Genetic linkage mapping and QTL identification for salinity tolerance in Indian mustard(Brassica juncea L.Czern and Coss.)using SSR markers

Soil salinity is one of the major environmental constraints that limits crop yield and nearly 7%of the total area worldwide is affected by salinity.Salinity-induced oxidative stress causes membrane damage during germination and seedling growth.Indian mustard is a major oilseed crop in India and its production and productivity are severely affected by salt stress.Breeding Brassica cultivars for salinity tolerance by conventional means is very difficult and time-consuming.Therefore,understanding the molecular components associated with salt tolerance is needed to facilitate breeding for salt tolerance in Brassica.In this investigation,quantitative trait loci(QTLs)associated with salt tolerance were identified using F_(2:3)mapping population developed from a cross between CS52(salinity tolerant)and RH30(salinity sensitive).Parents and F_(2:3)were evaluated under controlled and salinity stress conditions for 14 morpho-physiological traits for two consecutive generations(F2 and F_(2:3)),explaining proportion of the phenotypic variance under control condition.Simple sequence repeat(SSR)markers were used for mapping studies.A genetic linkage map based on 42 simple sequence repeats(SSRs)markers was constructed covering 2298.5 cM(Haldane)to identify the loci associated with salt tolerance in Brassica juncea.Forty-one SSRs showing polymorphism in the parents(CS52 and RH30)were mapped on 8 linkage groups(C1–C8).One marker(nga 129)did not map to any of the linkage group and was excluded from mapping.Linkage group 5(C5;317.9 cM)was longest and linkage group 1(C1,255.0 cM)was shortest.Further,we identified 15 QTLs controlling 8 traits using F_(2:3)population.These QTLs explained 12.44–60.63%of the phenotypic variation with a LOD score range of 3.62–5.97.Out of these QTLs,QMI4.1 related to membrane injury showed 51.28%phenotypic variance with a LOD score of 3.34.QTL QBYP8.1 related to biological yield per plant showed 60.63%phenotypic variance at a LOD score of 3.62.The highest LOD score of 5.97 was recorded for QTL related to seed yield per plant(QSYP4.1).Major QTLs were QTL for biological yield per plant(QBYP8.1),QTL for siliquae per plant(QSP4.1),QTL for primary branches(QPB4.1),QTLs for seed per siliqua(QSS4.1,QSS4.2),QTL for seed yield per plant(QSYP4.1),and QTL for membrane injury(QMI8.1)which showed more than 50%phenotypic variance.These QTLs identified in our study need to be confirmed in other populations as well so that these can be used in marker-assisted selection and breeding to enhance salt tolerance in Brassica juncea.

看护培养和子叶部位对儿菜(Brassica juncea Coss.var.gemmifera Lee et Lin)再生芽发生的影响

以‘种都1号儿菜’(BrassicajunceaCoss.var.gemmiferaLeeetLincv.Zhong-DouNo.1)无菌子叶为材料,对看护培养效果及子叶部位对再生芽发生的影响进行了研究。结果表明,子叶可以直接分化产生再生芽。在接种子叶的同时,接种已有芽点分化的子叶外植体,可以提早再生芽的发生,并能显著提高芽再生率,但对平均再生芽数无明显影响。带子叶柄的完整子叶,其芽再生率最高,并极显著高于不带子叶柄的子叶叶片,但平均再生芽数以不带子叶柄的完整子叶叶片最高;子叶柄培养未能获得再生芽。

铝对芥菜(Brassica juncea Coss)幼苗根系形态和叶内抗氧化系统的影响

研究了沙培条件下铝对两个芥菜品种‘雪里蕻’和‘花芥菜’幼苗根系形态和叶生理特性的影响。结果表明,1mg·L-1和10mg·L-1Al3+在短时间内能够促进两种芥菜根系生长,并降低花芥菜叶内MDA的含量。随着铝处理浓度的增加和处理时间的延长,芥菜根系生长受到抑制,叶内MDA含量增加,同时POD和CAT活性升高,但不同品种芥菜叶内AsA、GSH含量和SOD活性对铝胁迫的反应不同。研究结果说明低浓度铝在短时期内对芥菜生长有促进作用,但处理时间的延长会抑制芥菜的生长,在铝胁迫下芥菜体内抗氧化酶活性升高可以提高其铝适应性。

Assembly and marker analysis of mitochondrial genomes provide insights into origin,evolution and spread of Brassica juncea(L.)Czern.et Coss.

