Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

Genome evolution in Arabidopsis/Brassica: conservation and divergence of ancient rearranged segments and their breakpoints

Since the tetraploidization of the Arabidopsis thaliana ancestor 30-35 million years ago (Mya), a wave of chromosomal rearrangements have modified its genome architecture. The dynamics of this process is unknown, as it has so far been impossible to date individual rearrangement events. In this paper, we present evidence demonstrating that the majority of rearrangements occurred before the Arabidopsis-Brassica split 20-24 Mya, and that the segmental architecture of the A. thaliana genome is predominantly conserved in Brassica. This finding is based on the conservation of four rearrangement breakpoints analysed by fluorescence in situ hybridization (FISH) and RFLP mapping of three A. thaliana chromosomal regions. For this purpose, 95 Arabidopsis bacterial artificial chromosomes (BACs) spanning a total of 8.25 Mb and 81 genetic loci for 36 marker genes were studied in the Brassica oleracea genome. All the regions under study were triplicated in the B. oleracea genome, confirming the hypothesis of Brassica ancestral genome triplication. However, whilst one of the breakpoints was conserved at one locus, it was not at the two others. Further comparison of their organization may indicate that the evolution of the hexaploid Brassica progenitor proceeded by several events, separated in time. Genetic mapping and reprobing with rDNA allowed assignment of the regions to particular Brassica chromosomes. Based on this study of regional organization and evolution, a new insight into polyploidization/diploidization cycles is proposed.     

Comparison of a Brassica oleracea genetic map with the genome of Arabidopsis thaliana

Brassica oleracea is closely related to the model plant, Arabidopsis thaliana. Despite this relationship, it has been difficult to both identify the most closely related segments between the genomes and determine the degree of genome replication within B. oleracea relative to A. thaliana. These difficulties have arisen in part because both species have replicated genomes, and the criteria used to identify orthologous regions between the genomes are often ambiguous. In this report, we compare the positions of sequenced Brassica loci with a known position on a B. oleracea genetic map to the positions of their putative orthologs within the A. thaliana genome. We use explicit criteria to distinguish orthologous from paralogous loci. In addition, we develop a conservative algorithm to identify collinear loci between the genomes and a permutation test to evaluate the significance of these regions. The algorithm identified 34 significant A. thaliana regions that are collinear with >28% of the B. oleracea genetic map. These regions have a mean of 3.3 markers spanning 2.1 Mbp of the A. thaliana genome and 2.5 cM of the B. oleracea genetic map. Our findings are consistent with the hypothesis that the B. oleracea genome has been highly rearranged since divergence from A. thaliana, likely as a result of polyploidization.     

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.     

Contrasting genome organisation: two regions of the Brassica oleracea genome compared with collinear regions of the Arabidopsis thaliana genome.

Brassica crop species are of worldwide importance and are closely related to the model plant Arabidopsis thaliana for which the complete genome sequence has recently been established. We investigated collinearity of marker order by comparing two contrasting regions of the Brassica oleracea genome with homologous regions of A. thaliana. Although there is widespread replication of marker loci in both A. thaliana and B. oleracea, we found that a combination of genetic markers mapped in B. oleracea, including RFLPs, CAPS, and SSRs allowed comparison and interpretation of medium-scale chromosomal organisation and rearrangements. The interpretation of data was facilitated by hybridising probes onto the whole A. thaliana genome, as represented by BAC contigs. Twenty marker loci were sampled from the whole length of the shortest B. oleracea linkage group, 06, and 21 from a 30.4-cM section of the longest linkage group, 03. There is evidence of locus duplication on linkage group 06. Locus order is well conserved between a putative duplicated region of 10.5 cM and a discrete region comprising 25 cM of A. thaliana chromosome I. This was supported by evidence from seven paralogous loci, three of which were duplicated in a 30.6-cM region of linkage group 06. The pattern of locus order for the remainder of linkage group 06 and the sampled section of linkage group 03 was more complex when compared with the A. thaliana genome. Although there was some conservation of locus order between markers on linkage group 03 and approximately 9 cM of A. thaliana chromosome I, this was superimposed upon a complex pattern of additional loci that were replicated in both A. thaliana and B. oleracea. The results are discussed in the context of the ability to use collinear information to assist map-based cloning.     

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.     

Comparative physical mapping of segments of the genome of Brassica oleracea var. alboglabra that are homoeologous to sequenced regions of chromosomes 4 and 5 of Arabidopsis thaliana

Due to their relatedness to Arabidopsis thaliana (Arabidopsis), the cultivated Brassica species represent the first group of crops with which to evaluate comparative genomics approaches to understanding biological processes and manipulating traits. We have constructed a high-quality binary BAC library (JBo) from genomic DNA of Brassica oleracea var. alboglabra, in order to underpin such investigations. Using the Arabidopsis genome sequence and clones from the JBo library, we have analysed aspects of gene conservation and microsynteny between a 222 kb region of the genome of Arabidopsis and homoeologous segments of the genome of B. oleracea. All 19 predicted genes tested were found to hybridize to clones in the JBo library, indicating a high level of gene conservation. Further analyses and physical mapping with the BAC clones identified allowed us to construct clone contig maps and analyse in detail the gene content and organization in the set of paralogous segments identified in the genome of B. oleracea. Extensive divergence of gene content was observed, both between the B. oleracea paralogous segments and between them and their homoeologous segment within the genome of Arabidopsis. However, the genes present show highly conserved collinearity with their orthologues in the genome of Arabidopsis. We have identified one example of a Brassica gene in a non-collinear position and one rearrangement. Some of the genes not present in the discernible homoeologous regions appear to be located elsewhere in the B. oleracea genome. The implications of our findings for comparative map-based cloning of genes from crop species are discussed.     

