大白菜部分形态性状的QTL定位与分析

应用352个标记位点的大白菜AFLP和RAPD图谱和一套栽培品种间杂交获得的重组自交系群体,采用复合区间作图的方法对大白菜9个形态性状进行QTL定位及遗传效应研究。在14个连锁群上检测到50个QTL:其中控制株型的QTL有5个;控制株高的QTL有6个;控制开展度的QTL有5个;控制最大叶长的QTL有7个;控制最大叶宽的QTL有4个;控制叶形指数的QTL有6个;控制中肋长的QTL有7个;控制中肋宽的QTL有4个;控制抽苔的QTL有6个。另外,估算了单个QTL的遗传贡献率和加性效应。这将为大白菜品种改良中形态性状的分子标记辅助选择提供理论依据。     

Analysis of genomic DNA methylation and gene expression in Chinese cabbage ( Brassica rapa L. ssp. pekinensis ) after continuous seedling breeding

Vernalization plays a key role in the bolting and flowering of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plants can switch from vegetative to reproductive growth and then bolt and flower under low temperature induction. The economic benefits of Chinese cabbage will decline significantly when the bolting happens before the vegetative body fully grows due to a lack of the edible value. It was found that continuous seedling breeding reduced the heading of Chinese cabbage and led to bolt and flower more easily. In the present study, two inbred lines, termed A161 and A105, were used as experiment materials. These two lines were subjected to vernalization and formed four types: seeds-seedling breeding once, seedling breeding twice, seedling breeding thrice and normal type. Differences in plant phenotype were compared. DNA methylation analysis was performed based on MSAP method. The differential fragments were cloned and analyzed by qPCR. Results showed that plants after seedling breeding thrice had a loosen heading leaves, elongated center axis and were easier to bolt and flower. It is suggested that continuous seedling breeding had a weaker winterness. It was observed that genome methylation level decreased with increasing generation. Four differential genes were identified, short for BraAPC1, BraEMP3, BraUBC26 and BraAL5. Fluorescent qPCR analysis showed that expression of four genes varied at different reproduction modes and different vernalization time. It is indicated that these genes might be involve in the development and regulation of bolting and flowering of plants. Herein, the molecular mechanism that continuous seedling breeding caused weaker winterness was analyzed preliminarily. It plays an important guiding significance for Chinese cabbage breeding.     

Linkage Map Construction and Quantitative Trait Loci Analysis for Bolting Based on a Double Haploid Population of Brassica rapa

Early bolting of Chinese cabbage （Brassica rapa L.） during spring cultivation often has detrimental effects on the yield and quality of the harvested products. Breeding late bolting varieties is a major objective of Chinese cabbage breeding programs. In order to analyze the genetic basis of bolting traits, a genetic map of B. rapa was constructed based on amplified fragment-length poiymorphism （AFLP）, sequence-related amplified poiymorphism （SRAP）, simple sequence repeat （SSR）, random amplification of polymorphic DNA （RAPD）, and isozyme markers. Marker analysis was carried out on 81 double haploid （DH） lines obtained by microspore culture from F1 progeny of two homozygous parents： B. rapa L. ssp. pekinensis （BY） （an extra-early bolting Chinese cabbage line） and B. rapa L. ssp. rapifera （MM） （an extra-late bolting European turnip line）. A total of 326 markers including 130 AFLPs, 123 SRAPs, 16 SSRs, 43 RAPDs and 14 isozymes were used to construct a linkage map with 10 linkage groups covering 882 cM with an average distance of 2.71 cM between loci. The bolting trait of each DH line was evaluated by the bolting index under controlled conditions. Quantitative trait loci （QTL） analysis was conducted using multiple QTL model mapping with MapQTL5.0 software. Eight QTLs controlling bolting resistance were identified. These QTLs, accounting for 14.1% to 25.2% of the phenotypic variation with positive additive effects, were distributed into three linkage groups. These results provide useful information for molecular marker-assisted selection of late bolting traits in Chinese cabbage breeding programs.     

