通过白菜型油菜和埃塞俄比亚芥的Ar和Cc基因组导入创造甘蓝型油菜新材料

油菜是目前我国主要种植的油料作物之一,但现有的种质资源限制了产量的进一步提高。本研究采取了一种新的育种方式来增加甘蓝型油菜的种质资源,即通过远缘杂交结合分子标记辅助选择的方式将白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc对现有的甘蓝型油菜品种的基因组(AnAnCnCn)进行部分替换。通过对五倍体杂交后代(ArAnBcCcCn)进行染色体选择,找到了染色体数目为38的材料。为了和现有的甘蓝型油菜进行区分,得到的新材料被认定为甘蓝型油菜新材料。实验结果表明,得到的部分甘蓝型油菜新材料具有基本正常的减数分裂过程、正常的花粉萌发以及胚囊发育过程,这说明甘蓝型油菜新材料达到了遗传平衡。分子标记分析表明:甘蓝型油菜新材料的约50%的基因组被白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc替换,并且这些甘蓝型油菜新材料之间具有丰富的遗传多样性。因此,白菜型油菜的Ar基因组和埃塞俄比亚芥的Cc基因组导入对于丰富现有的甘蓝型油菜种质资源具有明显的效果。     

Structural features and expression analysis of a linear mitochondrial plasmid in rapeseed (Brassica napus L.)

A linear plasmid molecule about 11 kb in length is present in the mitochondria of some varieties of rapeseed (Brassica napus L.). This plasmid can be inherited from the male parent, through the pollen, as well as by the usual maternal route, although the main mitochondrial genome is maternally inherited in rapeseed. We determined the complete nucleotide sequence of this plasmid DNA and clarified its genetic organization. The length of the linear plasmid is 11,640 bp. At the termini of the plasmid molecule are inverted repeats of 327 bp. The GC content of the plasmid DNA is 30.9%; thus, the plasmid is quite AT-rich compared to the main mitochondrial genome in higher plants. The plasmid has six ORFs, two of which encode a phage-type DNA polymerase and a phage-type RNA polymerase, respectively. RT-PCR analyses revealed that all six ORFs are transcribed, and all four ORFs on the minus strand are probably cotranscribed from a single promoter located in the terminal inverted repeat. We also show here that at least three of the six ORFs are translated into proteins in rapeseed mitochondria, and expressed at relatively high levels in flowers, as shown by Western analysis. These results suggest that this linear DNA molecule is able to replicate as an autonomous replicon and to express the genes it carries in rapeseed mitochondria.     

Identification of the A and C genomes of amphidiploid Brassica napus (oilseed rape).

A genetic linkage map consisting of 399 RFLP-defined loci was generated from a cross between resynthesized Brassica napus (an interspecific B. rapa x B. oleracea hybrid) and "natural" oilseed rape. The majority of loci exhibited disomic inheritance of parental alleles demonstrating that B. rapa chromosomes were each pairing exclusively with recognisable A-genome homologues in B. napus and that B. oleracea chromosomes were pairing similarly with C-genome homologues. This behaviour identified the 10 A genome and 9 C genome linkage groups of B. napus and demonstrated that the nuclear genomes of B. napus, B. rapa, and B. oleracea have remained essentially unaltered since the formation of the amphidiploid species, B. napus. A range of unusual marker patterns, which could be explained by aneuploidy and nonreciprocal translocations, were observed in the mapping population. These chromosome abnormalities were probably caused by associations between homoeologous chromosomes at meiosis in the resynthesized parent and the F1 plant leading to nondisjunction and homoeologous recombination.     

Genetic diversity of allozymes in turnip (Brassica rapa L. var. rapa) from the Nordic area

Genetic diversity and relationships based on isozymes were studied in 31 accessions of turnip (Brassica rapa L. var. rapa). The material included varieties, elite stocks, landraces and older turnip of slash-and-burn type from the Nordic area. A total of 9 isozyme loci and 26 alleles were studied. The isozyme systems were ACO, DIA, GPI, GOT, PGM, PGD and SKD. The level of heterozygosity was reduced in the landraces, but it was high for the variety group 'Ostersundom'. Turnip has a higher genetic variation than other crops within B. rapa and than in other species with the same breeding system. The genetic diversity showed that 18.7% of the genetic variation was within the accessions, and the total H tau value was 0.358. Gpi-I and Pgd-I showed the lowest variation compared with the other loci. The cluster analysis revealed five clusters, with one main cluster including 25 of the 31 accessions. The dendrogram indicated that the variety group 'Ostersundom' clustered together whereas the variety group 'Bortfelder' was associated with country of origin. The landraces were spread in different clusters. The 'slash-and-burn' type of turnip belonged to two groups.     

