Resynthesis of rapeseed (Brassica napus L.): A comparison of sexual versus somatic hybridization

Sexual and somatic Brassica napus hybrids produced from the same parental plants were compared. Sexual crosses between a white-flowered, self-compatible broccoli selection (B. oleracea var. italica, cc genome) as the maternal parent and a flowering pak choi accession (B. chinensis, aa genome) yielded one unique spontaneous hybrid and four hybrids through embryo rescue. Thirty-nine somatic hybrids were recovered from a protoplast fusion experiment. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Sexual and somatic hybrids exhibited differences in leaf morphology, flower colour, flowering habit, and organellar inheritance. Sexual hybrids were all fertile amphidiploids (2n = 38, aacc) following spontaneous chromosome doubling. All somatic hybrids had high nuclear DNA contents; most were probably hexaploids (aaaacc or aacccc) from the fusion of three portoplasts. Two initially sterile hexaploid (aaaacc) regenerates eventually set selfed seed after the loss of the putative extra aa genome following regrowth from axillary buds. A bias toward inheritance of B. chinensis chloroplasts was observed with somatic hybrids.     

亚麻荠油脂相关转录因子CsLEC2基因家族的鉴定及表达分析

对亚麻荠CsLEC2家族进行全基因组鉴定、半定量RT-PCR、qRT-PCR,检测CsLEC2基因的时空表达特性并通过转录组数据预测CsLEC2下游靶基因,以解析CsLEC2调控亚麻荠种子油脂合成和积累等生物学功能。结果表明,在亚麻荠基因组中共鉴定到3个亚麻荠CsLEC2基因（CsLEC2.1、CsLEC2.2和CsLEC2.3）。蛋白理化性质和高级结构分析显示,亚麻荠CsLEC2蛋白具有与拟南芥AtLEC2相似的理化性质,且其二级结构的主体也相似。系统进化分析表明,亚麻荠CsLEC2蛋白与模式植物拟南芥AtLEC2蛋白及拟南芥琴亚种AlLEC2蛋白亲缘关系最近。qRT-PCR结果显示亚麻荠CsLEC2在未成熟的种子中高表达。转录组数据分析显示下游基因CsWRI1和CsOLE3高表达,推测CsLEC2可能直接上调CsWRI1和CsOLE3的转录表达,参与油脂代谢调控。     

Sexual hybridization between Capsella bursa‐pastoris (L.) Medik (♀) and Camelina sativa (L.) Crantz (♂) (Brassicaceae)

The development of transgenic oilseed Camelina sativa (2n = 40) and the potential for hybridization with its weedy relative Capsella bursa‐pastoris (2n = 36) necessitates a careful evaluation of the reproductive compatibility between the species. Here, we conducted over 1800 crosses (emasculation and manual pollination) to examine the ability of 10 Canadian C. bursa‐pastoris (♀) accessions to hybridize with five accessions of C. sativa (♂). Seven hybrids were confirmed among 586 putative hybrids screened with species‐specific markers, indicating a hybridization rate of 1.5 hybrids per 10 000 ovules pollinated. All seven hybrids had intermediate DNA content compared to their parents, were morphologically distinct, had low (1.9%) pollen fertility and failed to produce selfed or backcrossed seed. Given the abundance of C. bursa‐pastoris along field margins, hybrids will likely be generated in the wild, but they will be unable to establish lineages unless fertility is restored. The large number of crosses and the diversity captured by the use of multiple accessions resulted in strong statistical power and a high degree of confidence in the estimated hybridization rate.     

Hybridization between Camelina sativa (L.) Crantz (false flax) and North American Camelina species

The potential for gene flow between Camelina sativa, a promising edible and industrial oil crop in Canada, and its wild North American relatives C. alyssum, C. microcarpa and C. rumelica subsp. rumelica, was investigated. Sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was used to differentiate Camelina species and/or accession‐specific nucleotide markers and identify interspecific F₁ hybrids. ITS analysis identified hybrids in progeny of C. alyssum × C. sativa, C. microcarpa accession 36010 × C. sativa and C. sativa × C. rumelica subsp. rumelica. Seed set on C. alyssum × C. sativa F₁ and F₂ progeny was similar to the parents; few seeds were produced on hybrid progeny of C. microcarpa accession 36010 × C. sativa and C. sativa × C. rumelica subsp. rumelica. The study provided evidence that should the species have sympatric distributions and overlapping flowering periods, gene flow between C. sativa and its wild North American relatives is possible and that it would most likely occur with C. alyssum given the high fertility of the F₁ hybrids recovered.     

