Molecular Mapping and Character Tagging in Brassica juncea—I. Degree,Nature and Linkage Relationship of RFLPs and Their Association With Quantitative Traits

For molecular mapping and character tagging in Indian mustard (Brassica juncea), cultivar Varuna and exotic collection BEC 144 were chosen on the basis of morphological and molecular differences. High degree of RFLP was detected between them using genomic DNA clones of Pst! subgenomic library as probes. Of the 48 probe — enzyme combinations, 89.5% were polymorphic. Majority of the probes revealed duplicate loci and a high frequency of null alleles (71.4%). Segregation analysis in the F² population revealed significant deviation from expected 1:2:1 ratio for 32% of the markers. Using the computer package MAPMAKER, 15 markers could be placed in six linkage groups which covered a total length of 173.9 eM. Based on single factor analysis of variance, three significant marker-quantitative trait associations viz. BJG 59a primary branches/plant, BJG 42Gb - secondary branches/plant and BJG 433-days to f1owering could be identified. The BEC 144 alleles at the quantitative trait loci (QTL) in the marked genomic regions enhanced the trait expression. The putative gene action at these loci was found to be non-additive.     

Ethylene Production during Development of Mustard (Brassica juncea) and Canola (Brassica napus) Seed

An open, continuous flow system was used to investigate ethylene production during degreening of maturing seed of mustard (Brassica juncea cv Cutlass and cv Lethbridge 22A) and canola (Brassica napus cv Westar and cv Alto). Isolated mustard seed evolved higher amounts of ethylene than those of canola, and this was particularly evident both early in embryogeny and later during the desiccation phase of seed maturation. The silique walls produced negligible amounts of ethylene in both species. The concentrations of ethylene surrounding seed as they matured within siliques were significantly higher in mustard than in canola, and this interspecies difference was greatest during the seed desiccation phase. In mustard, a 4-fold increase in silique internal ethylene levels was apparent during desiccation. In comparison, only a moderate increase in silique-derived ethylene occurred in canola.     

Evaluation of Microsatellite Markers for Genome Mapping in Indian Mustard (Brassica juncea L)

Microsatellites are robust markers for genome mapping, gene tagging and marker assisted selection. The genus Brassica, having a large and complex genome, requires such type of markers for various applications in genetics and breeding. A set of 202 microsatellite markers were used to screen two parental genotypes of Indian mustard (Brassica juncea) namely, ‘Varuna’, an indigenous cultivar and BEC144, an exotic collection from Poland, of which 36 (17.8%) were informative and usable for segregation analysis. The polymorphic markers detected heterozygosity in advanced generation recombinant inbred lines (RILs) developed earlier from the cross Varuna × BEC144 with a varying frequency that ranged from 0% to 23.5%. Normal Mendelian segregation for majority of microsatellite markers was observed. Eleven markers showed significant deviation from the expected 1:1 segregation ratio. Twelve markers were assigned to six different linkage groups of Indian mustard genome map. The level of polymorphism between the parents and the percentage of useful informative markers as observed in this study, suggested that many more markers are needed to achieve a reasonable coverage of mustard genome. This is the first report on the evaluation of microsatellite markers for genome mapping in B. juncea.     

Molecular Mapping of Loci Affecting the Contents of Three Major Fatty Acids in Indian Mustard (Brassica juncea L)

The fatty acid constituents of mustard oil are palmitic, stearic, oleic, linoleic, linolenic and erucic acids. With the objective of mapping loci influencing the content of these fatty acids, a population of F₆ generation recombinant inbred lines (RILs) derived from an inter-varietal cross of mustard was analyzed. Transgressive variation was evident for all the six fatty acids analysed irrespective of the levels of differences between the parents. The frequency distribution was normal for the linolenic acid, linoleic acid and stearic acid contents, while deviation from normality was observed for the other three fatty acids. The content of erucic acid was negatively correlated with the contents of all other fatty acids, which were positively correlated. Based on single marker analysis and interval mapping, two loci each for linoleic, linolenic and erucic acids were mapped to marker intervals on three linkage groups. Position of log of odds ratio (LOD) peaks suggested presence of common, linked and independently segregating loci for the fatty acid contents. The percentage of phenotypic variance explained by individual quantitative trait loci (QTLs) ranged from 10.5 to 19.5%, whereas the cumulative action of loci detected for different traits accounted for 16.3 to 27.6% of the variance. The additive effect for an individual locus ranged from 1.09 to 4.33. Presence of the favourable alleles at both the contributing loci in most of the RILs with a high linolenic acid content and of the unfavourable alleles in the lines with a low linolenic acid content indicated the possibility of pyramiding useful genes from phenotypically similar parental lines.     

