Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

Traditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid breeding.     

Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa ssp. pekinensis)

A restriction fragment length polymorphism (RFLP) – based linkage map of Chinese cabbage (Brassica rapa ssp. pekinensis) (2n=20) including two agronomic traits, clubroot resistance and orange-yellow pigmentation, was constructed using doubled haploid parents. The total linkage distance was 735 cM; 63 loci were distributed into ten linkage groups. Clubroot resistance of the parental line T136-8 to the current pathotype, race 2, was predominantly controlled by a single dominant gene that originated from European turnip. The locus for clubroot resistance by the dominant major gene (CRa) was mapped on linkage group 3, and RFLP loci HC352b and HC181 were located 3 cM and 12 cM from it, respectively. The locus HC352b was identified by a 4.4 Kb Eco R I fragment, which segregated for null allele. The absence of an allelic fragment in HC352b could be interpreted by deletion in the resistance source; homozygotes for CRa could be efficiently selected by detecting null types for the marker. Orange-yellow pigmentation expressed in head inner leaves and petals was governed by a single recessive gene. The locus (Oy) for the pigmentation was mapped on linkage group 1, being located 17–19 cM from three RFLP loci that were closely linked to each other. The linkage analysis for clubroot resistance and unique pigmentation revealed some informative RFLP markers. Identification of molecular markers for clubroot resistance and other agronomically important traits would provide useful information in breeding programs of Chinese cabbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

大白菜NPR家族基因鉴定及表达模式分析

为明确大白菜NPR转录辅助因子在大白菜基因组中的数量、结构和表达特点,揭示NPR在水杨酸信号途径调控大白菜生长发育过程中的作用.本研究利用拟南芥资源信息库TAIR10和芸薹属基因组网站Brassica Database鉴定大白菜基因组中的NPR家族成员,并对其蛋白和基因特性进行生物信息学分析.鉴定出大白菜基因组中包括1...     

小白菜小孢子胚状体诱导影响因素研究

以10个小白菜杂交种为试材,采用游离小孢子培养方法,研究影响小孢子胚状体形成因素的结果表明,不同品种胚状体的诱导率不同;NLN培养基中大量元素减半有利于小孢子胚诱导,其诱导率达到100%;培养基中添加0.05 mg/L的6-BA和0.2 mg/L的NAA可以提高胚状体诱导率;活性炭和琼脂糖对诱导胚状体发生和发育有促进作用,可使子叶胚的比例达90%以上;花蕾经4℃低温预处理24 h后的成胚率显著提高。     

RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

茄子抗冷细胞变异体的RAPD分析及自交一代株系的抗冷性鉴定

以茄子品系E－9903花药低温胁迫培养获得的抗冷细菌变异体为试材，用SDS方法从茄子幼嫩叶片提取DNA，选用40个长度为10个碱基的寡聚核苷酸（OPK01－OPK20，OPJ01－OPJ20）作为RAPD引物进行PCR扩增反应，并测定抗冷细胞变异体自交一代株系的冷害指数。结果表明：以OPK12为PCR反应引物时RAPD扩增谱带在抗冷性植株与同品系的对照株之间存在多态性差异，抗冷细胞变异体自交一代株系的冷害指数显著低于同品系的对照。     

Selection for resistance to Plasmodiophora brassicae Wor. in oriental subspecies of Brassica rapa L.

Summary Selection for resistance to Plasmodiophora brassicae Wor. in oriental groups of Brassica rapa L.Two hundred and sixty-five cultivars of leafy, oriental bassicas were tested for resistance to 18 collections of Plasmodiophora brassicae, the causal agent of clubroot. The tests were conducted in the greenhouse at low and high level inoculum concentrations. Eleven cultivars of B. rapa pe-tsai, five cultivars of B. rapa pak-choy and three cultivars of B. rapa choy-sum consistently segregated for resistance at the lower concentration of inoculum (1000 spores/ml). All 265 cultivars were susceptible at the higher concentration (1 000 000 spores/ml). Three cultivars were used in pedigree and recurrent selection schemes for increased resistance. After three cycles of selfing resistant individuals, significantly more resistant S₃ lines were derived from each cultivar. Lines derived from two cultivars. Chinese White and PI 257236, continued to improve with each cycle of selection and demonstrated increased resistance to higher levels of inoculum (up to 1 000 000 spores/ml) New cultivars based on intercrosses of S₂ resistant individuals also had significantly better resistance than the original cultivar. After two cycles of selection in the third cultivar, PI 419007, resistance did not increase and its S₂ mass did not differ significantly from the original cultivar. Evidence that indicates resistance is pathotype-non-differential and offers an alternative to major gene, pathotype-differential types of resistance currently being introduced to the leafy oriental brassicas from other Brassica rapa groups.     