大白菜骨干自交系的苗期抗病性评价

为明确大白菜骨干自交系的抗病性,本研究于2012-2014年,对课题组保存和创制的203个大白菜自交系进行了霜霉病、病毒病、黑腐病、黄萎病和根肿病的苗期抗性评价。结果显示,高抗上述病害的自交系分别有7、9、0、31和12个;只抗其中一种病害的自交系82个;兼抗两种病害的有61个,兼抗三种病害的自交系有28个,兼抗四种病害的自交系有4个。自交系11-234、04-622、12-85、13-108和09-894综合抗病性最优。此外,春大白菜、夏大白菜和秋大白菜三种生态类群间,以及四种叶球抱合类群间的抗病性表现出明显差异。     

Genetic and non-genetic factors affecting anther culture of Brussels sprouts (Brassica oleracea var. gemmifera)

Summary Eight inbred lines of Brussels sprouts and ten F₁ hybrids derived from them were tested for their response to anther culture. From 5–19 plants per genotype were tested, and each plant was tested on 3–6 separate occasions. Results from the inbred lines were broadly similar to those from the F₁ hybrids, despite the inbreds producing fewer buds and having a higher frequency of anther deformities. The maximum embryo yield from an inbred line was 215 embryos per 100 anthers, and from a hybrid was 275. From estimation of the variance components it was calculated that, for both inbreds and hybrids, about half the total variation was genetic whereas variation due to plants within genotypes and to occasions within plants were each about 13% of the total. The narrow sense heritability of responsiveness to anther culture (estimated by the proportion of variation between inbred lines which was genetic) was 0.48, and there was partial dominance for this character. In three cases the hybrid outyielded the better inbred, and this heterosis may well be due to dispersed dominant genes.     

Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.     

茄子氮素利用效率相关性状的配合力分析

在筛选氮素利用高效茄子自交系的基础上,利用Griffing-Ⅱ设计,在两个氯素水平下(450kg/hm2争不施氮)分析了7个茄子自交系氮素利用效率相关性状的配合力.结果表明,两个氮素水平下,氮效率受加性效应和非加性效应的共同控制;氮素吸收总量和氮素利用效率则以加性遗传为主.氮高效育种中,自交系609和749在两个氮素水平下均有较高的氮效率一般配合力.与高氮处理相比,低氮条件下氮素利用效率相关性状的广义遗传力有所增大,而狭义遗传力多为递减趋势.     

不结球白菜自交不亲和S单元型的鉴定研究

依据甘蓝SRK基因保守序列设计SRK特异引物并进行验证,通过PCR-RFLP法分析16份不结球白菜自交系材料的SRK基因的多态性,根据其差异鉴定不结球白菜S单元型及自交不亲和性.结果表明:16个不结球白菜自交系SRK基因共产生6种不同的带型,其中有1个是强自交不亲和系,4个是弱自交不亲和系,且与田间测定的亲和指数相符,说明利用SRK基因的多态性鉴定不结球白菜纯合自交系的自交不亲和性和S单元型是可行的.同时对部分材料的SRK基因的核苷酸序列进行分析验证,表明相同类型SRK基因序列一致性较高,且SRK基因存在明显的单核苷酸多态性,为进一步自交不亲和性的研究奠定了基础.     

四倍体大白菜雄性不育系资源的创制

目前国内外对二倍体大白菜雄性不育系的创制已进行了深入的研究,利用雄性不育系技术育种已相对成熟,而四倍体大白菜育种仍以突破稔性、提高结籽率的自交系选育为主,四倍体雄性不育系的研究尚无相关报道。本研究通过从二倍体大白菜细胞质雄性不育系中筛选含新型甘蓝型油菜CMS基因的资源(CMS后36高)为不育源,依靠人工秋水仙素加倍,经倍性鉴定筛选和高代稳定四倍体自交系D571、D574连续多代回交转育,成功获得了CMS-D571、CMS-D574两个四倍体大白菜细胞质雄性不育系,该不育系具有不育度达100%、结籽率高、遗传稳定的特点。该研究证实细胞质雄性不育性也可在四倍体大白菜育种上应用,可以作为创制四倍体大白菜细胞质雄性不育系的方法,为四倍体大白菜开展雄性不育系育种奠定了物质基础。     

Comparison of methods for calculating the heritability of adult field resistance to yellow rust and grain yield in spring wheat