Molecular markers for seed colour in Brassica juncea.

A detailed RFLP map was used to map QTLs associated with seed colour in Brassica juncea using a doubled-haploid population derived from a cross between a black/brown-seeded cultivar and a yellow-seeded breeding line. Segregation analysis suggested that seed colour was under control of 2 unlinked loci with duplicate gene action. However, QTL analysis revealed 3 QTLs, SC-B4, SC-A10 and SC-A6, affecting seed colour. The QTLs were consistent across environments, and individually explained 43%, 31%, and 16%, respectively, and collectively 62% of the phenotypic variation in the population. Digenic interaction analysis showed that closest flanking locus of QTL SC-B4, wg7b6cNM, had strong epistasis with the locus wg5a1a, which is tightly linked to QTL SC-A6. The interaction of these 2 loci explained 27% of the phenotypic variation in the population, while the whole model explained 84%. In a multiple regression model, the effects of QTL SC-A10, as well as its interaction with other loci, were non-significant, whereas the effects of loci wg7b6cNM and wg5a1a and their interaction were significant. Ninety-eight percent of the DH lines carried the expected alleles of loci wg7b6cNM and wg5a1a for seed colour, confirming that only these 2 loci were linked to seed colour in B. juncea. Four additional digenic interactions significantly affected seed colour, and all 5 digenic interactions were consistent across environments.     

RFLP Mapping in Soybean: Association between Marker Loci and Variation in Quantitative Traits

Quantitative trait loci (QTLs) have been mapped to small intervals along the chromosomes of tomato (Lycopersicon esculentum), by a method we call substitution mapping. The size of the interval to which a QTL can be mapped is determined primarily by the number and spacing of previously mapped genetic markers in the region surrounding the QTL. We demonstrate the method using tomato genotypes carrying chromosomal segments from Lycopersicon chmielewskii, a wild relative of tomato with high soluble solids concentration but small fruit and low yield. Different L. chmielewskii chromosomal segments carrying a common restriction fragment length polymorphism were identified, and their regions of overlap determined using all available genetic markers. The effect of these chromosomal segments on soluble solids concentration, fruit mass, yield, and pH, was determined in the field. Many overlapping chromosomal segments had very different phenotypic effects, indicating QTLs affecting the phenotype(s) to lie in intervals of as little as 3 cM by which the segments differed. Some associations between different traits were attributed to close linkage between two or more QTLs, rather than pleiotropic effects of a single QTL: in such cases, recombination should separate desirable QTLs from genes with undesirable effects. The prominence of such trait associations in wide crosses appears partly due to infrequent reciprocal recombination between heterozygous chromosomal segments flanked by homozygous regions. Substitution mapping is particularly applicable to gene introgression from wild to domestic species, and generally useful in narrowing the gap between linkage mapping and physical mapping of QTLs.     

芥菜类作物的遗传多样性

芥菜类是我国重要的经济作物,遗传资源十分丰富。现就目前国内外芥菜类作物遗传多样性研究的现状进行综述,并着重阐述了芥菜类作物的分类、分布以及遗传多样性研究方法,如形态学分析法、同工酶分析法、RAPD法和RFLP法。同时对芥菜类作物遗传多样性研究存在的问题和今后的研究方向进行了探讨。     

Callus Formation and Morphogenesis of Cultured Mustard (Brassica juncea) Protoplasts

Protoplasts from mustard seedling cotyledons were suspended in Nitsch medium and cultured at 26℃ under low intensity illumination. When colonies were observed in the cultures, fresh medium (3% sucrose replaced mannitol) was added by equalvolume. When protoplasts developed into small callus the cultures were transfered to modified MS agar medium, in which some of them produced roots while an others gave rise to green spots. Shoot-differentiation experiment is in progress.     

应用因子分析法研究茎瘤芥(榨菜)性状间的关系

应用因子分析法可把23份茎瘤芥品种资源的15个数量性状集约在5个主因子上,采用正交因子和斜交因子模型分析了性状间的遗传作用关系,并探讨了各因子间的关系及其生物学意义。     

提高榨菜离体培养植株再生频率

采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg／L6．BA 0．2mg／L2,4-D的组合较为适宜,其不定芽再生频率可达50％,且外植体以下胚轴为好：而CPPU和2,4-D的适宜组合为1．5mg／L 0．2mg／L,其不定芽再生频率高达66．67％,最适外植体为带柄子叶。同时,研究结果显示,添加0．25～1mg／L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。     

Transport and Partitioning of Lead in Indian Mustard (Brassica juncea) and Wheat (Triticum aestivum)