Mitigation using a tandem construct containing a selectively unfit gene precludes establishment of Brassica napus transgenes in hybrids and backcrosses with weedy Brassica rapa

Transgenic oilseed rape ( Brassica napus ) plants can interbreed with nearby weedy Brassica rapa , potentially enhancing the weediness and/or invasiveness of subsequent hybrid offspring. We have previously demonstrated that transgenic mitigation effectively reduces the fitness of the transgenic dwarf and herbicide-resistant B. napus volunteers. We now report the efficacy of such a tandem construct, including a primary herbicide-resistant gene and a dwarfing mitigator gene, to preclude the risks of gene establishment in the related weed B. rapa and its backcross progeny. The transgenically mitigated and non-transgenic B. rapa  ×  B. napus interspecific hybrids and the backcrosses (BC₁) with B. rapa were grown alone and in competition with B. rapa weed. The reproductive fitness of hybrid offspring progressively decreased with increased B. rapa genes in the offspring, illustrating the efficacy of the concept. The fitness of F₂ interspecific non-transgenic hybrids was between 50% and 80% of the competing weedy B. rapa , whereas the fitness of the comparable T₂ interspecific transgenic hybrids was never more than 2%. The reproductive fitness of the transgenic T₂ BC₁ mixed with B. rapa was further severely suppressed to 0.9% of that of the competing weed due to dwarfism. Clearly, the mitigation technology works efficiently in a rapeseed crop–weed system under biocontainment-controlled environments, but field studies should further validate its utility for minimizing the risks of gene flow.     

Conserved patterns of chromosome pairing and recombination in Brassica napus crosses.

The patterns of chromosome pairing and recombination in two contrasting Brassica napus F1 hybrids were deduced. One hybrid was from a winter oilseed rape (WOSR) x spring oilseed rape cross, the other from a resynthesized B. napus x WOSR cross. Segregation at 211 equivalent loci assayed in the population derived from each hybrid produced two collinear genetic maps. Alignment of the maps indicated that B. napus chromosomes behaved reproducibly as 19 homologous pairs and that the 19 distinct chromosomes of B. napus each recombined with unique chromosomes from the interspecific hybrid between Brassica rapa and Brassica oleracea. This result indicated that the genomes of the diploid progenitors of amphidiploid B. napus have remained essentially unaltered since the formation of the species and that the progenitor genomes were similar to those of modern-day B. rapa and B. oleracea. The frequency and distribution of crossovers were almost indistinguishable in the two populations, suggesting that the recombination machinery of B. napus could cope easily with different degrees of genetic divergence between homologous chromosomes. Efficient recombination in wide crosses will facilitate the introgression of novel alleles into oilseed rape from B. rapa and B. oleracea (via resynthesized B. napus) and reduce linkage drag.     

The identification of quantitative trait loci that control the paternal inheritance of a mitochondrial plasmid in rapeseed (Brassica napus L.)

Some varieties of Brassica napus (rapeseed) and B. rapa contain a liner mitochondrial plasmid that is unique in that it can be inherited from the male parent through the pollen. We found that two rapeseed cultivars, Norin 16 and Westar, showed different rates of plasmid inheritance from the paternal parent (78.8% and 27.5%, respectively). To identify nuclear genes controlling the inheritance of the plasmid, we carried out quantitative trait locus (QTL) analyses using F(2) populations derived from a cross between these two cultivars. The F1 plants transmitted the plasmid from the paternal plant at a frequency of approximately 60%; the transmission rates of the F2 lines varied greatly, from 0 to 100%, with an average of 68.2%. A genetic map was constructed based on the segregation of 175 loci in the 102 F2 plants. A total of 22 linkage groups were obtained, all of which could be assigned to the 19 rapeseed chromosomes. The total map length was 1374.7 cM, with an average distance of 7.9 cM between the markers. We found that three quantitative trait loci for plasmid paternal transfer, qPpt1, qPpt2 and qPpt3, located on chromosomes A5, C2 and C9, respectively, were significantly linked to the transmission frequency, whose the logarithm of odds (LOD) score were 4.97, 3.49 and 3.57, respectively. Their explained phenotypic variances were 25.0%, 22.2% and 37.1%, respectively. These results suggest that the paternal inheritance of the mitochondrial plasmid is controlled by a relatively small number of nuclear genes.     

RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值

采用RFLP和AFLP标记对来自中国和欧美的7份甘蓝型油菜和22份白菜型油菜进行了遗传多样性分析。在这29份材料中,166个酶-探针组合和2对AFLP引物共检测到1477个RFLP标记和183个AFLP标记。RFLP数据显示以拟南芥EST克隆作探针比用油菜基因组克隆做探针能检测到更多的多态性位点,且采用EcoR Ⅰ或BamH Ⅰ酶切比HindⅢ酶切多态性好,白菜型油菜和甘蓝型油菜中基因的拷贝数平均都为3个左右。UPGMA聚类分析表明中国白菜型油菜的遗传多样性比甘蓝型油菜和欧美白菜型油菜丰富,欧美甘蓝型油菜与欧美白菜型油菜聚为一类,而与中国甘蓝型油菜差异更大。中国白菜型油菜丰富的遗传多样性为中国甘蓝型油菜的改良提供了宝贵的资源,揭示了利用白菜型油菜A基因组和甘蓝型油菜A基因组间亚基因组杂种优势的可能性。     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

油菜3个栽培种发芽期耐盐性评价

对油菜种质资源进行耐盐性评价是培育耐盐油菜品种的基础性工作。本研究在室内分别采用3种盐(NaCl)浓度(86 mmol/L、170 mmol/L、256 mmol/L)对芥菜型油菜(Brassica juncea)、白菜型油菜(B.rapa)和甘蓝型油菜(B.napus)等3个栽培种的203份种质资源进行盐胁迫,结果表明盐胁迫对各栽培种的发芽率有不同程度抑制作用,白菜型油菜表现最好,甘蓝型油菜次之,芥菜型油菜最差。基于发芽率筛选出14份优异种质。进一步利用梯度浓度盐(NaCl)溶液对其进行胁迫,发现各栽培种发芽率均随着盐浓度的升高呈下降趋势,符合Boltzmann曲线,但白菜型油菜下降最为缓慢。通过比较盐胁迫下各栽培种6个性状值(胚轴长、根长、总鲜重、地上部分鲜重、总叶绿素含量和电导率)后发现,白菜型油菜发芽期耐盐性优于甘蓝型油菜和芥菜型油菜。对170 mmol/L NaCl胁迫下14份种质各性状值的主成分分析表明,白菜型油菜Br2、甘蓝型油菜Bn1和Bn3为最耐盐种质,可用于耐盐油菜品种的培育。     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Identification of repetitive, genome-specific probes in crucifer oilseed species.

Direct amplification of minisatellite DNA by PCR (DAMD PCR) was used to amplify and subsequently clone several fragments of DNA from crucifer species. The PCR-derived fragments of DNA were generated using known minisatellite core sequences as PCR primers. Southern hybridization of these putative minisatellite DNA fragments revealed that many were genome-specific; they hybridized with high affinity only to the genomic DNA of the species from which they were cloned. The DNA fragments were believed to be dispersed in the genome, based on smear-like hybridization signals on EcoRI-, BamHI-, and HindIII-digested genomic DNA. Genome-specific probes were specifically isolated from Brassica rapa (A genome), Brassica nigra (B genome), and Sinapis alba in addition to several other crucifer species. The sequence of a B. rapa specific probe (pBr17.1.3A) contained a minisatellite region that could be divided into three tandem repeats; each repeat contained between two and five subrepeats and each subrepeat shared a highly conserved core region of 29 bp. This minisatellite sequence also hybridized with high affinity to the A genome species B. napus and B. juncea. This research showed that dispersed, genome-specific probes can be isolated using DAMD PCR and that these probes could be used to detect and quantify alien DNA present in progeny from intergeneric or interspecific crosses.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.     

Divergent patterns of allelic diversity from similar origins: the case of oilseed rape (Brassica napus L.) in China and Australia.

Oilseed rape (Brassica napus) in Australia and China have similar origins, with introductions from Europe, Canada, and Japan in the mid 20th century, and there has been some interchange of germplasm between China and Australia since that time. Allelic diversity of 72 B. napus genotypes representing contemporary germplasm in Australia and China, including samples from India, Europe, and Canada, was characterized by 55 polymorphic simple sequence repeat (SSR) markers spanning the entire B. napus genome. Hierarchical clustering and two-dimensional multidimensional scaling identified a Chinese group (China-1) that was separated from "mixed group" of Australian, Chinese (China-2), European, and Canadian lines. A small group from India was distinctly separated from all other B. napus genotypes. Chinese genotypes, especially in the China-1 group, have inherited unique alleles from interspecific crossing, primarily with B. rapa, and the China-2 group has many alleles in common with Australian genotypes. The concept of "private alleles" is introduced to describe both the greater genetic diversity and the genetic distinctiveness of Chinese germplasm, compared with Australian germplasm, after 50 years of breeding from similar origins.     

