Improved genome annotation of Brassica oleracea highlights the importance of alternative splicing

Brassica oleracea has been developed into many important crops,including cabbage,kale,cauliflower,broccoli and so on.The genome and gene annotation of cabbage(cultivar JZS),a representative morphotype of B.oleracea,has been widely used as a common reference in biological research.Although its genome assembly has been updated twice,the current gene annotation still lacks information on untranslated regions(UTRs)and alternative splicing(AS).Here,we constructed a high-quality gene annotation(JZSv3)using a full-length transcriptome acquired by nanopore sequencing,yielding a total of 59452 genes and 75684 transcripts.Additionally,we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference,and found that 3843 out of 11908 differentially expressed genes(DEGs)underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress.Meanwhile,we also identified many AS genes,including BolLHCB5 and BolHSP70,that displayed distinct expression patterns within variant transcripts of the same gene,highlighting the importance of JZSv3 as a pivotal reference for AS analysis.Overall,JZSv3 provides a valuable resource for exploring gene function,especially for obtaining a deeper understanding of AS regulation mechanisms.

花椰菜(Brassica oleracea var.botrytis)抗黑腐病差异表达cDNA片段的克隆及功能的初步研究

采用花椰菜抗黑腐病近等基因系C731（感病系）和C712（抗病系）作为材料，利用cDNA—AFLP技术，研究了花椰菜黑腐病抗性在黑腐病菌侵染和非侵染条件下基因表达的情况．获得了两个阳性克隆M2和M6．Northern杂交和点杂交进一步证明M2是组成型表达的cDNA片段，只是在病菌侵染与非侵染条件下表达丰度不同．而M6是差异表达的cDNA片段．Southern杂交表明M6 cDNA片段在花椰菜基因组中是单拷贝序列．随后的序列分析发现M6cDNA片段与拟南芥1号染色体的BACF19P19中66665～66813bp有84％同源性，该片段编码拟南芥的2A6蛋白的部分序列．推测的氨基酸序列与拟南芥的2A6蛋白部分序列有91％同源性．用H2O2作为外源分子胁迫处理的初步功能分析发现：M6cDNA片段受H2O2诱导，在诱导早期16～24h高度表达，其在叶片中的积累呈逐渐上升趋势．根据序列分析和初步的功能分析的结果推测：M6cDNA片段可能就是编码花椰菜中类2A6蛋白的部分基因片段．是参与花椰菜抗病反应信号途径的相关调控基因片段．

花椰菜(Brassica oleracea var.botrytis)黑腐病抗性基因同源序列分离及克隆的研究

通过从 NBS 保守序列设计简并引物 PCR 的方法,以花椰菜(Brassica oleracea vat.botrytis)抗、感黑腐病的近等基因系为材料,分离得到 NBS-LRR 型抗性基因同源序列,并获得1个克隆,命名为 RGA330-7.Southern 杂交表明,该片段在近等基因系中存在明显的多态性,且该片段在抗黑腐病基因位点至少存在3个以上类似 RGA330-7的同源拷贝.序列分析结果认为该克隆与 NBS-LRR 型抗性基因的部分 CDSs 有很高的同源性,说明该片段属于 NBS-LRR 型.系统进化分析该序列与甘蓝型油菜的2个抗病同源序列归为一类,很可能这3个不同来源的抗性基因同源序列同属于一种抗性基因家族.因此推测该序列与花椰菜抗黑腐病基因紧密相关,为进一步克隆花椰菜抗黑腐病基因提供了可靠的候选基因,对分子标记辅助抗性育种具有重要意义。

与花椰菜(Brassica oleracea ssp.botrytis)抗黑腐病基因连锁的RAPD标记

利用 RAPD技术 ,在花椰菜 ( Brassica oleracea ssp.botrytis)抗感黑腐病的近等位基因系之间进行 1 0碱基随机引物的筛选 .34 5条 RAPD引物扩增的产物表明 ,有 7条引物在近等基因系中呈多态性 .进一步的重复实验表明 ,只有引物 OP2 2 4能够产生稳定的多态性 .为了确定扩增产物 OP2 2 4/1 6 0 0与抗黑腐病基因的连锁程度 ,利用 F2分离群体进行检测 ,结果表明 :花椰菜种质 C71 2抗黑腐病生理种 BJ的抗性由一对显性基因控制 .RAPD标记OP2 2 4/1 6 0 0与抗病基因连锁 ,其遗传距离为 ( 4 .5± 1 .37) c