Several methods are available for estimating heritability in disomic species, including parent-offspring regression, realized heritability, intraclass correlations of recombinant inbred lines, and diallel-cross analysis. Estimates were obtained by these various methods for a set of eight bread wheat (Triticum aestivum) lines adapted to the East African highlands, which had been intercrossed and selfed in a half-diallel arrangement to give F₁, F₂ and F₃ generations, and F₆ recombinant inbred lines. Significant genetic variation existed among parents and crosses for both grain yield and yellow rust resistance in all generations. Based on the heritability calculated from the analysis of F₆ recombinant inbred lines, analysis of the F₂ diallel crosses was recommended for determining the heritability of both characters in early segregating generations. The results also suggest that a form of tandem selection may be effective in developing locally adapted germplasm which combines high grain yield with yellow rust resistance. Received: 15 February 1999 / Accepted: 11 March 1999     

Identification of candidate genes for fusarium yellows resistance in Chinese cabbage by differential expression analysis

Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans is an important disease of Brassica worldwide. To identify a resistance (R) gene against Fusarium yellows in Chinese cabbage (Brassica rapa var. pekinensis), we analyzed differential expression at the whole genome level between resistant and susceptible inbred lines using RNA sequencing. Four hundred and eighteen genes were significantly differentially expressed, and these were enriched for genes involved in response to stress or stimulus. Seven dominant DNA markers at putative R-genes were identified. Presence and absence of the sequence of the putative R-genes, Bra012688 and Bra012689, correlated with the resistance of six inbred lines and susceptibility of four inbred lines, respectively. In F₂ populations derived from crosses between resistant and susceptible inbred lines, presence of Bra012688 and Bra012689 cosegregated with resistance, suggesting that Bra012688 and Bra012689 are good candidates for fusarium yellows resistance in Chinese cabbage.     

抗根肿病大白菜的花药培养

大白菜是中国北方的重要蔬菜之一,根肿病是危害大白菜生产世界性病害,利用花药培养可以大大加速抗根肿病大白菜杂交育种工作的进程。对33份抗根肿病大白菜品种(品系)进行花药培养,有24个品种诱导出胚,品种诱导率为72.7%,以东方皇冠×C11的诱导率最高,为1.86胚/蕾。对大部分基因型来说,在培养基中添加0.4 g/L谷氨酰胺的诱导效果最好。研究还发现生长在温室中的供体植株更容易诱导出胚状体。变绿的子叶型胚转到B5+0.2 mg/L BA+0.1 mg/L NAA+0.1%活性炭的胚分化培养基上可诱导生芽。     

大白菜-结球甘蓝5号和8号单体异附加系的筛选和鉴定

为建立成套的大白菜-结球甘蓝异附加系,以大白菜-结球甘蓝异源三倍体（AAC,2n=3x=29）与二倍体大白菜（AA,2n=2x=20）回交世代BC1为材料,对其进行细胞学观察并从中筛选单体异附加系。结果表明,BC1群体植株发生了染色体数目变异,变异范围为2n=20～29,其中以2n=23～24的非整倍体植株居多,占总观察株数的51．44％;对2个2n=21的植株进行核型分析,初步鉴定为大白菜-结球甘蓝5号和8号单体异附加系,分别命名为CO-5—1和CO-8—2。     

Genetic analysis of organogenesis in the cotyledons of zygotic embryos of sunflower (Helianthus annuus L.)

Crosses were made between five cytoplasmic male-sterile and five restorer sunflower inbred lines. Twenty-five F₁ hybrids and their parents were studied for their organogenesis ability in a randomized block design with four replications. Each replication per genotype consisted of ten petri dishes with four expiants. Regeneration medium consisted of full MS medium modified by the addition of hormones and solidified with 6 g/l agar. Statistical analysis showed that both general and specific combining abilities were significant for all of the organogenesis parameters studied, and both showed several significant positive or negative values. General combining ability values were usually higher than those of specific combining ability, indicating the importance of additive genetic control for organogenesis parameters in sunflower. Narrow-sense heritability for the number of explants producing shoots and roots was 65.8%, which suggests that organogenesis of currently inferior inbred lines in sunflower should be improved in a crossing program.     

