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.

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.

Variation and Statistic Analysis of Agronomic Traits of Recombinant Inbred Lines of Brassica juncea L.

Correlation analysis and principal component analysis( PCA) were conducted for some agronomic traits of 139 recombinant inbred lines of Brassica juncea L. The results showed that under the environmental conditions in Guiyang,the flowering time,number of seeds per silique,plant height and thousand seed weight differed significantly among different lines. The results of principal component analysis showed that the lines could be classified into three groups. The results of Pearson correlation tests showed that the plant height was positively correlated with the flowering time( P < 0. 05),and the thousand seed weight was negatively correlated with the flowering time and number of seeds per silique( P < 0. 01).

The influence of Lactobacillus plantarum fermentation in selenium-enriched Brassica napus L.:changes in the nutritional constituents,bioactivities and bioaccessibility

Selenium(Se)-enriched Brassica napus L.is a valuable organic Se supplement.In this study,the fermentation broth enriched with organic Se(FFS)was prepared using Lactobacillus plantarum to ferment the substrate of Se-enriched Brassica napus L.Significant increases were observed after fermentation in total sugars,reducing sugars,soluble proteins,total phenolic content(TPC),and total flavonoid content(TFC).The organic Se was retained at a concentration of 54.75 mg/g in the freeze-dried sample.Principal component analysis and cluster analysis showed good separation between the FFS and unfermented(FS)groups.Fragrant 2-ethyloxetane had the highest content among all volatiles,while sinapine had the highest content among all phenolic compounds.The fermentation process showed remarkable improvement in the abundance and concentration of volatile compounds and phenolic contents,making FFS exhibit strong antioxidant activity and inhibitory capacity againstα-glucosidase activity.The bioaccessibility of phenolic compounds was significantly greater in FFS compared to FS.ADMET analysis revealed that the majority of phenolic compounds contained in FFS did not exhibit mutagenicity toxicity,hepatotoxicity,skin sensitization,or blood-brain barrier penetration,indicating a favorable level of biosafety.Overall,our study provides a new insight into the further utilization of Se-enriched Brassica napus L.in foods.

Effect of Foliar Fertilizer Spraying at Seedling Stage on the Growth Physiological Characteristics and Yield of Brassica napus L.

In order to select a suitable foliar fertilizer for Brassica napus L.at the seedling stage,using‘Fengyou 958’as the material,different foliar fertilizers including BR,Se,Si,BR+Si,BR+Se,Se+Si and BR+Se+Si were sprayed at the seedling stage to study their effect on the physiological characteristics,growth and yield of Brassica napus L..The results showed that the growth,chlorophyll content,soluble sugar content,soluble protein content,and yield of different treatments at the budding stage improved compared with the control.The effect of the Si+Se treatment was the best,followed by the Se and Si treatments.The chlorophyll content of the flower decreased continuously during the whole flowering period,and the chlorophyll content of the mature silique peel was higher than that of the seed.The content of soluble sugar in flowers was the highest in the early flowering stage,and the content of soluble sugar in leaves at the flowering stage was higher than that at the budding stage.The soluble sugar content in the harvested silique peel decreased gradually with the increase of time,and that in the silique peel and seed was similar at 35 d after pollination.The soluble protein content in the silique peel and seed decreased gradually at the mature stage,and the soluble protein content in the silique peel was higher than that in the seed at the same stage.Si+Se foliar fertilizer spraying at the seedling stage can promote the growth and yield of Brassica napus L.and can be applied in Brassica napus L.production.

Development of a KASP Marker on Chromosome A05 for Seed Oil Content of Brassica napus

In order to identify the molecular markers that can be widely used in the breeding of Brassica napus L.varieties with high seed oil content under different genetic backgrounds,we developed a Kompetitive Allele Specific PCR(KASP)marker for seed oil content on the basis of the results from available studies.The verification in the F_(2) population showed that the marker was closely linked to the quantitative trait locus(QTL)for oil content on chromosome A05.The findings helped to breed the‘Fengyou’varieties with high seed oil content in the middle reaches of the Yangtze River.

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

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.

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。

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。

甘蓝型黄籽油菜粒色基因候选区间的QTL-Seq定位及连锁标记开发

甘蓝型油菜黄籽性状遗传来源较为复杂且易受环境条件影响,基因定位和分子标记开发难度大.相比于传统QTL定位,结合二代深度测序等生信方法直接对甘蓝型油菜黄籽基因进行染色体定位、基因挖掘和标记开发,将有助于黄籽遗传机理的研究.利用甘蓝型油菜黄籽临保系63-2和黑籽67-1配制杂交组合,构建F 2粒色分离群体.遗传分析表明:该群体中黄籽性状受1对隐性主效基因控制;以法国甘蓝型油菜Darmor-bzh基因组为参考,利用集团分离分析法和全基因组重测序将黄籽性状基因定位于C03染色体6.1~9.0 Mb置信区间内,同时基于基因组变异和聚丙烯酰胺凝胶电泳验证获得与粒色基因紧密连锁的SSR标记4个和InDel标记5个.

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.