The release of mitochondrial genome sequences provides the basis for characterizing interspecific and intraspecific variation in Brassica mitochondrial genomes.However,few B.juncea(mustard)mitochondrial genomes have been published.We assembled the mitochondrial genomes of three B.juncea subspecies and compared them with previously published genomes.The genomes were phylogenetically classified into A,B,C,and Bna clades.Two variant sites,a transversion(C→A)at nt 79,573 and a 31-bp copy-number variation between nts 65,564 and 65,596,were identified.Based on these variant sites,mitotype-specific sequence markers were developed to characterize the variation among worldwide 558 B.juncea accessions.Three mitochondrial genome types(mitotypes MT1–MT3)were identified.In terms of geographical distribution,MT1 and MT2 accessions were distributed mainly to the north and MT3 to the south of 34°N.Root mustards carried only MT1,leaf and stem mustards carried mainly MT3,and seed mustards carried all three mitotypes,implying that the mitotypes underwent selection during B.juncea domestication.A new form of oil mustard evolved by hybridization between two gene pools in southwest China.

重金属污染土壤的植物修复研究 Ⅲ.金属富集植物Brassica juncea对锌镉的吸收和积累

采用温室盆栽试验研究了印度芥菜对土壤中锌镉污染的忍耐、积累能力 ,以检验这种植物修复Zn、Cd污染土壤的可能性及其潜力。在加入Zn 5 0 0和 1 0 0 0mgkg- 1 的土壤中 ,印度芥菜生长 66天后 ,叶片中积累Zn的平均浓度分别达 2 80和 662mgkg- 1 ,地上部带走的Zn分别为每盆 2 1 95和 341 2 μg。在加入Cd 2 0 0mgkg- 1 的土壤中生长的印度芥菜 ,叶片中积累Cd浓度为 1 61mgkg- 1 ,地上部带走的Cd为每盆 381 μg。和普通植物相比 ,印度芥菜更能将Zn和Cd从根运输到地上部。Zn 5 0 0mgkg- 1 处理的土壤在种植印度芥菜后其NH4NO3提取的Zn显著高于不种植物的处理 ;土壤添加Cd 2 0 0mgkg- 1 的处理NH4NO3提取的Cd也显著高于不种植物的处理 ,可能的原因是植物根分泌出特殊的分泌物 ,专一性地螯合溶解根系附近的难溶态Zn和Cd,从而提高土壤溶液中的浓度。印度芥菜对Zn、Cd有较强的忍耐和富集能力 ,是Zn、Cd污染土壤修复有潜力的植物。

三种类型油菜(Brassica spp.)和野芥菜(B.juncea var.gracilis Tsen et Lee)杂交亲和性及F_1的适合度——潜在基因转移的研究

通过人工去雄授粉 ,采用荧光显微镜观察了 3种类型栽培油菜花粉在两地采集的野芥菜柱头上的萌发生长情况 ,结合杂交后的结实率 ,探讨了 3种类型油菜和野芥菜杂交的亲和性 .结果表明 ,甘蓝型油菜和芥菜型油菜与野芥菜的亲和性都非常高 ,亲和性指数达 10 .0以上 ,而白菜型油菜和野芥菜的亲和性较低 ,亲和指数小于 0 .2 .子一代的适合度研究结果表明 ,芥菜型油菜向野芥菜杂交一代的适合度没有降低 ,而甘蓝型及白菜型和野芥菜杂交一代的适合度明显下降 ,表现在花粉活力降低 ,结实率极低 .上述结果表明 ,白菜型油菜和野芥菜的基因转移可能性最小 ,甘蓝型居中 ,而芥菜型极易向野芥菜发生基因转移 .表 4参