根癌农杆菌介导白细胞介素-4基因转化甘蓝(Brassica oleracea L.)研究

利用根癌农杆菌介导法，以白细胞介素-4（interleukin-4，IL-4）基因转化中甘11号甘蓝。本研究对可能影响il-4转化率的外植体类型、外植体的预培养时间、农杆菌的侵染时间以及外植体与农杆菌的共培养时间进行了优化。试验结果表明：il-4基因对带1-2mm子叶柄的子叶的转化率显著高于下胚轴；带柄子叶在预培养1d、侵染3min、共培养1d时，转化效果最好，转化率达23．3%;下胚轴在预培养1-3d，侵染3～5min，共培养1-2d时，转化效果较好，转化率最高可达13．3％。经PCR检测及PCR-Southem杂交，初步证明目的基因il-4已经转入甘蓝的再生植株。转il-4基因甘蓝的PCR阳性再生植株已经开花并产生了后代。

Molecular Cloning and Sequence Analysis ofBoPGIP2 Gene from Brassica oleracea L. var. alboglabra

PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences.

花椰菜(Brassica oleracea var.botrytis L.)凝集素的细胞凝集和糖抑制作用的研究

花椰莱(Brassica oleracea var. botrytis L.)凝集素能凝集兔、大鼠和小鼠的红细胞,其中对兔的红细胞的凝集活性最高,最低凝集浓度为0.68μg/ml。但不凝集我们所测试的其它16种红细胞。浓度为0.1mg/ml时,花椰菜凝集素也能凝集小鼠艾氏腹水瘤细胞,S_(180)肉瘤细胞,大鼠W_(256)肿瘤细胞、人的MGC_(80-3)胃癌细胞、小鼠和大鼠的脾脏淋巴细胞、骨髓细胞以及牛精子细胞,而不凝集人的Hela细胞。该凝集素对免的红细胞的凝集活性可被L—鼠李糖和D—树胶醛糖所抑制,最低抑制浓度分别为33.3m mol/L和16.7m mol/L。

BnaWRKY75 positively regulates the resistance against Sclerotinia sclerotiorum in ornamental Brassica napus

With the development of tourism at home and abroad,Rapeseed(Brassica napus)has become an important ornamental plant.However,its ornamental value at the inflorescence stage is greatly reduced by Sclerotinia sclerotiorum.Identification of important genes in the defense responses is critical for molecular breeding,which is an important strategy for controlling the disease.In this study,we isolated a B.napus WRKY transcription factor gene,BnaWRKY75.BnaWRKY75 was found to encode a nucleus-localized protein and exhibited relatively high expression in the stems.Arabidopsis thaliana transgenic plants expressing BnaWRKY75 showed enhanced resistance to S.sclerotiorum,and both ProBnaWRKY75:GUS and gene expression analyses showed that BnaWRKY75 was highly responsive to S.sclerotiorum infection,indicating the involvement of BnaWRKY75 in response to this infection.Furthermore,overexpression(OE)of BnaWRKY75 in B.napus significantly enhanced the resistance to S.sclerotiorum,whereas the resistance was reduced in RNAi transgenic B.napus plants.Moreover,the BnaWRKY75-OE B.napus plants exhibited constitutive activation of salicylic acid-,jasmonic acid-,and ethylene-mediated defense responses and the inhibition of both H_(2)O_(2)and O_(2)·^(-)accumulation in response to this pathogen.By contrast,BnaWRKY75-RNAi plants showed a reverse pattern,suggesting that BnaWRKY75 is involved in hormonal signaling pathways and in the control of reactive oxygen species accumulation.In conclusion,these data indicate that BnaWRKY75,a regulator of multiple defense responses,positively regulates resistance against S.sclerotiorum,which may guide the improvement of resistance in rapeseed.

Transformation of insect-resistant gene into cauliflower (Brassica oleracea L.var. botrytis)

Cowpea Trypsin Inhibitor (CpTI) gene was transferred into the cotyle dons and hypocotyls of cauliflower by Agrobacterium-mediated transformation met hod. The best selective concentration of kanamycin (kan) was 15 mg L-1. The con centration of carbencillin (carb) was 500 mg L-1. 14 transgenic cauliflower pla nts were obtained. The putative transformants were assayed by PCR and Southern b lotting analysis. The results indicated that CpTI gene was transferred into caul iflower successfully.