Lead (Pb) contamination in soils is a serious concern because it can be taken up by crops and then transferred through the food chain, posing a potential risk to human health. Indian mustard (Brassica juncea) and wheat (Triticum aestivum) are important crop species known to accumulate heavy metals in their tissues. This study aimed at understanding the transport and accumulation of Pb in these two species and the risk associated with consumption of these foods, which would help us in mitigating accumulation of Pb in edible tissues. The plants were grown at different Pb concentrations for the entire life cycle, and the partitioning of the metal to different tissues was examined. The results showed that plant species differ widely in their ability to transport and accumulate Pb in different tissues. In B. juncea, there was significant accumulation of Pb in both siliques and seeds, whereas most of the Pb in wheat was concentrated in the vegetative tissues and less to the flag leaf and reproductive tissues. In both species, although seed Pb concentrations exceeded acceptable limits, dietary intake did not exceed acceptable limits in most treatments, indicating that more studies on Pb transport and redistribution in crop species is necessary.     

叶用芥菜细胞质雄性不育相关基因orf 220的分子特性

采用榨菜细胞质雄性系为不育源,通过变种间杂交和回交的方法,转育叶用芥菜细胞质雄性不育种质。根据Brassica nap型细胞质雄性不育相关基因off222设计兼并引物,利用特异引物PCR方法,分别在不育源、F1、BC2以及BC3世代中扩增出一条600～700bp大小的特异条带,而在叶用芥菜保持系中无此条带,具细胞质基因遗传特性。进一步序列分析表明,此特异条带大小为663bp,具有起始密码子和终止密码子,编码220个氨基酸,定名为off220。同时,off220推导的氨基酸序列存在两个跨膜区,氨基端与Oenothera berteriana中的COXⅢ(线粒体细胞色素氧化酶复合体亚基)、萝卜中的ATP8(线粒体ATP酶复合体亚基)以及向日葵中的ORFB(ATP酶复合体α亚基相关)蛋白氨基端高度同源。RT-PCR方法分析表明,off220基因的表达为组成性表达,无器官特异性。     

Association of RFLP markers and biomass heterosis in trigenomic hybrids of oilseed rape ( Brassica napus x B. campestris)

Laboratory screening with DNA-based markers and field measurements of biomass production were carried out on each of the 120 trigenomic hybrids, obtained by interspecific hybridization between Brassica napus (AACC) and Brassica campestris (A'A'). The objective of this study was to elucidate the relationship between molecular markers and biomass heterosis of the interspecific hybrid between B. napus and B. campestris, which has been explored practically in rapeseed production for many years. The experiment was first carried out on 65 trigenomic hybrids in 1999. The average over-mid-parent heterosis of biomass production was around 30%, and the highest value was 175.4%. In the following year, the observation was expanded to 120 trigenomic hybrids and the best average over-mid-parent heterosis was 93%. A total of 1,477 DNA fragments, generated by Southern hybridization with 50 Brassica cDNA clones and 25 Arabidopsis EST clones, was scored across their parental lines. One hundred and twenty six and 215 fragments were identified as significantly associated with biomass production respectively in the 2 successive years. Using these active markers, a statistical model to resolve the heterosis is proposed and a new way to make use of the subgenomic heterosis is also discussed.     

聚乙二醇（PEG）浸种对茎瘤芥老化种子活力及其幼苗生长的影响

茎瘤芥品种‘永安小叶’老化种子经聚乙二醇（PEG）浸种后,其种子活力和幼苗生长与PEG浓度有一定的相关性,不同浓度PEG浸种后的茎瘤芥种子膜透性均降低,种子发芽率、发芽势、发芽指数和活力指数增大,幼苗根干重、茎干重和根长也均增大,幼苗叶片中丙二醛（MDA）含量降低。幼苗生长与种子活力的变化趋势一致,PEG浓度小于30%时呈增大趋势,大于30%时呈减小趋势,但均大于不做PEG处理的;幼苗叶片中MDA含量与种子膜透性变化趋势一致,PEG浓度小于25%时,呈降低趋势,大于25%时则呈升高趋势,但这些指标均低于不做PEG处理的。     

Seed Coat Microsculpturing Is Related to Genomic Components in Wild Brassica juncea and Sinapis arvensis

It has been reported that wild Brassica and related species are widely distributed across Xinjiang, China, and there has been an argument for species identification. Seed coat microsculpturing (SCM) is known to be an excellent character for taxonomic and evolutionary studies. By identifying collections from Xinjiang, China, and combining SCM pattern, flow cytometry, and genome-specific DNA markers as well as sexual compatibility with known species, this study aimed to detect potential relationships between SCM and genomic types in wild Brassica and related species. Three wild collections were found to be tetraploid with a SCM reticulate pattern similar to B. juncea, and containing A and B genome-specific loci, indicating relatively high sexual compatibility with B. juncea. The others were diploid, carrying S-genome-specific DNA markers, and having relatively high sexual compatibility with Sinapis arvensis. Moreover, their SCM was in a rugose pattern similar to that of S. arvensis. It was suggested that SCM, as a morphological characteristic, can reflect genomic type, and be used to distinguish B-genome species such as B. juncea from the related S. arvensis. The relationship between SCM and genomic type can support taxonomic studies of the wild Brassica species and related species.     