基于非变性聚丙烯酰胺凝胶电泳的菜心SCoT分析

以菜心(Brassica rapa var. parachinensis)抗小菜蛾品种Caixin65和感小菜蛾品种Caixin69及6份F2株系为材料,利用非变性聚丙烯酰胺凝胶电泳检测菜心抗虫株系的SCoT多态性,同时进行SCoT多态性的非变性聚丙烯酰胺凝胶电泳检测效率、遗传多样性分析和差异条带克隆分析。结果表明,非变性聚丙烯酰胺凝胶能检测出亲本与子代间的SCoT多态性和遗传多样性变化,条带数量较多且清晰,提高了SCoT标记检测效率。选取亲本的10条非变性聚丙烯酰胺凝胶差异片段克隆测序,获得感虫序列4条、抗虫序列6条,GENSCAN预测其中8条具有启动子、终止子、阅读框等基因结构序列。同源性检索分析表明,感虫序列分别与泛素羧基末端水解酶mRNA序列、大白菜克隆序列、白菜线粒体丙酮酸载体蛋白序列、甘蓝基因组编码未知蛋白的HDEM序列同源,抗虫序列分别与白菜RNA假尿苷合酶4线粒体mRNA序列、京水菜线粒体DNA序列、白菜未知蛋白mRNA序列、白菜4-香豆酸：辅酶A连接酶mRNA序列、大白菜克隆序列、哈茨木霉mRNA序列同源。本研究提高了SCoT标记清晰度、遗传多样性检测水平和差异片段克隆的精准性,使得SCoT成为批量克隆差异片段的高效工具,有助于挖掘SCoT功能性标记信息,开展初步的功能基因组学研究,提高优异株系筛选鉴定效率,加快育种进程,为菜心抗虫性机制的进一步研究提供理论基础。     

Amphidiploid Brassica juncea contains conserved progenitor genomes.

To perform a detailed study of genome evolution in the natural Brassica amphidiploid B. juncea, we have constructed two linkage maps based on RFLP (restriction fragment length polymorphism) markers; one generated from a cross between a resynthesized B. juncea (a chromosome doubled interspecific B. rapa x B. nigra hybrid) and a natural B. juncea cultivar, the other from a cross between two B. juncea cultivars. By using a common cultivar in both crosses, the two maps could be unambiguously integrated. All loci exhibited disomic inheritance of parental alleles in the natural x resynthesized cross, showing that B. rapa chromosomes paired exclusively with their A-genome homologues in B. juncea and that B. nigra chromosomes likewise paired with their B-genome homologues. The maps derived from the two crosses were also perfectly collinear. Furthermore, these maps were collinear with maps of the diploid progenitor species (B. nigra and B. rapa) produced using the same set of RFLP probes. These data indicate that the genome of B. juncea has remained essentially unchanged since polyploid formation. Our observations appear to refute the suggestion that the formation of polyploid genomes is accompanied by rapid change in genome structure.     

Regional association analysis delineates a sequenced chromosome region influencing antinutritive seed meal compounds in oilseed rape

This study describes the use of regional association analyses to delineate a sequenced region of a Brassica napus chromosome with a significant effect on antinutritive seed meal compounds in oilseed rape. A major quantitative trait locus (QTL) influencing seed colour, fibre content, and phenolic compounds was mapped to the same position on B. napus chromosome A9 in biparental mapping populations from two different yellow-seeded × black-seeded B. napus crosses. Sequences of markers spanning the QTL region identified synteny to a sequence contig from the corresponding chromosome A9 in Brassica rapa. Remapping of sequence-derived markers originating from the B. rapa sequence contig confirmed their position within the QTL. One of these markers also mapped to a seed colour and fibre QTL on the same chromosome in a black-seeded × black-seeded B. napus cross. Consequently, regional association analysis was performed in a genetically diverse panel of dark-seeded, winter-type oilseed rape accessions. For this we used closely spaced simple sequence repeat (SSR) markers spanning the sequence contig covering the QTL region. Correction for population structure was performed using a set of genome-wide SSR markers. The identification of QTL-derived markers with significant associations to seed colour, fibre content, and phenolic compounds in the association panel enabled the identification of positional and functional candidate genes for B. napus seed meal quality within a small segment of the B. rapa genome sequence.