Cytogenetics and germplasm enrichment in Brassica allopolyploids in China

This paper reviews research advances in cytogenetics and germplasm innovation in Brassica allopolyploids, particularly oilseed rape(Brassica napus), in China. Three naturally evolved Brassica allotetraploid species are cytologically stable but tend to preferentially lose several chromosomes from one subgenome when induced by alien chromosome elimination. A-subgenome is extracted from B. napus, and the ancestral Brassica rapa was restituted after the total loss of C-subgenome chromosomes. Genome-wide genetic and epigenetic alterations were observed in both natural and synthetic Brassica allotetraploids. B. napus was subjected to extensive interspecific hybridization with landraces of B. rapa and Brassica juncea, which exhibit abundant phenotype variations, to widen the genetic diversity in breeding and select numerous elite germplasm resources and cultivars; these cultivars include the representative Zhongyou 821, which also parented numerous other varieties. Novel B. napus genotypes were obtained using Brassica trigenomic hybrids and allohexaploids(2 n=54, AABBCC) by combining subgenomes from extant allotetraploids and diploids as bridge. Alien additions, substitutions, and translocations of the B. napus genome were developed by intergeneric/intertribal sexual and somatic hybridizations with several crucifers. Furthermore, mitochondrial DNA recombination promoted the production of novel cytoplasmic male sterile lines.     

Identification of soft rot resistance loci in Brassica rapa with SNP markers

Soft rot caused by Pectobacterium carotovorum(Pc) is a devastating disease of Brassica rapa, causing substantial reductions in crop yield and quality. Identifying genes related to soft rot resistance is the key to solving this problem.To characterize soft rot resistance, we screened a soft rot-susceptible Chinese cabbage(A03), a resistant pakchoi(‘Huaguan’), and a resistant mutant(sr). An F2 population was generated by crossing susceptible Chinese cabbage A03and resistant pakchoi ‘Huaguan’ to id...     

Global Analysis of Cytosine Methylation and Proteome Under Cold Treatment in Brassica napus

Cytosine methylation/demethylation plays pivotal roles in regulating gene expression at a genome-wide level. However, limited reports are available to reveal correlating changes of cytosine methylation and proteomic expression in Brassica napus so far. Therefore, in the present study, global cytosine methylation and proteome were analysed in B. napus after cold treatment by methylation-sensitive amplified polymorphism （MSAP） and two-dimensional protein electrophoresis technology （2-DE）. The results showed that the lowered genome-wide DNA methylation status was revealed after cold treatment, and about 0.88% of discrepancy in DNA methylation was detected between the non-flowering and flowering plants after cold treatment. Moreover, the 52 significantly up-regulated proteins emerged in comparison with the 36 down-regulated proteins, as well as the 14 proteins exclusively detected in the flowering plants. Intriguingly the 8 specifically expressed proteins in the non-flowering plants disappeared in the flowering plants with cold treatment. Therefore, these present data proved that the correlating changes of cytosine methylation and proteomic expression were evidenced under cold treatment in B. napus.     

Identification of quantitative trait loci and candidate genes controlling seed pigments of rapeseed

Rapeseed (Brassica napus L.) is an important source of edible vegetable oil and feed protein; however, seed pigments affect the quality of rapeseed oil and the feed value of the residue from oil pressing. Here, we used a population of rapeseed recombinant inbred lines (RILs) derived from the black-seeded male parent cultivar Zhongyou 821 and the yellow-seeded female parent line GH06 to map candidate genes controlling seed pigments in embryos and the seed coat. We detected 94 quantitative trait loci (QTLs) for seed pigments (44 for embryos and 50 for seed coat), distributed over 15 of the 19 rapeseed chromosomes. These included 28 QTLs for anthocyanidin content, explaining 2.41–44.66% of phenotypic variation; 24 QTLs for flavonoid content, explaining 2.41–20.26% of phenotypic variation; 16 QTLs for total phenol content, accounting for 2.74–23.68% of phenotypic variation; and 26 QTLs for melanin content, accounting for 2.37–24.82% of phenotypic variation, indicating that these traits are under multigenic control. Consensus regions on chromosomes A06, A09 and C08 were associated with multiple seed pigment traits, including 15, 19 and 10 QTLs, respectively, most of which were major QTLs explaining >10% of the phenotypic variation. Based on the annotation of the B. napus “Darmor-bzh” reference genome, 67 candidate genes were predicted from these consensus QTLs regions, and 12 candidate genes were identified as potentially involved in pigment accumulation by RNA-seq and qRT-PCR analysis. These preliminary results provide insight into the genetic architecture of pigment biosynthesis and lay a foundation for exploring the molecular mechanisms underlying seed coat color in B. napus.     