芥菜型油菜(Brassica Juncea)杂种优势利用的研究

1973年,本院发现芥菜型油菜雄性不育株后,经连续几年研究,1975年育成了欧新 A 不育系,欧新 B 保持系和74243—6恢复系,实现了芥菜型“三系”配套。多年来连续测定表明,育成的芥菜型“三系”表现育性稳定,欧新 A 不育系的不育株率达100%,单株自交结实指数平均在0.000—0.103之间。恢复株率为99.54～100%,F_1杂种有较强的苗期优势,也有种子产量和含油量优势。比芥菜型地方良种昆明高棵增产19.2～34.8%,含油量提高6.58～8.23%。芥菜型“三系”的育成,填补了我国芥菜型杂优利用的空白,势将推动芥菜型油菜杂优利用的研究。

芥菜(Brassica juncea L.)小孢子胚发生和植株再生

对芥菜(Brassica juncea L.)的7种基因型进行游离小孢子培养试验,从供体母株取2～4mm长的花蕾,分离小孢子并将其置于NLN-82液体培养基中薄层培养,先在33℃,后移到25℃继续暗培养,3周后统计形成的球形期至子叶期胚.将成熟的小孢子胚转移到无激素MS琼脂培养基直接或经二次分化得到小孢子植株.7种基因型均观察到细胞分裂,6种基因型获得了小孢子胚,3种基因型得到了再生植株.各基因型小孢子胚胎发生频率差异很大;产量最高的“成都大头菜”,平均每蕾小孢子胚产量为10.0个.

Effects of Bacillus amyloliquefaciens Biofertilizer on Brassica juncea var. multiceps Growth and N_2O Emission from Soil

In this study, Bacil us amyloliquefaciens A3 was continual y incubated in shake fIasks contalning wastewater from sweet potato starch production as an ef-fective biofertiIizer for cuItivation of Brassica juncea var. multiceps（XueIihong）. Based on pot experiments in the greenhouse, the effects of chemical fertiIizers （CN）, biofertiIizer （BF）, inactivated broth （BI）, starch wastewater （SW） and the combination of biofertiIizer and chemical fertiIizer （BC） on the yield, NO3- content and NO2- con-tent of XueIihong, soiI physicochemical properties and N2O emission were investi-gated. The resuIts showed that the yield of XueIihong in BC and CN treatments was improved by five times compared with CK; BF and SW treatments had insignifi-cant impact on the yield of XueIihong. Compared with CN treatment, BCL treatment exhibited simiIar improving effects on the yield of XueIihong, in which NO3- content of XueIihong and soiI was reduced by 16.4%-73.6% and 22%-29%, which reduced the risk of nitrogen eIuviations in soiI; average N2O fIux （FPV30） in BCL treatment was reduced by 58.3%-73.1% compared with CN treatment. In concIusion, B. amy-loliquefaciens is a feasibIe Iow-cost biofertiIizer for sustalnabIe vegetabIe farming with a great potential for starch wastewater utiIization.

芥菜型油菜（Brassica juncea）游离细胞培养及体细胞无性系的建立

以芥菜型油菜子叶和下胚轴来源的愈伤组织为材料，进行悬浮细胞振荡培养，通过诱导愈伤组织及分化试验表明：用乳白色且结构紧密的愈伤组织进行悬浮振荡培养能获得较正常的单细胞；在悬浮振荡培养的第６～７天内及时过滤并将单细胞转入浅层液体分正培养，可高频率产生意伤组织；愈伤组织的质量对芽的分化有重要影响，不同基因型来源的愈伤组织其植株再生频率有很大差异。通过试验在云南芥菜型油菜中发现了一批易再生材料并建立了体细胞无性系。