High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis

Brassica rapa (syn. Brassica campestris) ssp. chinensis is an important vegetable crop, but it is relatively recalcitrant to microspore culture. One genotype each of B. rapa ssp. chinensis var. communisand var. utilis were used formicrospore culture. Embryo production of3.8–42.4 embryos/bud was obtained. A high rate of plant regeneration directly from microspore-derived embryos without subculture was achieved by an improved protocol involving replacement of culture media and reduction of sucrose concentrations after 48 h of induction,among other modifications. More than 70%of regenerated plants were spontaneous diploids. Some spontaneous tetraploid plants were also obtained from isolated microspores of both genotypes tested. These tetraploids may be directly exploited a snew varieties in a Brassica rapabreeding programme. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

Identification of RAPD markers linked to linolenic acid genes in rapessed

Summary The inheritance of the low linolenic acid content (derivated from mutant lines) in rapeseed was investigated. Molecular techniques of gene mapping through RAPD markers were applied on a microspore-derived progeny from a high × low linolenic acid F₁ hybrid. Bulked segregant analysis made it possible to test rapidly number of RAPD primers. Two linkage groups of 6 markers (72.7 cM and 75.6 cM) were determined. Each corresponded to a major QTL which explained 24% and 30.7% of the total phenotypic variation of the linolenic acid content. It was confirmed that two independant mutations were implied in the low linonenic acid content.     

Identification of RAPD markers linked to recessive genes conferring siliqua shatter resistance in Brassica rapa

Shattering of siliquae causes significant seed loss in canola (Brassica napus) production worldwide. There is little genetic variation for resistance to shatter in canola and, hence, the trait has been studied in B. rapa. Previous studies have shown two randomly segregating recessive genes to be responsible for shatter resistance. Three random amplified polymorphic DNA markers were identified as being linked to shatter resistance using bulked segregant analysis in a F₃B. rapa population. The population was derived from a cross between a shatter‐susceptible Canadian cultivar and a shatter‐resistant Indian line. Of the three markers, RAC‐3₉₀₀ and RX‐7₁₀₀₀ were linked to recessive sh1 and sh2 alleles, and SAC‐20₁₃₀₀ was linked to both dominant Sh1 and Sh2 alleles. The common marker for the dominant wild‐type allele for the two loci was explained to have resulted from duplication of an original locus and the associated markers through chromosome duplication and rearrangements in the process of evolution of the modern B. rapa from its progenitor that had a lower number of chromosomes. Segregation data from double heterozygous F₃ families, although limited, indicated the markers were not linked to each other and provided further evidence for the duplication hypothesis.     

一个新的白菜苗期TuMV-C4抗性主效QTL定位及连锁分子标记开发

为寻找与TuMV抗性基因紧密连锁的分子标记,选用高抗病毒病大白菜自交系91-112和高感病毒病自交系T12-19以及由二者为双亲构建的包含100个株系的DH群体为材料,通过SSR和InDel标记的遗传分析,在A09上定位了一个新的与大白菜苗期TuMV-C4抗性相关的主效QTL位点BrTuA09。在此基础上,针对该QTL位点所在的标记区间,根据作图群体双亲的重测序结果,设计合成27对引物,其中11个InDel在双亲间具有多态性,且条带单一、扩增稳定;连锁分析发现,11个InDel标记均被定位在A09连锁群上BrTuA09的置信区间。利用BC1群体进行标记验证发现,这些标记对高抗单株选择的准确率均达到78%以上,可应用于分子标记辅助选择,为大白菜TuMV抗病分子育种奠定了良好的基础。     

与大白菜抗霜霉病基因连锁的分子标记研究

【研究目的】 研究与大白菜抗霜霉病基因紧密连锁的DNA分子标记。【方法】利用高抗自交系‘660’和高感自交系‘654B’及其杂交F2群体162个单株为材料,采用构建抗、感病池,利用BSA法筛选了87对SSR引物,35对拟南芥抗霜霉病相关基因的特异引物,其中特异引物RPP13P2和RPP131-2R在抗感病亲本和抗感病池中扩增出一条多态性片段RPP13MK,利用这对引物对F2代单株构建的分享群体进行扩增,验证标记RPP13MK与目的基因的连锁关系,Mapmaker 3.0软件计算遗传距离。【结果】通过F2单株验证后证明RPP13MK与抗霜霉病基因紧密连锁,其遗传距离为5.6 cM。【结论】获得了一个与大白菜抗霜霉病基因紧密连锁的分子标记RPP13MK。     

Allelic variation of a clubroot resistance gene (Crr1a) in Japanese cultivars of Chinese cabbage (Brassica rapa L.)