芥菜型油菜优良性状导入甘蓝型油菜研究

分析了芥菜型油菜独具的优良性状,提出了通过种间杂交将芥菜型油菜优良基因导入甘蓝型油菜的基本程序,并对所获得的甘蓝型油菜黄籽超高油分、细胞质雄性不育恢复系、极早熟、高油酸和矮杆抗倒等新种质进行了介绍。     

甘蓝型油菜和诸葛菜属间杂种高世代材料形态学和细胞学分析(英文)

[目的]进一步揭示甘蓝型油菜和诸葛菜属间远缘杂种高世代(F8～F10)材料的遗传变异规律。[方法]以甘蓝型油菜和诸葛菜属间杂种高世代群体为材料,对甘蓝型油菜和诸葛菜远缘杂交后代表型偏白菜型油菜变异群体进行形态、细胞学方面的研究。[结果]形态学分析表明,杂种后代叶型、叶色、株型、早花性状偏向白菜型油菜,而二级分枝数、角果长度和千粒重与甘蓝型油菜Oro更为接近;细胞学研究表明,这些杂种高世代材料细胞中已不含诸葛菜染色体,多数植株都为少于38条染色体的亚倍体;与白菜型油菜杂种的减数分裂配对结果显示,这些亚倍体后代细胞中丢失的可能是来自C基因组的染色体,随着世代的增加,体细胞染色体数目都有向甘蓝型油菜2n=38升高和回归的趋势。[结论]该研究为揭示杂种后代亚倍体中丢失染色体的来源及形态学改变的原因奠定基础。     

甘蓝型油菜和诸葛菜属间杂种高世代材料形态学和细胞学分析

[目的]进一步揭示甘蓝型油菜和诸葛菜属间远缘杂种高世代(F8～F10)材料的遗传变异规律。[方法]以甘蓝型油菜和诸葛菜属间杂种高世代群体为材料,对甘蓝型油菜和诸葛菜远缘杂交后代表型偏白菜型油菜变异群体进行形态、细胞学方面的研究。[结果]形态学分析表明,杂种后代叶型、叶色、株型、早花性状偏向白菜型油菜,而二级分枝数、角果长度和千粒重与甘蓝型油菜Oro更为接近;细胞学研究表明,这些杂种高世代材料细胞中已不含诸葛菜染色体,多数植株都为少于38条染色体的亚倍体;与白菜型油菜杂种的减数分裂配对结果显示,这些亚倍体后代细胞中丢失的可能是来自C基因组的染色体,随着世代的增加,体细胞染色体数目都有向甘蓝型油菜2n=38升高和回归的趋势。[结论]该研究为揭示杂种后代亚倍体中丢失染色体的来源及形态学改变的原因奠定基础。     

叶面喷施含钾有机肥对油菜薹品质与产量的影响

为探究钾肥对不同类型油菜品种菜薹产量和品质的影响,以甘蓝型油菜品种‘徽油48’和白菜型油菜品种‘皖油13’为材料,采用叶面喷施不同用量含钾有机肥“富万钾”的方式,研究其对不同类型油菜品种主薹和侧薹的外观、口感及菜薹产量的影响。结果表明,甘蓝型油菜品种的菜薹产量和品质优于白菜型油菜品种,每公顷喷施750 g“富万钾”最有利于提高菜薹品质和产量。常规栽培措施下,‘徽油48’和‘皖油13’的菜薹总产量分别可达4 714.94和3 568.80 kg·hm^-2,‘徽油48’较‘皖油13’增产32.12%。与常规栽培措施相比,每公顷喷施750 g“富万钾”可显著提高菜薹总产量,其中‘徽油48’与‘皖油13’分别提高了14.92%和23.92%。同时,叶面喷施“富万钾”可显著提高菜薹的品质,其中‘徽油48’主薹和侧薹的SPAD值、茎叶比、茎的糖度、胡萝卜素含量以及侧薹叶的糖度,分别显著提高9.88%和15.66%、34.03%和69.54%、21.21%和10.70%、27.20%和19.04%、22.88%,纤维素含量降低9.04%;‘皖油13’主薹和侧薹的茎叶比、茎的糖度...     