体积排阻高效液相色谱-电感耦合等离子体质谱法同时测定印度芥菜(Brassica Juncea)中镉铜锌形态

建立了体积排阻高效液相色谱-电感耦合等离子体质谱法同时测定印度芥菜中镉、铜、锌形态分析方法。为了防止巯基化合物氧化反应的发生,样品采取液氮保护并在-70℃保存,样品分析全流程采取氮吹防氧化措施。在镉、铜、锌胁迫下,诱导产生植物螯合肽(PCs)。在叶片和根部均检测到3种元素的4种形态,植物螯合肽(PC)3-Cd(Cu,Zn)、植物螯合肽(PC)2-Cd(Cu,Zn)、谷胱甘肽(GSH)-Cd(Cu,Zn)、半胱胺酸(Cys)-Cd(Cu,Zn)及其在植物体内的分布规律。结合植物不同部位Cd、Cu、Zn分布规律初步推断,Cd、Cu、Zn在与GSH及Cys的结合上存在竞争。

Brassica juncea×Brassica barrelieri F_1杂种的获得及其亲本染色体的同源性研究

采取胚胎挽救技术,培养103个子房获得4个Brassica juncea×Brassica barrelieri F_1杂种植株。杂种形态介于两亲本之间,花期长,分枝多,花粉高度不育,减数分裂表现异常。F_1花粉母细胞的减数分裂中期(MI),来自两亲本物种的染色体按染色体组彼此分开,集中分布成10/18状,呈现罕见的染色体组界沟。10与18条染色体之间最多见3个二价体联会。并观察到染色体组内的次级联会现象。根据染色体组间和组内联会的特点,作者认为两个亲本物种的染色体组构型可以表示为aabbccdefg(B.barrelieri)和bcdhhhiiijjkklmnop(B.juncea)。研究表明,(1)两个亲本物种在形态和染色体的同源性等方面具有较大差异,B.barrelieri的分类地位值得进一步商榷。(2)两个亲本存在一定的遗传同源性并具有遗传物质交流的可能性。(3)第一次减数分裂中期染色体组隔沟的出现以及染色体有规律的分布显示两个物种间的基因重组主要发生在部分同源的染色体之间,无同源性的染色体之间的重组将十分困难。

Canonical Correlation Analysis of Agronomic Characters of Brassica juncea in Western China

[Objective] The study aimed at exploring the relationship among the agronomic characters of B. juncea in western China, in order to provide scientific basis for the breeding of B. juncea in western China. [Method] 39 B. juncea materials from western China were used for the canonical correlation analysis, and canonical correlations between each pair of the four ecological character （containing 18 variables） were verified, including yield characters （5 variables）, caulis characters （6 variables）, branch characters （3 variables） and pod characters （3 variables）. [Result] Yield per plant of B. juncea in western China suffered a tremendous influence from effective pod number per plant while was not significantly affected by the total pod number per plant, seed number per pod and 1 000-seed weight; the most important character related with the yield character of B. juncea in western China was caulis character, followed by the branch character and pod character; yield characters, caulis characters, branch characters and pod characters of B. juncea in western China were closely correlated. [Conclusion] In order to improve the yield characters of B. juncea in western China, caulis characters should be focused on, followed by branch characters and pod characters; rapeseed varieties with high performance in total pod number per plant and effective pod number per plant should be chosen through the perspectives of effective branch number, plant height, pod number of main inflorescence, fruit stalk number of main inflorescence and other traits, while rapeseed varieties with high performance in seed number per pod and 1 000-seed weight should be chosen through the perspectives of beak length and other traits.