Chromosomal mapping of <Emphasis Type="Italic"> Brassica oleracea </Emphasis>based on ESTs from <Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: complexity of the comparative map

Expressed sequence tags (ESTs) from the Arabidopsis thaliana sequencing project were used to construct a genetic RFLP map for Brassica oleracea. Of the 110 A. thaliana ESTs tested, 95 were found to be informative RFLP probes in map construction. In total, 212 new loci corresponding to the 95 ESTs were added to the existing genetic map of B. oleracea. The enriched map covers all nine basic linkage groups and confirms that the chromosomes of B. oleracea and A. thaliana are similar in linear organization. However, varying levels of sequence conservation between the chromosomes of B. oleracea and A. thaliana were detected in different regions of the genomes. Long conserved regions encompassing entire chromosome arms in both genomes were identified; these are probably shared by descent. On the other hand, extensive rearrangements were observed in numerous chromosome regions, producing a mosaic of A. thaliana-like segments in the genome of Brassica. The presence of extensive chromosome duplication in A. thaliana was taken into consideration in the construction of the comparative maps of B. oleracea and A. thaliana.     

Comparative Genome Mapping in Brassica

A Brassica nigra genetic linkage map was developed from a highly polymorphic cross analyzed with a set of low copy number Brassica RFLP probes. The Brassica genome is extensively duplicated with eight distinct sets of chromosomal segments, each present in three copies, covering virtually the whole genome. Thus, B. nigra could be descended from a hexaploid ancestor. A comparative analysis of B. nigra, B. oleracea and B. rapa genomes, based on maps developed using a common set of RFLP probes, was also performed. The three genomes have distinct chromosomal structures differentiated by a large number of rearrangements, but collinear regions involving virtually the whole of each the three genomes were identified. The genic contents of B. nigra, B. oleracea and B. rapa were basically equivalent and differences in chromosome number (8, 9 and 10, respectively) are probably the result of chromsome fusions and/or fissions. The strong conservation of overall genic content across the three Brassica genomes mirrors the conservation of genic content observed over a much longer evolutionary span in cereals. However, the rate of chromosomal rearrangement in crucifers is much higher than that observed in cereal genomes.     

Novel insights into seed fatty acid synthesis and modification pathways from genetic diversity and quantitative trait Loci analysis of the Brassica C genome

Natural genetic variation in fatty acid synthesis and modification pathways determine the composition of vegetable oils, which are major components of human diet and renewable products. Based on known pathways we combined diversity and genetic analysis of metabolites to infer the existence of enzymes encoded by distinct loci, and associated these with specific elongation steps or subpathways. A total of 107 lines representing different Brassica genepools revealed considerable variation for 18 seed fatty acid products. The effect of genetic variation within a single biochemical step on subsequent products was demonstrated using a correlation matrix of scatterplots, and by calculating relative step yields. Surprisingly, diploid Brassica oleracea segregating populations had a similar range of variation for individual fatty acids as across the whole genepool. This allowed identification of 22 quantitative trait loci (QTL) associated with activity in the plastid, early stages of synthesis, desaturation, and elongases. Four QTL were assigned to early stages of synthesis, seven to subpathway specific or general elongase activity, one to ketoacyl acyl-carrier protein synthetase, and two each to fatty acid desaturase and either desaturase or fatty acyl-carrier protein thioesterase. An additional 10 QTL had distinct effects but were not assigned specific functions. Where contrasting behavior in more than one subpathway was detected, we inferred QTL specificity for particular combinations of substrate and product. The assignment of enzyme function to QTL was consistent with the known position of some Brassicaeae candidate genes and collinear regions of the Arabidopsis (Arabidopsis thaliana) genome.     

Gametic embryos of maize as a target for biolistic transformation: comparison to immature zygotic embryos

The aim of the present study was to determine the suitability of maize gametic embryos of three ETH genotypes as a target for biolistic transformation. We studied parameters considered essential for a successful transformation, such as the frequency of secondary embryo formation, their regeneration ability and the transient transgene expression. Transformable zygotic embryos of one of the ETH genotypes were used as positive control. Our results indicate that gametic embryos can potentially be transformed by particle bombardment, since they responded positively to all the studied parameters, although with lower efficiencies than the zygotic embryos. In particular, differences were found in the rate of secondary embryogenesis and the density of transformed cells.     

Physical organization of the major duplication on Brassica oleracea chromosome O6 revealed through fluorescence in situ hybridization with Arabidopsis and Brassica BAC probes.