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility （CMS） lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-IA, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-IB, and the abortive process of SaNa-IA were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-IA was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-IA aborted in the pollen mother cell （PMC） stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-IA is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.     

Association mapping of morphological and physiological-biochemical traits in Brassica rapa L. species

The search of associative relations between 258 SSR and S-SAP molecular markers and chromosomal loci determining morphological and physiological-biochemical traits of the accessions from the core collection of genetic resources of Brassica rapa L. has been carried out. The revealed SSR and S-SAP molecular markers can serve as an efficient genetic and breeding tool for the mass screening of collection accessions of this species, including prebreeding investigations, and are able to facilitate the transfer of revealed alleles into important breeding lines and varieties.     

23份油菜的抗氧化酶特性及过氧化物同工酶分析

对23份油菜叶片的抗氧化酶活性(SOD、CAT和POD)和丙二醛质量摩尔浓度(MDA)进行测定,用聚丙烯酰胺凝胶电泳法分析其过氧化物酶同工酶,明确酶谱的特征及分布,并用聚类分析的方法分析各品种间的亲缘关系。结果表明,在温和的环境中,白菜型油菜相比甘蓝型油菜有更高的抗氧化酶活性和更低的脂质过氧化水平,说明白菜型油菜的抗逆性强于甘蓝型油菜,特别是白菜型油菜的过氧化物酶活性平均值比甘蓝型油菜的10倍还高。POD同工酶分析共发现67条酶带,白菜型和甘蓝型油菜都存在其特征谱带,不同品种在带型分布及显色强度上存在差异。白菜型油菜Largo、Prisma-B和Prisma-Y的p4酶带和甘蓝型油菜类似,表明这3个材料和甘蓝型油菜有较近的亲缘关系。聚类分析表明,在类平均距离4.98处可将23份油菜分为2大类:白菜型油菜和甘蓝型油菜;在类平均距离1.32处,可进一步分为4类。     

Cloning and Molecular Identification of A Fatty Acid Desaturase 2 Gene in a and C Genome of Brassica Species

The fatty acid desaturase 2 (fad2) gene was proven to be a major locus for high oleic acid (C18:1). Brassica napus is an amphidiploid species originating from a spontaneous hybridization of Brassica rapa and Brassica oleracea. B. napus contains multiple copies in genome for most of the genes, including fad2 genes. The research cloned nine fad2 genes from 3 varieties of B. rapa and 3 varieties of B. oleracea, respectively. Alignment of the nine fad2 sequences from B. rapa and B. oleracea detect-ed 6 single nucleotide polymorphic sites, which resulted in 6 amino-acid substitutions. The nucleotide substitutions at position 743 bp in the fad2-A gene and position 947 bp in the fad2-C gene were used as 3’ end of al ele-specific primers. In use of the al-lele-specific primers to amplify fad2 gene, we could identify if the fad2 gene originated from A genome or C genome. Besides, the research found that fad2 genes in C genome are more conserved in evolutionary process than those in A genome. The fad2 expression data reported in this study revealed that fad2-A from B. rapa was not only expressed in siliques same as fad2-C from B. oleracea, but also expressed in a high level in stems. Not even the less, fad2 gene from B. napus was expressed higher in roots and flowers. Al these results provided evidences that fad2, though it was expressed differently in B. rapa and B. oleracea, but it was regulated by the same approach in B. napus.     

Effect of CaCl2 treatment on the changing of drought related physiological and biochemical indexes of Brassica napus

Some experiments revealed that the Ca²⁺ has a relationship with drought resistance. In this paper, some physiological and biochemical indices were studied in order to analyze the effect of Ca²⁺ treatment on the drought resistance of Brassica napus. The physiological and biochemical experiments revealed that the proline content and the soluble sugar contents in the Ca²⁺-treated B. napus were much higher than those of water-treated B. napus. However, the content of malonaldehyde in the Ca²⁺ treated B. napus was lower than that of control. The transpiration and stomatal conductance analysis show that the transpiration and stomatal conductance in the Ca²⁺-treated materials were lower than those of water-treated materials. Further research revealed that the stomata hatch of Ca²⁺-treated materials was markedly less than that of water-treated materials, which indicated that the closure of the stomata was the main reason for the decrease of transpiration and stomatal conductance. These authors contributed equally to this article. __________ Translated from Journal of Huazhong Agricultural University, 2007, 26(5): 607–611 [译自: 华中农业大学学报]     

5-Aminolevulinic acid alleviates herbicide-induced physiological and ultrastructural changes in Brassica napus