Study on the Relationship Between the Ploidy Level of Microspore-Derived Plants and the Number of Chloroplast in Stomatal Guard Cells in Brassica oleracea

The relationship between the ploidy level of microspore-derived plants and chloroplast number in stomatal guard cells was studied in cabbage, broccoli, and Chinese kale. In the experiment, distribution statistics analysis and t-test were used to perform statistical analysis on chloroplast number of different ploidy level in those stomatal guard cells mentioned above, and morphology identifying and chromosome counting were used to test accuracy of counting chloroplast number in stomatal guard cells. The chloroplast average number in stomatal guard cells was very similar among the different leaf positions on the same plant and among significantly among the different ploidy the different locations in the same stoma in the same variety. All the leaf, while the chloroplast number varied distributions of the chloroplast number in different ploidy stoma were normal distribution fitted. A correlation has been established between ploidy and chloroplast number in the stomatal guard cells. In every single stoma of microspore-derived plants, the chloroplast number for a haploid should not be more than 10, diploids 11 to 15, and polyploids more than 15. The accuracy of this method for identification of different ploidy plants was 93.93%. Furthermore, the accuracy of this method was reliable and did not vary with the plants growth conditions. Therefore, the chromosome ploidy of plants derived from microspore culture in cabbage, broccoli, and Chinese kale can be identified by simply counting the chloroplast number in stomatal guard cells.

Plant Male Sterility Induced by Anti-Gene CYP86MF in Brassica oleracea var. italica

An anti-gene CYP86MF was introduced into hypocotyls of broccoli （Brassica oleracea L.var. italica Plenck） with Agrobacterium tumefaciens, and the transgenic plants were obtained by kanamycin selection. The results of PCR, Southern blot and Northern blot indicated that the anti-CYP86MF has been integrated into chromosome of the transgenic plant. And also, plants with hypogenetic stamina or ungerminated pollen were observed. The transgenic male sterility plant could fructify via artificial pollination with normal pollen. Thus it was proved that the pistil of male sterility plant was normally developed, and the sterility originated from anti-CYP86MF.

Genome-wide identification and analysis of cytokinin dehydrogenase/oxidase(CKX) family genes in Brassica oleracea L.reveals their involvement in response to Plasmodiophora brassicae infections

Cytokinins are a class of phytohormones that promote cell division and differentiation and are thought to affect plant immunity to multiple pathogens.However,a comprehensive analysis of cytokinin dehydrogenase/oxidase(CKX)family genes in cabbage has not been reported.In this study,a total of 36 CKX genes were identified using a genome-wide search method.Phylogenetic analysis classified these genes into three groups.They were distributed unevenly across nine chromosomes in B.oleracea,and 15 of them did not contain any introns.The results of colinearity analysis showed that 36 CKX gene in Arabidopsis was present in several copies in the Brassica oleracea genome.An analysis of cisacting elements indicated that all genes possessed at least one stress or hormone responsive cis-acting element.A heatmap of CKX gene expression showed the patterns of expression of these genes in various tissues and organs.Three genes(Bol028363,Bol031036 and Bol018140)were relatively highly expressed in all of the investigated tissues under normal conditions,showing the expression profile of housekeeping genes.Generally,the expression patterns of CKX genes in Jingfeng 1 and Xiangan 336 were quite different under the same treatment.Notably,three genes(Bol020547,Bol028392 and Bol045724)were significantly down-regulated and up-regulated in the susceptible and resistant material,respectively,after inoculation,which may indicate their crucial roles in resistance to clubroot disease.The results provide insights for better understanding the roles of CKX genes in the B.oleracea–P.brassicae interaction.