Genetic analysis of resistance to clubroot (Plasmodiophora brassicae Woron) in two Brassica oleracea groups (ssp. acephala and ssp. botrytis) through diallel analysis

Clubroot, caused by Plasmodiophora brassicae Woron, is a major disease of cruciferous crops and few sources of resistance have been detected and genetically studied in the Brassica oleracea species. In this study, two diallel mating systems using resistant kale lines and susceptible cauliflower lines were performed. Resistance was assessed from a disease index evaluated on young plants artificially inoculated with local isolates of the parasite. Both general and specific combining ability effects (GCA and SCA) and partly reciprocal effects were demonstrated. Resistance inheritance was, however, characterized by a predominance of additive genetic effects (GCA). Three groups of parent lines could be separated; kale lines with very high resistance, kale lines with intermediate resistance and susceptible cauliflower lines. In the two kale groups, two genetically different resistance types were suggested and various recurrent selection procedures are proposed following the diallel results.     

甘蓝型油菜和芸芥属间杂交的亲和性研究

为了利用芸薹属近缘属的基因资源进行油菜品种改良,探索了甘蓝型油菜×芸芥的属间杂交亲和性。以甘蓝型油菜湘油15号、中油821和J-2为母本,三营芸芥和东郊芸芥为父本,进行人工远缘杂交试验。结果表明：甘蓝型油菜与芸芥属间杂交亲和性较低,杂交亲和性的强弱与亲本材料有关,不同品种的甘蓝型油菜与不同品种的芸芥属间杂交亲和性（真杂种苗数/授粉花蕾数）不同,湘油15号与三营芸芥的亲和程度最高,为0.036,J-2和东郊芸芥的亲和程度最低,为0.014。     

结球甘蓝雌蕊调控因子SPT与HEC1的克隆及相互作用分析

为研究SPT和HECs及其相互作用对甘蓝雌蕊发育的影响, 以结球甘蓝自交不亲和系E1为材料, 提取雌蕊总RNA, 根据拟南芥中SPT和HEC1基因设计引物, 采用同源克隆的方法克隆SPT基因, 其序列1085 bp, 开放阅读框(ORF) 1062 bp; HEC1基因ORF 696 bp。通过cDNA推导得到的氨基酸序列表明, SPT编码353个氨基酸残基, 预测分子量为37.67 kD, pI为6.83; HEC1编码231个氨基酸残基, 预测分子量为25.26 kD, pI为10.2。分析表明, 两基因在各器官中均表达, 但SPT在果实和雌蕊中表达量最高, 而HEC1在根和花蕾中的表达量最高。为检测两者的相互作用, 构建原核表达质粒pCold I-SPT和pGEX-HEC1, Pull-down试验表明两蛋白能够在体外相互作用。同时构建pGBKT7-SPT、pGADT7-HEC1及互换载体pGBKT7-HEC1和pGADT7-SPT酵母表达载体, 分别转化酵母Y2HGold和Y187感受态细胞后均未出现自激活和毒性现象, 融合后的二倍体酵母均能在SD/–Trp–Leu–Ade–His/X-α-gal/AbA板上长出蓝色菌斑, 表明SPT和HEC1能够相互作用激活下游的HIS3、AUR1-C、MEL1和ADE2报告基因, 酵母双杂交试验结果与Pull-down检测一致, 说明SPT和HEC1能够相互作用以调控雌蕊的发育。     

春甘蓝抽薹的生理与相关基因转录分析

本研究以高代自交获得的4份具有不同抽薹时间且遗传稳定的春甘蓝品系为材料,在观察其园艺学性状的基础上,分析了抽薹性状与可溶性蛋白和可溶性糖含量之间的关系,结果显示,耐抽薹春甘蓝品系的可溶性蛋白和可溶性糖含量低于不耐抽薹的,抽薹前春甘蓝的可溶性蛋白和可溶性糖含量也均低于抽薹后的.进一步的转录分析结果表明,5个与植物抽薹开花...     

利用酵母双杂交法检测甘蓝SCR与SRK之间的相互作用

为深入研究甘蓝S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白(SCR)间相互识别的分子机理,以具有典型自交不亲和性的甘蓝D3为材料,利用巢式PCR分别扩增SRK胞外域(eSRK)和SCR,通过酵母双杂交检测二者之间的相互作用。并利用同源重组技术将eSRK与GAL4报告基因DNA活化域融合(pGADT7eSRK)以及SCR与GAL4报告基因DNA结合域融合(pGBKT7SCR)。两种重组质粒分别转化酵母Y187和Y2HGold后未出现自激活现象。融合的二倍体酵母(pGADT7eSRK× pGBKT7SCR)能在选择性固体培养基(SD/–Ade/–His/–Leu/–Trp/X-a-Gal/AbA)上生长,并且菌落呈蓝色。结果表明, eSRK与SCR蛋白能够相互结合, 为深入研究二者作用位点成功建立了一个技术平台。     