Expression of Nitrilases in Brassica juncea var. tumida Tsen in Root Galls Caused by Plasmodiophora brassicae

Five commonly-used reference genes： ACT （actin）, UBE （ubiquitin-conjugating enzyme）, RPL2 （ribosomal protein L2）, BRP II （RNA polymerase II subunit）, and NADH （nicotinamide adenine dinucleotide） were examined using geNorm software as reference genes for RT-qPCR. Among the tested reference genes, ACT and UBE were the most stable in all samples. In parallel, expression analysis of nitrilases in Brassica juncea var. tumida, was performed to preliminarily investigate the molecular interactions between nitrilase and clubroot development at 10, 15, 20, 25, 30, and 40 d postinoculation （dpi） with a suspension of resting spores of Plasmodiophora brassicae. The results showed that different gene expressions of nitrilases were regulated during the initial periods of clubroot development. The expression level of BjNIT1 increased sharply from 20 to 40 dpi in infected roots while there were no remarkable changes in healthy roots. From 15 to 30 dpi, the expression levels of BjNIT2 and BjNIT4 in infected roots were lower than those in non-infected roots. Finally, BjNIT2 in treatment was down approximately to control at 40 dpi. Our results suggest that BjNIT1, which promoted overproductions of auxin, might be involved in P. brassicae infection of B. juncea.

KCR、HCD基因在高芥酸芥菜型油菜(Brassica Juncea)生育期内的量化表达

研究应用常规RT-PCR(Reverse transcription polymerase chain reaction)的技术和QuantumRNA18STM内标对芥菜型高芥酸油菜生育期内芥酸合成基因KCR、HCD基因的表达进行量化分析,了解芥酸的合成与功能基因量化表达的关系,为芥菜型油菜的高芥酸品种的选育提供理论依据。实验表明,KCR、HCD基因在高芥酸芥菜型油菜整个生育期中均有表达,即不但在种子发育期表达,在全生育期内叶中也表达,并且显示芥酸合成基因表达量的变化与油菜的发育时期相关。同时也表明具有内标的常规RT-PCR技术可实现对芥酸合成基因的量化表达分析。

Isolation and functional analysis of a Brassica juncea gene encoding a com-ponent of auxin efflux carrier

Polar auxin transport plays a divergent role in plant growth and developmental processes including root and embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsis pin gene family was believed to encode a component of auxin efflux carrier (G(?)lweiler et al, 1998). Based on the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpin1), which encoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level with AtPINl and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homology to AtPIN3). Hydrophobic analysis showed similar structures between BjPINl and AtPIN proteins. Presence of 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp) in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blot analysis indicated that Bjpin1 was expressed in most of the tissues tested, with a relatively higher level of transcript in flowers and a lower level in root tissues. Promoter-reporter gene fusion studies further revealed the expression of Bjpin1 in the mature pollen grains, young seeds, root tip, leaf vascular tissue and trace bundle, stem epidermis, cortex and vascular cells. BjPINl was localized on the plasma membrane as demonstrated through fusion expression of green fluorescent protein (GFP). Auxin efflux carrier activity was elevated in transgenic Arabidopsis expressing BjPIN1.

A Pin gene families encoding components of auxin efflux carriers in Brassica juncea

Based on the sequence information of Arabidopsis PIN1, two cDNAs encoding PIN homologues fromBrassica juncea, Bjpin2 and Bjpin3, were isolated through cDNA library screening. Bjpin2 and Bjpin3encoded proteins containing 640 and 635 amino acid residues, respectively, which shared 97.5% identities witheach other and were highly homologous to Arabidopsis PIN1, PIN2 and other putative PIN proteins. BjPIN2and BjPIN3 had similar structures as AtPIN proteins. Northern blot analysis indicated that Bjpin2 wasexpressed in stem, leaf and floral tissues, while Bjpin3 was expressed predominantly in stem and hypocotyls.Two promoter fragments of pin genes, Bjpin-X and Bjpin-Z, were isolated by 'genome walking' techniqueusing primers at 5'-end of pin cDNA. Promoter-gus fusion studies revealed the GUS activities driven byBjpin-X were at internal side of xylem and petal; while those driven by Bjpin-Z were detected at leaf vein,epidermal cell and cortex of stem, vascular tissues and anther. Results of the pin genes with differentexpression patterns in B. juncea suggested the presence of a gene family.

