Jinjin Jiang

Use of digital gene expression to discriminate gene expression differences in early generations of resynthesized Brassica napus and its diploid progenitors

BackgroundPolyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. Brassica napus L. is one of the most economically important polyploid oil crops and has been broadly studied as an example of polyploid crop. RNA-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids.ResultsWe examined the global gene expression patterns of the first four generations of resynthesized B. napus (F1–F4), its diploid progenitors B. rapa and B. oleracea, and natural B. napus using digital gene expression analysis. Almost 42 million clean tags were generated using Illumina technology to produce the expression data for 25959 genes, which account for 63% of the annotated B. rapa genome. More than 56% of the genes were transcribed from both strands, which indicate the importance of RNA-mediated gene regulation in polyploidization. Tag mapping of the B. rapa genome generated 19023, 18547, 24383, 20659, 18881, 20692, and 19955 annotated genes for the B. rapa, B. oleracea, F1–F4 of synthesized B. napus, and natural B. napus libraries, respectively. The unambiguous tag-mapped genes in the libraries were functionally categorized via gene ontological analysis. Thousands of differentially expressed genes (DEGs) were identified and revealed the substantial changes in F1–F4. Among the 20 most DEGs are DNA binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs suggested approximately 120 biological pathways.ConclusionsThe systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized B. napus. This information broadens our understanding of the mechanisms of B. napus polyploidization and contributes to molecular and genetic research by enriching the Brassica database.

Protoplast fusion for crop improvement and breeding in China

Protoplast fusion offers an opportunity for circumventing barriers to sexual reproduction and allows for gene transfer of nuclear and cytoplasmic genomes to enrich the gene pool of cultivated species. Moreover, protoplast fusion effectively generates novel germplasm for elite breeding of conventional crosses and promotes crop improvement in existing cultivars. Over the past few decades, protoplast fusion in China has considerably progressed not only for food crops but also for economic plants. In this review, we present and discuss the development of somatic hybrids in wheat, rapeseed, citrus, and cotton, some of which are already in use or have potential for future commercial use in China. For example, an elite salt- and drought-resistant line, Shangrong No. 3, has already been registered as a commercial wheat cultivar. Some other hybrids have been found to have disease resistance as well as modified fatty acids, high oil and protein contents, novel cytoplasmic male sterility, and numerous other desirable agronomic characteristics that are useful for further breeding.

Phenolic composition analysis and gene expression in developing seeds of yellow- and black-seeded Brassica napus.

Breeders have focused on yellow-seeded Brassica napus (rapeseed) for its better quality compared with the black-seeded variety. Moreover, flavonoids have been associated with this kind of rapeseed. In this study, we applied lipid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS(n)) to compare flavonoids in developing seeds of natural black-seeded B. napus and yellow-seeded introgression lines selected from progenies of B. napus-Sinapis alba somatic hybrids. Aside from the most abundant phenolic compounds (sinapine and sinapic acid) and 1, 2-disinapoylglucose, 16 different flavonoids were identified and quantified, including (-)-epicatechin, five monocharged oligomers of (-)-epicatechin ([DP 2](-), [DP 3](-), [DP 4] [DP 2](-) B2 and [DP 2](-) B5), quercetin, kaempferol, isorhamnetin-dihexoside, kaempferol-sinapoyl-trihexoside, isorhamnetin-sinapoyl-trihexoside, isorhamnetin-hexoside-sulfate, and isorhamnetin-3-O-glucoside. Most of the flavonoids accumulated with seed development, whereas some rapidly decreased during maturation. The content of these flavonoids were lower in the yellow-seeded materials than in the black seeds. In addition, variations of insoluble procyanidin oligomers and soluble phenolic acids were observed among both rapeseed varieties. Transcriptome changes of genes participating in the flavonoid pathway were discovered by quantitative reverse transcription polymerase chain reaction analysis. Consistent with flavonoid changes identified by high performance liquid chromatography analysis, the expression of most genes in the flavonoid biosynthetic pathway was also downregulated.