大白菜(Brassica pekinensis)与甘蓝(B.oleracea)的种间试管受精

通过大白菜胚珠与甘蓝花粉的试管授粉和受精得到了杂种种子。杂交一代的植株形态和花粉发育情况及根尖细胞染色体数均表明为种间杂种。杂种植株具有父母本的特征,某些性状介于双亲之间;花粉粒发育不正常;根尖细胞染色体数为2n=19。本研究为其他十字花科植物的远缘杂交中克服不亲和性提供了依据。

Nuclear and chloroplast genome diversity revealed by low-coverage whole-genome shotgun sequence in 44 Brassica oleracea breeding lines

Whole-genome shogun sequence(WGS)data generated by next-generation sequencing(NGS)platforms are a valuable resource for crop improvement.We produced 5–6×WGS coverage of 44 Brassica oleracea breeding lines representing seven subspecies/morphotypes:cabbage,broccoli,cauliflower,kailan,kale,Brussels sprout,and kohlrabi to systematically evaluate the nuclear and chloroplast(Cp)diversity in the 44 B.oleracea breeding lines.We then exploited the impact of low-coverage NGS by evaluating nuclear genome diversity and assembly,annotation of complete chloroplast(Cp)genomes and 45 S nuclear ribosomal DNA(45S nrDNA)sequences,and copy number variation for major repeats.Nuclear genome diversity analysis has revealed a total of 496463 SNPs and 37493 indels in the nuclear genome across the 44 accessions.Interestingly,some SNPs showed subspecies enrichment at certain chromosomal regions.The assembly of complete Cp genomes contained 153361–153372 bp with 37 variants including SNPs and indels.The 45S nrDNA transcription unit was 5802 bp long with a total of 31 SNPs from the 44 lines.The phylogenetic tree inferred from the nuclear and Cp genomes coincided and clustered broccoli,cauliflower,and kailan in one group and cabbage,Brussels sprout,kale,and kohlrabi in another group.The morphotypes diverged during the last 0.17 million years.The Cp genome diversity reflected the unique cytoplasm of each subspecies,and revealed that the cytoplasm of many breeding lines was replaced and intermingled via inter-subspecies crosses during the breeding process instead.The polymorphic Cp markers provide a classification system for the cytoplasm types in B.oleracea.Furthermore,copy numbers of major transposable elements(TEs)showed high diversity among the 44 accessions,indicating that many TEs have become active recently.Overall,we demonstrated a comprehensive utilization of low-coverage NGS data and might shed light on the genetic diversity and evolution of diverse B.oleracea morphotypes.

BnaC03.BIN2 regulates plant height by affecting the main inflorescence length and first effective branch height in Brassica napus L.

Rapeseed(Brassica napus L.)is one of the main oil crops in the world,and increasing its yield is of great significance for ensuring the safety of edible oil.Presently,improving rapeseed plant architecture is an effective way to increase rapeseed yield with higher planting density.However,the regulatory mechanism of rapeseed plant architecture is poorly understood.In this study,a dwarf rapeseed mutant dwarf08(df08)is obtained by ethyl methane sulfonate(EMS)-mutagenesis.The decrease in plant height of df08 is mainly caused by the reduction in main inflorescence length and first effective branch height and controlled by a single semi-dominant gene.The hybrid plants(F1)show a semi-dwarf phenotype.Through map-based cloning and transgenic assay,we confirm that the nonsynonymous single nucleotide variant(SNV)(C to T)in BnaC03.BIN2,which is homologous with Arabidopsis(Arabidopsis thaliana)BIN2,is responsible for the dwarfism of df08.BnaC03.BIN2 interacts with BnaBZR1/BES1 and involves in brassinosteroids(BRs)signal transduction.Proline to Leucine substitution in 284(P284L)enhances the protein stability of BnaC03.bin2-D,disrupts BRs signal transduction and affects the expression of genes regulating cell division,leading to dwarfism of df08.This study provides a new insight for the mechanism of rapeseed plant height regulation and creates an elite germplasm that can be used for genetic improvement of rapeseed architecture.

Molecular mechanism of flowering time regulation in Brassica rapa:similarities and differences with Arabidopsis

Properly regulated flowering time is pivotal for successful plant reproduction.The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions.Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops.Flowering time is one of the most important agricultural traits of B.rapa crops because of its influence on yield and quality.The transition to flowering in B.rapa is regulated by several environmental and developmental cues,which are perceived by several signaling pathways,including the vernalization pathway,the autonomous pathway,the circadian clock,the thermosensory pathway,and gibberellin(GA)signaling.These signals are integrated to control the expression of floral integrators BrFTs and BrSOC1s to regulate flowering.In this review,we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B.rapa and compare this to what is known in Arabidopsis.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax(Linum usitatissimum L.)is a versatile crop and its seeds are a major source of unsaturated fatty acids.Stearoyl-acyl carrier protein desaturase(SAD)is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.However,the function of SAD orthologs from L.usitatissimum has not been assessed.Here,we found that two LuSAD genes,LuSAD1 and LuSAD2,are present in the genome of L.usitatissimum cultivar‘Longya 10’.Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.Interestingly,ectopic expression of LuSAD2 in A.thaliana caused altered plant architecture.Similarly,the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.Furthermore,we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels,as well as reducing membrane damage.These findings not only broaden our knowledge of the LuSAD functions in plants,but also offer promising targets for improving the quantity and quality of oil,and the abiotic stress tolerance of oil-producing crops,through molecular manipulation.