The close relationship between Brassica oleracea and Arabidopsis thaliana has been used to explore the genetic and physical collinearity of the two species, focusing on an inverted segmental chromosome duplication within linkage group O6 of B. oleracea. Genetic evidence suggests that these segments share a common origin with a region of Arabidopsis chromosome 1. Brassica oleracea and Arabidopsis bacterial artificial chromosome probes have been used for fluorescence in situ hybridization analysis of B. oleracea pachytene chromosomes to further characterize the inverted duplication. This has been highly effective in increasing the local resolution of the cytogenetic map. We have shown that the physical order of corresponding genetic markers is highly conserved between the duplicated regions in B. oleracea and the physical lengths of the regions at pachytene are similar, while the genetic distances are considerably different. The physical marker order is also well conserved between Arabidopsis and B. oleracea, with only one short inversion identified. Furthermore, the relative physical distances between the markers in one segment of B. oleracea and Arabidopsis have stayed approximately the same. The efficacy of using fluorescence in situ hybridization, together with other forms of physical and genetic mapping, for elucidating such issues relating to synteny is discussed.     

白菜和甘蓝基因组转座子表达及其对基因调控的潜在影响

转座子或转座元件是大多数真核生物基因组的主要组成成分。甘蓝(Brassica oleracea)基因组比白菜(B. rapa)大主要是转座子的扩增差异造成的。然而, 这两个芸薹属近缘物种转座子表达水平以及对基因的调控和功能的影响目前还不清楚。文章对白菜和甘蓝叶、根、茎3个器官的转录组数据进行了初步分析。结果显示, 转座子的表达量很低, 转录组reads中有1%来自转座子的转录本; 转座子的表达存在器官差异, 且不同类别和家族的转座子表达量相差很大, 相同类别和同一家族的转座子在白菜和甘蓝基因组中的表达活性也不相同。进一步鉴定到转录读出的LTR反转座子, 其与下游基因距离小于2 kb的有41个, 小于100 bp的有9个, 这些LTR的转录读出很可能通过正义或反义的转录本激活或干扰下游基因的表达。同时, 具有转录读出的intact LTR比solo LTR具有更强的读出活性。通过深入分析转座子的插入位点发现, 白菜基因组中转座子插入基因内部的频率比甘蓝基因组中的高; 与反转座子相比, DNA转座子更偏向于插入或保留在基因的内含子当中。这些结果为认识转座子对其他蛋白编码基因的影响提供了基础。     

Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the β -glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.     

The distribution of transgene insertion sites in barley determined by physical and genetic mapping

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.     

转基因植物中T-DNA整合的分子特征及表达

植物中不同转基因方法转化外源基因的T-DNA整合特征既具有共性,又具有特性,使得转基因的遗传在各独立转化体间呈现多样性,另外多种遗传因子和限制因素使受体植物中外源基因的表达存在下降,甚至出现基因沉默等复杂现象。本文主要对农杆菌介导及裸露DNA直接转化转基因植物中T-DNA的分子特征和转基因表达的影响因子进行了介绍和概述。转化体中转基因的遗传稳定性和表达主要取决于转基因在植物基因组中的整合位置、拷贝数及组成结构。因而,通过对具有表达水平各异的转化体进行深入的遗传分析和分子生物学研究以及转化体之间进行的比较研究,将对转基因技术自身的完善、定点整合以及更有效的利用转基因技术都具有十分重要的意义。     

十字花科蔬菜基因组含量的测定与分析

以我国14种重要十字花科蔬菜为材料,利用流式细胞术测定基因组合量.其中青苤蓝、乌塌菜、雪里蕻、芥蓝的基因组含量属首次报道.本试验数据与国外文献提供的相关数据对比,发现青萝卜、结球甘蓝、青花菜和根用芥菜的基因组含量与已报道数值基本吻合,而大白菜、花椰菜的基因组含量值与报道数据存在差异.造成同一物种基因组含量值差异的因为可能是品种的不同,也可能与生长环境或测定时参考标准选用等因素不同有关.     

Molecular characterisation and chromosomal localisation of a telomere-like repetitive DNA sequence highly enriched in the C genome of Brassica

The aim of this work was to find C genome specific repetitive DNA sequences able to differentiate the homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica, in order to assist in the physical identification of B. napus chromosomes. A repetitive sequence (pBo1.6) highly enriched in the C genome of Brassica was cloned from B. oleracea and its chromosomal organisation was investigated through fluorescent in situ hybridisation (FISH) in B. oleracea (2n = 18, CC), B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC) genomes. The sequence was 203 bp long with a GC content of 48.3%. It showed up to 89% sequence identity with telomere-like DNA from many plant species. This repeat was clearly underrepresented in the A genome and the in situ hybridisation showed its B. oleracea specificity at the chromosomal level. Sequence pBo1.6 was localised at interstitial and/or telomeric/subtelomeric regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus 18 to 24 chromosomes hybridised with pBo1.6. The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful for the determination of the chromosomal location of transgenic DNA in genetically modified oilseed rape.