Intertribal somatic hybrids between Brassica napus and Camelina sativa with high linolenic acid content

Intertribal somatic hybrids of Brassica napus and Camelina sativa were developed by protoplast electrofusion. Hybrid identity of the regenerants was determined using flow cytometric analysis of nuclear DNA content and simple sequence repeat (SSR) marker analysis. Three hybrids exhibited specific bands for B. napus and C. sativa. These hybrids showed intermediate leaf, flower and seed morphology compared with the two parental species. The seeds of these three hybrids had a modified fatty acid profile, indicating higher level of linolenic and eicosanoic acids than those of B. napus. Our results suggest that somatic hybridization offers opportunities for transferring entire genomes between B. napus and C. sativa in improving rapeseed breeding.

Phenotypic variation in progenies from somatic hybrids between Brassica napus and Sinapis alba

A number of variant phenotypes, such as long siliques, high density of pods, increased seed number per pod and yellow seed color resembling Sinapis alba, were selected from intergeneric somatic hybrids between Brassica napus and Sinapis alba through successive backcrosses. Resistance to Sclerotinia sclerotiorum among 24 BCF4 lines was also tested by inoculation with mycelia, and four lines showed stronger resistance than the resistant rapeseed variety ‘Zhongshuang 9’. Comparative anatomy studies on mature seed revealed that the seed coat pigments were mainly distributed in the palisade layer, which is considerably thinner in S. alba compared to B. napus. The area index of protein bodies in cotyledon cells was highest in S. alba, lowest in B. napus and intermediate in the intergeneric progenies. This study demonstrates that wide hybridization can enable exploitation of valuable trait diversity from S. alba for broadening the genetic basis for rapeseed breeding.

Analysis of flavonoids and hydroxycinnamic acid derivatives in rapeseeds (Brassica napus L. var. napus) by HPLC-PDA-ESI(-)-MSn/HRMS.

A comprehensive description of flavonoids and hydroxycinnamic acid derivatives in Brassica napus L. var. napus seeds is important to improve rapeseed quality. HPLC-PDA-ESI(-)-MS(n)/HRMS has been broadly applied to study phenolic compounds in plants. In the present study, crude phenolic compounds extracted from rapeseed were subjected to column chromatography, alkaline hydrolysis, and HPLC-PDA-ESI(-)-MS(n)/HRMS analysis. A total of 91 flavonoids and hydroxycinnamic acid derivatives were detected, including 39 kaempferol derivatives, 11 isorhamnetin derivatives, 5 quercetin derivatives, 6 flavanols and their oligomers, and 30 hydroxycinnamic acid derivatives. A total of 78 of these compounds were tentatively identified; of these, 55 were reported for the first time in B. napus L. var. napus and 24 were detected for the first time in the genus Brassica. This research enriches our knowledge of the phenolic composition of rapeseed and provides a reliable guide for the selection of rapeseed with valuable breeding potential.

FT‐IR and NMR study of seed coat dissected from different colored progenies of Brassica napus–Sinapis alba hybrids

BACKGROUND Yellow-seeded Brassica napus is preferred by breeders for economic reasons, and seed coat is the most obvious comparable character between yellow and black rapeseed. RESULTS Owing to an incomplete understanding of chemical components in yellow rapeseed coat, here we optimized a technique based on attenuated total reflectance-Fourier transform infrared (FT-IR) microspectroscopy to screen differences in chemical compositions of Brassica napus, Sinapis alba and four progeny lines with different yellow seed color derived from somatic hybrids between B. napus and S. alba. Nuclear magnetic resonance (NMR) analysis of seed coat from B. napus and S. alba represented two specific peaks representing components of lignin in B. napus, which were absent in the progenies. Also, the intensity of peaks related to fiber were stronger in B. napus than S. alba and yellow seed lines, indicating the differences of fiber and lignin contents in yellow-seeded lines and parent lines. Differences in the lipid, protein and carbohydrate features were identified between B. napus and S. alba, some of which were specifically inherited in progenies of the somatic hybrids. CONCLUSION FT-IR spectrometry and NMR provide a rapid, direct bioanalytical technique for exploring the changes in molecular chemistry within the canola seed coat and for selecting rapeseed materials with high quality, which can be further used for breeding or nutrition of human and animals.