Utilizing resequencing big data to facilitate Brassica vegetable breeding:tracing introgression pedigree and developing highly specific markers for clubroot resistance

Clubroot caused by Plasmodiophora brassicae is a devastating disease of Cruciferous crops.Developing cultivars with clubroot resistance(CR)is the most effective control measure.For the two major Brassica vegetable species B.rapa and B.oleracea,several commercial cultivars with unclear CR pedigrees have been intensively used as CR donors in breeding.However,the continuous occurrence of CR-breaking makes the CR pedigree underlying these cultivars one of the breeders'most urgent concerns.The complex intraspecific diversity of these two major Brassica vegetables has also limited the applicability of CR markers in different breeding programs.Here we first traced the pedigree underlying two kinds of CR that have been widely applied in breeding by linkage and introgression analyses based on public resequencing data.In B.rapa,a major locus CRzi8 underlying the CR of the commercial CR donor‘DegaoCR117’was identified.CRzi8 was further shown to have been introgressed from turnip(B.rapa ssp.rapifera)and that it carried a potential functional allele of Crr1a.The turnip introgression carried CRb^(c),sharing the same coding sequence with the CRb that was also identified from chromosome C07 of B.oleracea CR cultivars with different morphotypes.Within natural populations,variation analysis of linkage intervals of CRzi8,PbBa8.1,CRb,and CRb^(c)yielded easily resolved InDel markers(>20 bp)for these fundamental CR genes.The specificity of these markers was tested in diverse cultivars panels,and each exhibited high reliability in breeding.Our research demonstrates the value of the practice of applying resequencing big data to solve urgent concerns in breeding programs.

Turnip mosaic virus pathogenesis and host resistance mechanisms in Brassica

Turnip mosaic virus(TuMV)is a devastating potyvirus pathogen that infects a wide variety of both cultivated and wild Brassicaceae plants.We urgently need more information and understanding of TuMV pathogenesis and the host responses involved in disease development in cruciferous crops.TuMV displays great versatility in viral pathogenesis,especially in its replication and intercellular movement.Moreover,in the coevolutionary arms races between TuMV and its hosts,the virus has evolved to co-opt host factors to facilitate its infection and counter host defense responses.This review mainly focuses on recent advances in understanding the viral factors that contribute to the TuMV infection cycle and the host resistance mechanism in Brassica.Finally,we propose some future research directions on TuMV pathogenesis and control strategies to design durable TuMV-resistant Brassica crops.

Assignment of unanchored scaffolds in genome of Brassica napus by RNA-seq analysis in a complete set of Brassica rapa-Brassica oleracea monosomic addition lines

The economically valuable oil crop Brassica napus(AACC,2 n=38),which arose from interspecific hybridization between the diploid ancestors Brassica rapa(AA,2 n=20) and Brassica oleracea(CC,2 n=18),has a complex genome.More than 10% of the assembled sequences,most of which belong to the C subgenome,have not been anchored to the corresponding chromosome.Previously,a complete set of monosomic alien addition lines(MAALs,C1–C9) with each of the nine C-subgenome chromosomes added to the extracted A subgenome was obtained from the allotetraploid B.napus donor Oro,after the ancestral B.rapa(RBR Oro) genome was restored.These MAALs effectively reduced the complexity of the B.napus genome.Here,we determined the expression values of genes on unanchored scaffolds in the MAALs and RBR Oro.Then,multiple comparisons of these gene expression values were used to determine the affiliations of the nonanchored scaffolds on which the genes were located.In total,54.68%(44.11 Mb) of the 80.67 Mb of non-anchored scaffolds belonging to the C subgenome were assigned to corresponding C chromosomes.This work highlights the potential value of these MAALs in improving the genome quality of B.napus.