SSR (simple sequence repeats) and 14 MSAP (methylation sensitive amplified polymorphism), respectively. 33.74% new alleles in DNA methylation, but not in DNA sequence were detected in S0 after chromosome doubling of ACC. DNA profilling revealed a little genetic but much epigenetic differences among S0, S1 and S2 generations. Genetic alteration was relatively stable, because only 8.09% and 3.21% alleles inheriated from ACC were changed in S2 and S1, respectively. While on average of 52.44 ± 5.32% DNA methylation site inherited from ACC were detected in S1, and 41.52 ± 9.04% in S2 due to dramatic epigenetic variance among early generations. New DNA methylation sites occurred in S0 would inheritated into the successive generations, but the frequency was decreased because some new site might be recovered. It demonstrated that much DNA methylation but a little DNA sequence variance was occurred in hexaploid early generation.