Complete sequence of heterogenous-composition mitochondrial genome (Brassica napus) and its exogenous source

BackgroundUnlike maternal inheritance of mitochondria in sexual reproduction, somatic hybrids follow no obvious pattern. The introgressed segment orf138 from the mitochondrial genome of radish (Raphanus sativus) to its counterpart in rapeseed (Brassica napus) demonstrates that this inheritance mode derives from the cytoplasm of both parents. Sequencing of the complete mitochondrial genome of five species from Brassica family allowed the prediction of other extraneous sources of the cybrids from the radish parent, and the determination of their mitochondrial rearrangement.ResultsWe obtained the complete mitochondrial genome of Ogura-cms-cybrid (oguC) rapeseed. To date, this is the first time that a heterogeneously composed mitochondrial genome was sequenced. The 258,473 bp master circle constituted of 33 protein-coding genes, 3 rRNA sequences, and 23 tRNA sequences. This mitotype noticeably holds two copies of atp9 and is devoid of cox 2-2. Relative to nap mitochondrial genome, 40 point mutations were scattered in the 23 protein-coding genes. atp6 even has an abnormal start locus whereas tatC has an abnormal end locus. The rearrangement of the 22 syntenic regions that comprised 80.11% of the genome was influenced by short repeats. A pair of large repeats (9731 bp) was responsible for the multipartite structure. Nine unique regions were detected when compared with other published Brassica mitochondrial genome sequences. We also found six homologous chloroplast segments (Brassica napus).ConclusionsThe mitochondrial genome of oguC is quite divergent from nap and pol, which are more similar with each other. We analyzed the unique regions of every genome of the Brassica family, and found that very few segments were specific for these six mitotypes, especially cam, jun, and ole, which have no specific segments at all. Therefore, we conclude that the most specific regions of oguC possibly came from radish. Compared with the chloroplast genome, six identical regions were found in the seven mitochondrial genomes, which show that the Brassica family has a stable chloroplast-derived source.

Assessment of DNA methylation changes in tissue culture of Brassica napus

Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rapeseed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.

Morphological Structure and Transcriptome Comparison of the Cytoplasmic Male Sterility Line in Brassica napus (SaNa-1A) Derived from Somatic Hybridization and Its Maintainer Line SaNa-1B

SaNa-1A is a novel cytoplasmic male sterility (CMS) line in Brassica napus derived from progenies of somatic hybrids between B.napus and Sinapis alba, and SaNa-1B is the corresponding maintainer line. In this study, phenotypic differences of floral organs between CMS and the maintainer lines were observed. By microscope observation in different anther developmental stages of two lines, we found the anther development in SaNa-1A was abnormal since the tetrad stage, and microspore development was ceased during the uninucleate stage. Transcriptomic sequencing for floral buds of sterile and fertile plants were conducted to elucidate gene expression and regulation caused by the alien chromosome and cytoplasm from S. alba. Clean tags obtained were assembled into 195,568 unigenes, and 7811 unigenes distributed in the metabolic and protein synthesis pathways were identified with significant expression differences between two libraries. We also observed that genes participating in carbon metabolism, tricarboxylic acid cycle, oxidative phosphorylation, oxidation–reduction system, pentatricopeptide repeat, and anther development were downregulated in the sterile line. Some of them are candidates for researches on the sterility mechanism in the CMS material, fertility restoration, and improvement of economic traits in the maintainer line. Further research on the tags with expressional specificity in the fertile line would be helpful to explore desirable agronomic traits from wild species of rapeseed.