Quantitative genetic variation of metabolism in the nymphs of the sand cricket, Gryllus firmus, inferred from an analysis of inbred-lines

Compared with morphological and life history traits, quantitative genetic variation of metabolic and related traits in animals has been poorly studied. We used flow-through VCO(2) respirometry and simultaneous activity measurement on nymphs of the sand cricket (Gryllus firmus) from inbred lines to estimate broad-sense heritability of four metabolic variables. In addition, we measured a number of linear dimensions in the adults from the same inbred lines. There were significant multivariate effects of inbred lines for all traits and broad-sense heritability for physiological traits was 4.5%, 5.2%, 10.3% and 8.5% for average, resting, minimum and maximum CO(2) production in nymphs, respectively. Though the MANOVA indicated significant genetic variation among inbred lines in adult morphology, the broad-sense heritabilities were relatively low ranging from 0-18%. Our results indicate that the heritabilities of metabolic measures are large enough to potentially respond to selection.     

Genetic factors influencing regeneration ability in rye (Secale cereale L.). I. Immature inflorescences

Immature inflorescences of ten rye inbred lines (inbred degree S10 and S11) were cultured on solidified MS medium supplemented with 3.0 mg/dm³ of 2,4-D. According to their capability for callus production explants were classified into two groups : responsive (giving weak or intensive callus production) and non responsive (lack of callus formation). After transferring responsive material into hormone-free medium the regeneration of roots or shoots from the intensive growing callus was observed. Consistent differences between lines in the portion of explants with a certain response were found. They were divided into five groups reacting in the same way. Lines with different in-vitro response were crossed in an incomplete diallel. F₁, F₂ and F₃ generations were analyzed and the following conclusions drawn: the ability for plant regeneration from immature inflorescences in rye is determined by numerous loci, has a recessive character, and both callus production and regeneration suppression may be controlled by complementary genes.     

利用RNA干扰技术获得晚抽薹开花转基因大白菜

LEAFY基因在植物的成花转变中起重要作用。在拟南芥中,降低LEAFY基因的表达使开花延迟,并且增加分枝。利用RNAi技术降低大白菜LEAFY基因表达,获得晚抽薹开花的转基因大白菜。开花时转基因大白菜的株高明显低于对照,而叶片数、叶面积和侧枝数则高于对照;转基因植株的LEAFY基因表达明显降低,导致大白菜抽薹开花延迟。因此可以通过降低LEAFY基因表达来延迟需低温春化作用长日植物开花。也为利用生物技术获得晚抽薹转基因大白菜提供了理论依据。     

Genetic analysis of susceptibility to isoniazid-induced seizures in mice

Four sets of recombinant inbred lines of mice have been used to analyze genetic differences in acute toxicity of the drug, isonicotinic acid hydrazide. Standard inbred strains, their F₁ hybrids and recombinant inbred strains were all challenged with a single dose of the drug. The percent mortality of the different groups was analyzed to estimate heritability and the number of genes affecting resistance. The data indicated that resistance factors were dominant, heritability was moderate (.25-.37), and more than one gene was involved in each of four different sets of recombinant inbred lines. Possible approaches for identifying and mapping individual genes affecting resistance are discussed.     

Genome-wide identification of flowering time genes associated with vernalization and the regulatory flowering networks in Chinese cabbage

Flowering time (Ft) is the most important characteristic of Chinese cabbage with high leaf yields and late-flowering are favorable traits, while little knowledge on genes involved in Ft and the flowering mechanism in this crop. In this study, we conducted genome-wide RNA-seq analysis using an inbred Chinese cabbage ‘4004’ line in response to vernalization and compared the Ft gene expression with radish crop. A number of Ft genes which play roles in flowering pathways were performed quantitative RT-PCR analysis to verify the regulatory flowering gene network in Chinese cabbage. We found that a total of 223 Ft genes in Chinese cabbage, and 50 of these genes responded to vernalization. The majority of flowering enhancers were upregulated, whereas most flowering repressors were downregulated in response to vernalization as confirmed by RT-qPCR. Among the major Ft genes, the expression of BrCOL1-2, BrFT1/2, BrSOC1/2/3, BrFLC1/2/3/5, and BrMAF was strongly affected by vernalization. In reference to comparative RNA-seq profiling of Ft genes, Chinese cabbage and radish revealed substantially different vernalization response in particular GA flowering pathway. Thus, this study provides new insight into functional divergence in flowering pathways and the regulatory mechanisms in Brassicaceae crops. Further analysis of the major integrator genes between early and late-flowering inbred lines facilitates understanding flowering trait variation and molecular basis of flowering in Chinese cabbage.