Inheritance of Waxlessness in Kohlrabi (Brassica oleracea L. var. gongyloides) and its Utilization in Hybrid Seed Production

By genetic analysis of selfed progenies and reciprocal F₁, F₂ and B₁: populations of crosses of a waxless mutant and three waxy plants two recessive genes were identified in kohlrabi (Brassica oleracea L. var. gongyloides), causing waxlessness independently from each other. Gene a is responsible for glossy waxless and gene b tor dull waxless plants. To be able to form a wax layer a plant must have both genes in the dominant constitution, i.e. A.B. (“complementary gene effect). Some segregation results which deviate from expectation could indicate a potential linkage between one of the two wax loci and the incompatibility locus. Both genes can be used as markers m hybrid breeding of cole crops as well as in basic research.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Leptosphaeria maculans and Xanthomonas campestris pv. campestris

Summary Fifty-six landraces of Portuguese coles (Brassica oleracea l.), representing fourteen morphotypes, were screened for resistance to Leptosphaeria maculans at the cotyledon stage. Three isolates of the pathogen belonging to three pathogenicity groups were used in the screening. The mean disease interaction phenotype of the B. oleracea landraces with the L. maculans isolates confirmed their differences in pathogenicity. When compared to pathogenicity grouping using a B. napus differential set, a different classification was observed on B. oleracea for the three L. maculans isolates, suggesting possible differences in the genetic interaction between the two plant species and the pathogen. Most of the Portuguese cole landraces were found to be susceptible to all three isolates of L. maculans, although a few have shown intermediate interaction phenotype. Some accessions, on the other hand, elicit a differential reaction with the three isolates tested, and can be further selected to be used in host differential sets or in breeding for resistance to black leg.Fifty-four landraces of Portuguese coles, representing eleven morphotypes, were also screened in the field for resistance to Xanthomonas campestris pv. campestris. A good level of resistance to the bacterium was found in several of the morphotypes.Accessions of the Penca type, particularly, were identified as highly resistant. Landraces belonging to the same Portuguese cole group presented a similar range of mean interaction phenotype with the bacterium. Some Portuguese landraces of B. oleracea are potential sources for use in breeding programs for black rot resistance.     

甘蓝BoKIN1基因的ploy(A)位点的预测及其频率分析

多聚腺苷酸化(Polyadenylation)是真核生物中一种重要的pre-mRNA加工机制,是mRNA成熟的一个必经过程。为了解甘蓝BoKIN1基因的多聚腺苷酸化加工情况,本研究从甘蓝全基因组数据库中下载得到BoKIN1基因序列,并利用生物信息学手段分析了BoKIN1的基因结构和基因表达。研究结果显示:BoKIN1基因在花器官中表达最高;然后基于EST文库发现了3个ploy(A)位点,并计算了这些ploy(A)位点在premRNA加工过程中被选择的大致频率情况,结果显示BoKIN1基因在pre-mRNA加工过程中至少有6个ploy(A)位点可供选择加尾,其中AP4位点被选择加尾的频率最高,其次是AP5位点。因此选择AP4位点加尾后形成的mRNA是转录后加工的主要产物。通过本研究我们确定了甘蓝类BoKIN1基因6个选择性加尾位点及其3'UTR的主要产物和次要产物,可对于未来进一步利用这些3'UTR奠定基础。     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Peronospora parasitica and Plasmodiophora brassicae

Summary Forty-four landraces of Portuguese coles (Brassica oleracea L.), representing different morphotypes, were screened at the cotyledon stage for downy mildew resistance, caused by Peronospora parasitica. Two isolates of the pathogen, PHW 630 and PHW 828, were used in the experiment. The mean disease interaction phenotype varied among the landraces. Overall, the Portuguese coles have shown resistance to both isolates, particularly to isolate PHW 828. Some landraces were identified with differential reaction to the two P. parasitica isolates. Some of the Portuguese coles are, therefore, good sources of resistance genes to P. parasitica, and could be used in breeding programs or in the development of a downy mildew host differential set. Among the morphotypes tested, Couve Algarvia (ISA 207), Couve Galega (ISA 243 and 187) and Couve Glória de Portugal (ISA 84) have presented the highest resistance interaction. For the development of host differential sets, the landraces ISA 2 (Penca de Mirandela), ISA 35 (Couve de Valhascos) and ISA 600 (Penca Espanhola) could be further selected for the differentiation of P. parasitica isolates. They are resistant to PHW 828 and susceptible to PHW 643.The same morphotypes and landraces were also screened at the seedling stage for resistance to clubroot, caused by Plasmodiophora brassicae Woron.. Although some variation for resistance could be noticed in some the landraces tested, mainly Galega kale types, none of them presented a sufficient level of resistance as to be directly be useful in breeding for resistance to race 6 of P. brassicae.     