It is well known that application of 5-aminolevulinic acid(ALA) could promote the plant growth under abiotic stress in oilseed rape(Brassica napus L.). However, the specifics of its physiological and ultrastructural regulation under herbicide stress conditions are poorly understood. In the present study, alleviating role of ALA in B. napus was investigated under four levels of herbicide propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate(ZJ0273)(0, 100, 200 and 500 mg L–1) with or without 1 mg L–1 ALA treated for 48 or 72 h. Results showed that after 48 h of herbicide stress, the growth of rape seedlings was significantly inhibited with the successive increases of the ZJ0273 concentrations from 0 to 500 mg L–1, but this inhibition was obviously alleviated by exogenous application of ALA. However, when treatment time prolonged to 72 h, the recovery effects of ALA could not be evaluated due to the death of plants treated with the highest concentration of ZJ0273(500 mg L-1). Further, the root oxidizability and activities of antioxidant enzymes(superoxide dismutase, peroxidase and ascorbate peroxidase) were dramatically enhanced by the application of 1 mg L–1 ALA under herbicide stress. Therefore, plants treated with ALA dynamically modulated their antioxidant defenses to reduce reactive oxygen species(ROS) accumulation and malondialdehyde(MDA) content induced by herbicide stress. Additionally, exogenously applied ALA improved the ultrastructure's of chloroplast, mitochondria and nucleus, and induced the production of stress proteins. Our results suggest that ALA could be considered as a potential plant growth regulator for the improvement of herbicide tolerance through alleviation of the physiological and ultrastructural changes induced by the herbicide in crop production.     

芸薹属AC 基因组MTP1 基因的生物信息学分析

以拟南芥(Arabidopsis thaliana)AtMTP1基因序列为参考,利用白菜(Brassica rapa)、甘蓝(Brassica oleracea)、甘蓝型油菜(Brassica napus)全基因组数据对MTP1基因进行全基因组水平鉴定,通过Blastn比对和保守结构域分析,在芸薹属AC基因组中共鉴定到8个MTP1基因,其中白菜1个、甘蓝3个、甘蓝型油菜4个(A亚基因组3个、C亚基因组1个)。生物信息学分析结果表明,在蛋白质理化性质、二级结构、跨膜结构域、磷酸化位点等方面,3个物种中的MTP1基因间各有差异,并使用甘蓝型油菜转录组数据比较分析4个BnMTP1基因在不同组织部位的表达模式。     

Identification and genetic analysis of multiple P chromosomes of Agropyron cristatum in the background of common wheat

Agropyron cristatum, a wild relative of common wheat (Triticum aestivum L.), provides many desirable genetic resources for wheat improvement, such as tolerance to cold, drought, and disease. To transfer and utilize these desirable genes, in this study, two wheat-A. cristatum derivatives II-13 and II-23 were identified and analyzed. We found that the number of root tip cell chromosomes was 44 in both II-13 and II-23, but there were four and six P genome chromosomes in II-13 and II-23, respectively, based on genomic in situ hybridization (GISH). The chromosome configurations of II-13 and II-23 were both 2n=22II by the meiotic analysis of pollen mother cells (PMCs) at metaphase I, indicating that there were two and three pairs of P chromosomes in II-13 and II-23, respectively. Notably, wheat chromosome 7D was absent in derivative line II-13 while II-23 lacked chromosomes 4B and 7A based on SSR analysis combining fluorescence in situ hybridization (FISH) analysis with pAs1 and pSc119.2 as probes. Chromosomes 2P and 7P were detected in both II-13 and II-23. Another pair of P genome chromosomes in II-23 was determined to be 4P based on expressed-sequences tags-sequence tagged sites (EST-STS) markers specific to A. cristatum and FISH with probes pAcTRT1 and pAcpCR2. Overall, these results suggest that II-13 was a 7P (7D) substitution line with one pair of additional 2P chromosomes and II-23 was a multiple 4P (4B), 7P (7A) substitution line with one pair of additional 2P chromosomes. Moreover, we obtained six alien disomic addition lines and five alien disomic substitution lines by backcrossing. These new materials will allow desirable genes from A. cristatum to be used in common wheat.     

Analysis on Combining Ability for Characters of Male Sterile Lines in Rapeseed （ Brassica napus L.）

The male sterile line is very important in the hybrid breeding program of rapeseed. This study was conducted to evaluate the combining ability of many characters of male sterile lines in Brassica napus...