Clubroot resistance (CR) is an important trait in Chinese cabbage breeding worldwide. Although Crr1a, the gene responsible for clubroot-resistance, has been cloned and shown to encode the NLR protein, its allelic variation and molecular function remain unknown. Here, we investigated the sequence variation and function of three Crr1a alleles cloned from six CR F₁ cultivars of Chinese cabbage. Gain-of-function analysis revealed that Crr1a^Kinami90_a isolated from the cv. ‘Kinami 90’ conferred clubroot resistance as observed for Crr1a^G004. Because two susceptible alleles commonly lacked 172 amino acids in the C-terminal region, we investigated clubroot resistance in transgenic Arabidopsis harboring the chimeric Crr1a, in which 172 amino acids of the functional alleles were fused to the susceptible alleles. The fusion of the C-terminal region to the susceptible alleles restored resistance, indicating that their susceptibility was caused by the lack of the C-terminus. We developed DNA markers to detect the two functional Crr1a alleles, and demonstrated that the functional Crr1a alleles were frequently found in European fodder turnips, whereas they were rarely introduced into Japanese CR cultivars of Chinese cabbage. These results would contribute to CR breeding via marker-assisted selection and help our understanding of the molecular mechanisms underlying clubroot resistance.     

A Comparison of Genetic Segregation in Traditional and Microspore-Derived Populations of Brassica juncea L. Czern and Coss.

An isolated microspore culture procedure was used to produce doubled haploid lines of Brassica juncea from F¹ plants of reciprocal crosses between the cultivar‘RLM514’and a canola quality breeding line. The inheritance of two qualitative markers, seed color and leaf hairiness, was compared using traditional and microspore-derived populations from these crosses. Chi-square tests indicated that each trait is controlled by different sets of duplicate pairs of genes. Brown seeds or hairy leaves can result from the presence of either of two dominant alleles, whereas yellow seed or glabrous leaves are produced when alleles at both loci are recessive. The segregation of genes controlling seed color and leaf hairs in doubled haploid progeny did not differ significantly from that expected under random assortment, indicating that doubled haploids can be used in this species for genetic studies, and probably cultivar development as well.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

Disease response of resynthesized Brassica napus L. lines carrying different combinations of resistance to Plasmodiophora brassicae Wor.

Resistance responses of resynthesized Brassica napus lines to infection with Plasmodiophora brassicae were investigated. Lines that were derived from interspecific crosses between clubroot-resistant B. rapa and resistant B. oleracea exhibited very broad and effective resistance in both greenhouse and field tests. When clubroot resistance was introduced into resynthesized lines from the B. oleracea parent only, the plants were mainly susceptible. Interspecific hybrids from the most resistant parental genotypes, i.e. B. campestris ECD-04 and the B. oleracea cultivars ECD-15 or ‘Bohmerwaldkohf’, were used to initiate a B. napus resistance-breeding programme. These artificial rapeseed lines were resistant to isolates that were virulent on all B. napus differential lines and/or parental lines. Preliminary segregation analysis suggests that their resistance is due to at least two dominant and unlinked genes. In some cases progenies from selfed resynthesized plants exhibited resistance reactions that differed from those of the parental hybrid plant; this may have been the result of cytological instability.     

Effect of donor plant age and inflorescence age on microspore culture of Brassica napus L.

Summary Effect of age of donor plants and age of inflorescence on embryogenesis in microspore culture of B. napus was examined. Microspores isolated from buds of older plants had a higher embryo yield than those of younger ones. The effect of the age of inflorescence showed a different pattern. In older plants, a higher embryogenesis response was observed in microspores isolated from buds of new inflorescences, while in young plants, microspores isolated from buds of old inflorescences showed high embryo yield. These different responses were considered to be attributable to a difference in the developmental stage of pollen at the time of microspore isolation. Our results indicated that microspores collected from older inflorescences and older plants have sufficient embryogenic potential when the optimum developmental stage of pollen was used. Frequency of embryo to plant conversion was influenced by the size of embryos subcultured, but not by donor plant age or the age of the inflorescence.     

Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF₁ hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 ^°C after being transferred to solid regeneration mediumand an initial period of low temperature (2 ^°C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration.     

Identification of RAPD markers linked to the Ns locus in potato

Using the RAPD method and bulked segregant analysis we identified four RAPD markers linked to a dominant gene Ns, responsible for a hypersensitive reaction of potato (Solanum tuberosum L.) to potato virus S (PVS) infection. The markers OPE15⁵⁵⁰, OPJ13⁵⁰⁰, OPG17⁴⁵⁰ and OPH19⁹⁰⁰ were found to be closely linked to the Ns gene in diploid potato clones. They are situated at 2.6, 3.3, 4.6 and 6.6 cM from Ns, respectively. As a source of the gene, clone G-LKS 678147/60, which is known to carry Ns transferred from S. tuberosum ssp. andigena was used. These RAPD markers were not amplified in resistant tetraploid clones containing Ns derived from the clone MPl65 118/3, also having an andigenum origin. This suggests that there may be two separate loci of Ns in the sources identified, or different alleles with the same specificity at a single locus, or that the genetic background of tetraploids tested results in different RAPD amphlification patterns.