Production of somatic hybrids between S. tuberosum L. and late blight resistant Mexican wild potato species

Interspecific somatic hybrids between dihaploid breeding clones of potato, S. tuberosum and two accessions of wild Mexican species S. pinnatisectum and the hybrid line S. pinnatisectum × S. bulbocastanum were regenerated following electrofusion of mesophyll protoplasts to combine important agricultural traits of S. tuberosum and a high level of late blight resistance from selected wild accessions. In two fusion combinations 239 calli were regenerated; 162 from 195 calli analysed were identified as hybrids by means of isozyme analysis of peroxidases and, for some hybrid clones, by RAPD analysis. Depending on the fusion combination, 47–89 percent of the somatic hybrids had the expected ploidy level and 7–16 percent were mixoploids. Somatic hybrids were phenotypically intermediate as compared to their parents and some of them were able to be backcrossed sexually with potato. Fertility and crossability depended on combination and ploidy level of the somatic hybrids. In tests with detached leaves the wild partner clones had a high late blight resistance score of 8,6 and 8,9; the susceptible tuberosum-partners of 2,8 and 3,5, respectively. Nearly 25 percent of somatic hybrids had a resistance level of 6 or higher in the first year of assessment. The average resistance value of most somatic hybrids was lower than the average parental level. The reasons for variation in resistance values are discussed in connection with the practical application of fusion hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfer of resistance to race 2 of Plasmodiophora Brassicae from Brassica napus to cabbage (B. oleracea var. Capitata). I. Interspecific hybridization between B. napus and B. oleracea var. Capitata

Summary Interspecific hybridization between Brassica napus L. (2n=38, a₁a₁c₁c₁) and B. oleracea var. capitata L. (2x- and 4x-cabbage; 2n=2x=18, cc and 2n=4x=36, cccc) was carried out for the purpose of transferring clubroot disease resistance from the amphidiploid species to cabbage. Nineteen hybrids with three different chromosome levels (2n=28, a₁c₁c; 2n=37, a₁c₁cc and 2n=55, a₁c₁cccc) were obtained. The F₁ plants were mostly intermediate between the two parents but as the number of c genomes in the hybrids increased, the more closely the hybrids resembled the cabbage parent. All F₁ hybrids were resistant when tested against race 2 of Plasmodiophora brassicae wor. The complete dominance of resistance over susceptibility suggested that the gene(s) controlling resistance to this particular race of the clubroot pathogen is probably located on a chromosome of the a genome in Brassica.Contribution No. J654.     

Effect of genotype,environment and carbohydrate on anther culture response in head cabbage (Brassica oleracea L. convar. capitata (L.) Alef.)

Summary The effect of genotype, growing conditions for donor plants and type and concentration of carbohydrate in the culture medium was investigated for anther culture of head cabbage (white cabbage, savoy cabbage, pointed-headed cabbage). Strong genotypic effects on embryo formation from the cultured anthers were shown as well as superior embryo formation from anthers of field grown donor plants compared to plants grown in the greenhouse. When comparing 7, 10 and 13% sucrose in the medium, embryo response increased with increasing sucrose concentration. With maltose, which was generally inferior to sucrose as carbohydrate source for anther culture, the embryo response did not increase with maltose concentration above 10 per cent.     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

Genetic studies of powdery mildew resistance in common oat (avena sativa L.) I. Cultivars and breeding lines grown in Western Europe and North America

Common oat (Avena sativa L.) cultivars and breeding lines grown in Western Europe and North America were tested against twelve Erysiphe graminis f. sp. avenae mildew isolates collected in Germany and Denmark. These isolates were selected for their ability to produce differential response patterns permitting characterization of five oat mildew resistance (OMR) groups. From a total of 259 cultivars and lines tested 173 accessions showed susceptible responses, 48 accessions were characterized by susceptible or intermediate responses and 38 accessions revealed isolate-specific resistance response patterns. Eight cultivars and lines had resistance patterns of OMR group 1, six cultivars of OMR group 2, and eleven cultivars of OMR group 3. Two cultivars, each possessed resistance patterns corresponding to OMR groups 1 + 3 and 2 + 3 in combination, respectively. Three cultivars exhibited the response pattern of OMR group 3 in association with an additional unknown resistance. Eight cultivars and lines showed a resistance response pattern not yet detected in the documented OMR groups. This revised version was published online in July 2006 with corrections to the Cover Date.