Identification of MAM1s in Regulation of 3C Glucosinolates Accumulation in Allopolyploid Brassica juncea

Allopolyploid Brassica juncea is particularly enriched in sinigrin,a kind of 3C aliphatic glucosinolates(GSLs),giving rise to characteristic taste after picking.However,the molecular mechanism underlying 3C aliphatic GSLs biosynthesis in this species remains unknown.In this study,we genome-widely identified GSLs metabolic genes,indicating different evolutionary rate of GSLs metabolic genes between subgenomes of B.juncea.Eight methythioalkylmalate synthase(MAMs)homologs were identified from B.juncea,in which six MAM1s were located in chloroplast and the other two were not detected with any expression.Furthermore,BjMAM1-4,BjMAM1-5,and BjMAM1-6 displayed higher expression levels in leaves than other tissues.Silenced expression analysis revealed that BjMAM1-4 and BjMAM1-6 function in 3C and 4C aliphatic GSLs accumulation.The specificity of the substrate selection for the second cycle reaction is much lower than that of the first cycle,suggesting these genes may preferentially catalyze 3C aliphatic GSLs biosynthesis.Our study provides insights into the molecular mechanism underlying the accumulation of 3C aliphatic GSLs,thereby facilitating the manipulation of aliphatic GSLs content in B.juncea.

Effects of Barium Stress in Brassica juncea and Cakile maritima:The Indicator Role of Some Antioxidant Enzymes and Secondary Metabolites

Soil contamination by toxic trace metal elements,like barium(Ba),may stimulate various undesirable changes in the metabolic activity of plants.The plant responses are fast and with,direct or indirect,generation of reactive oxygen species(ROS).To cope with the stress imposed by the ROS production,plants developed a dual cellular system composed of enzymatic and non-enzymatic players that convert ROS,and their by-products,into stable nontoxic molecules.To assess the Ba stress response of two Brassicaceae species(Brassica juncea,a glycophyte,and Cakile maritime,a halophyte),plants were exposure to different Ba concentrations(0,100,200,300 and 500µM).The plants response was evaluated through their morphology and development,the determination of plant leaves antioxidant enzymatic activities and by the production of plants secondary metabolites.Results indicated that the two Brassicaceae species have the ability to survive in an environment containing Ba(even at 500µM).The biomass production of C.maritima was slightly affected whereas an increase in biomass B.juncea was noticed.The stress imposed by Ba activated the antioxidant defense system in the two species,noticed by the changes in the leaves activity of catalase(CAT),ascorbate peroxidase(APX)and guaicol peroxidase(GPX),and of the secondary metabolites,through the production of total phenols and flavonoids.The enzymatic response was not similar within the two plant species:CAT and APX seem to have a more important role against the oxidative stress in C.maritima while in B.juncea is GPX.Overall,total phenols and flavonoids production was more significant in the plants aerial part than in the roots,of the both species.Although the two Brassicaceae species response was different,in both plants catalytic and non-catalytic transformation of ROS occurs,and both were able to overcome the Ba toxicity and prevent the cell damage.

Karyotyping and identifying all of the chromosomes of allopolyploid Brassica juncea using multicolor FISH

Chromosome identification and karyotype using fluorescence in situ hybridization(FISH)provides a technical platform for genome and cytogenetic studies. Brassica juncea(brown mustard, 2n = 4× = 36; genome AABB) is an allopolyploid species that originated from a spontaneous hybridization of Brassica rapa and Brassica nigra and contains many valuable traits. In this study, a multicolor FISH procedure allowing the identification of all 18 chromosomal pairs was developed by two-step hybridizations with probes on the same metaphase chromosomes. The distribution patterns and chromosomal localizations of six repeat sequences(satellite repeat p Br STR, 5S r DNA, 45 S r DNA, B genome-specific repeat p BNBH35, and centromeric satellite repeats Cent Br1 and Cent Br2) on B. juncea chromosomes were characterized.Comparative karyotype analyses showed that the genome is relatively stable in comparison with its diploid progenitor species and revealed intraspecific karyotypic diversity among three accessions of B. juncea. This study provides valuable information about the genome evolution of B. juncea and a toolkit that will be helpful for chromosome identification.