Two Homologs of the FLOWERING LOCUS C Gene from Leaf Mustard (Brassica juncea)

Using the direct amplification of genomic DNA from two cultivars of leaf mustard (Brassica juncea), we obtained two homologs of the MADS-box gene FLOWERING LOCUS C(FLC), which regulates flowering time in arabidopsis. Nucleotide and deduced amino acid sequences of two cloned FLC fragments (from exon 2 to exon 7) were compared to the previously characterized FLC genes in arabidopsis and FLC homologs in other Brassicaceae species. The homolog AY266265 is an ortholog of the FLC3 gene from Brassica rapa (95% identity), whereas the function of the homolog AY268931 has not been established conclusively. The FLC gene and its homologs were used to compare the variability in the primary structures of exons and introns.     

茎瘤芥主要数量性状遗传力和遗传进度的初步研究

以23个茎瘤芥地方品种为试材估算了15个性状遗传力、遗传进度及遗传变异系数。结果表明各性状遗传力从大到小顺序为营养生长期、瘤茎膨大期、茎／叶、菜皮百分率、瘤茎产量、叶宽、瘤茎横径、出苗至瘤茎膨大始期、株鲜重、瘤茎纵径、叶长、菜形指数、开展度、瘤茎空心率、株高。瘤茎空心率、茎／叶、菜皮百分率和瘤茎产量的遗传变异较大,具有较大的选择潜力。在5％的选择率下,可获得较大的遗传进展。综合试验结果认为,瘤茎膨大期、营养生长期、茎／叶宜在早代进行一次性单株选择,菜皮百分率、瘤茎产量、叶宽等性状可适当加强早代选择。     

Detection and Mapping of Duplicate Loci in Brassica juncea

Restriction fragment length polymorphism (RFLP) analysis of Brassica juncea genome using 39 random homologous genomic DNA clones and chlorophyll a, b binding polypeptide (cab-3c) cDNA of tomato as probes revealed high degree of sequence duplication. The average number of hybridizing fragments per probe (8) was much higher than that earlier reported using cDNA probes in B. juncea. Null alleles observed for majority (56.2%) of the polymorphic duplicate loci suggested a significant role of insertion/deletion events in evolution of mustard genome. Distortion in segregation was evident in respect of only 9.6% of the segregating loci indicating that the mapping population used was relatively unbiased and thus can be used efficiently for genome mapping as well as for location of genes. Forty-nine polymorphic duplicate loci could be mapped to 15 linkage groups. Arrangement of these loci on different linkage groups revealed intra and inter-chromosomal duplications as well as duplication of chromosome blocks.Three of the eight cab loci could be mapped on three different linkage groups. Null allelic situation for seven of the cab loci suggested the role of DNA rearrangement in evolution of this multigene family in B. juncea.     

The EDTA Amendment in Phytoextraction of 134Cs From Soil by Indian Mustard (Brassica juncea)

Soil contamination with radiocaesium is a significant problem at any countries when a nuclear accident occurred. Recently, phytoextraction technique is developed to remediate the contaminated environment. However, the application is limited by the availability of the contaminant for root uptake. Therefore, a green house trial experiment of soil amendment with ethylene diamine tetraacetic acid (EDTA) has been conducted to examine ¹³⁴Cs availability for root uptake. Two groups of Indian mustard (Brassica juncea) were cultivated in ¹³⁴Cs contaminated soil. The soil in the first group was treated with EDTA amendment, while the other was not. Plant growth was observed gravimetrically and the ¹³⁴Cs concentration in soil as well as plants were determined using gamma spectrometry. The plant uptake capacity was determined as transfer factor (F_v), and the F_v values of 0.22 ± 0.0786 and 0.12 ± 0.039 were obtained for the soil treated with and without EDTA amendment, respectively. The phytoextraction efficiency of the plant cultivated in ¹³⁴Cs contaminated soil both with and without EDTA amendment was low. The EDTA amendment to the soil seems to enhance the ¹³⁴Cs availability for root uptake of Indian mustard and can still be considered to assist the field phytoremediation of contaminated soil.