Differentially Expressed Genes Related to Fertility Conversion in Thermo-sensitive Genic Male Sterile(TGMS) Lines of Brassica juncea

[Objective] This study was performed to screen functional genes related to the fertility conversion of thermo-sensitive genic male sterile （TGMS） lines of Brassica juncea L. [Method] A B. juncea TGMS line K121S was selected as the experimental material. The total RNAs were isolated from fertile and sterile pollens at different development stages, including mother cell stage, tetrad stage, tricellular pollen stage and maturity stage. DDRT-PCR was carried out to identify differentially expressed genes. [Result] A total of 44 differentially expressed cDNA fragments were identified with Dot blot. And seven candidate genes related to fertility conversion of K121S were screened out by BLASTN, including callose synthase gene, aldehyde dehydrogenase gene and RNA polymerase I transcription factor RRN3 gene which were differentially expressed at the transcriptional level, H＇-ATPase gene, fructose diphosphate aldolase -class I gene, teucine-rich repeat receptor-Jike serine/threonine- protein kinase gene and alkaline/neutral invertase gene, which were differentially expressed at the post-transcriptional level. [Conclusion] The results of this study will help to explain the molecular mechanism of thermo-sensitive genic male sterility of B. juncea.

Dissecting the genetic architecture of glucosinolate compounds for quality improvement in flowering stalk tissues of Brassica napus

Glucosinolates(GSLs) and their hydrolytic products contribute to the quality traits of rapeseed flowering stalk tissues, such as taste, flavor and anticarcinogenic properties(Glucoraphanin). However, little is known about the genetic mechanisms of GSL accumulation in rapeseed flowering stalks. In this study, the variation and genetic architecture of GSL metabolites in flowering stalk tissues were investigated for the first time among a panel of 107 accessions. All GSL compounds exhibited continuous and wide variations in the present population. Progoitrin,glucobrassicanapin and gluconapin were the most abundant GSL compounds. Five quantitative trait loci(QTL) significantly associated with three GSL compounds were identified by genome-wide association study. GRA_C04 was under selected during modern breeding, in which the ratio of lower GSL haplotype(HAP2) in the accessions bred before 1990(52.56%) was significantly lower than that after 1990(78.95%). Four candidate genes, BnaA01. SOT16, BnaA06. SOT17, Bna A06. MYB51a, and Bna A06. MYB51b, were identified in the GTL_A01 and 4OH_A06 regions.These findings provide new insights into GSL biosynthesis in flowering stalk tissues and facilitate quality improvement in rapeseed flowering stalks.

芥菜型油菜每角籽粒数QTL的上位性互作和环境互作分析

【目的】为揭示芥菜型油菜及芸薹属作物每角籽粒数形成的分子机理,提高和改良芥菜型油菜产量和育种工作奠定基础。【方法】研究以包含221个芥菜型油菜株系的重组自交系(recombinant inbred line,RIL)群体为材料,在5个环境条件下对每角籽粒数性状进行加性QTL、加性×加性上位互作及环境互作分析。【结果】(1)共检测到7个与每角籽粒数相关的加性QTL,主要分布在芥菜型油菜A02、A03、A05、A08、B02和B03等染色体上,其加性效应分布在(-11.6424)~4.5246之间,其中qSS2-71的加性效应和遗传率均最大,分别达到-11.6424和14.44%,其余6个加性QTL的加性效应和遗传率均较小;(2)检测到7对影响每角籽粒数的加性×加性QTL上位互作效应及其与环境的互作效应,上位性QTL互作效应值分布在(-4.9308)~4.1936之间,7对上位性QTL与不同环境互作的遗传力均接近0;(3)每角籽粒数性状的广义遗传率为80.98%,狭义遗传率为30.98%。【结论】综合分析,芥菜型油菜每角籽粒数受一定环境影响,但控制该性状的加性效应受环境影响较小,且其加性×加性上位性QTL互作效应不明显。

基于QTL和转录组测序鉴定甘蓝型油菜耐旱候选基因

干旱胁迫严重限制了甘蓝型油菜种植面积的扩大和产量的提升。耐旱性是由多基因控制的复杂数量性状,将QTL定位与转录组测序相结合,是鉴定甘蓝型油菜耐旱候选基因的有效手段。本研究对甘蓝型油菜干旱敏感品系三六矮和耐旱品系科里纳-2构建的F2:6和F2:8重组自交系群体幼苗进行正常灌溉和干旱胁迫处理,测定地上部鲜重、地上部干重、叶片相对含水量、丙二醛和可溶性糖含量,利用SSR和SNP多态性分子标记构建遗传连锁图谱,鉴定耐旱相关QTL和候选区间,结合耐旱材料No11和干旱敏感材料No28的转录组测序,筛选耐旱相关候选基因。研究结果表明:干旱胁迫使甘蓝型油菜幼苗地上部鲜重、地上部干重和叶片相对含水量下降,使叶片丙二醛和可溶性糖含量上升;耐旱相关QTL和候选区间分布于A01、A02、A06、A08、A09、A10、C02、C03、C04、C06和C09染色体;对耐旱材料和干旱敏感材料正常灌溉、干旱24 h、36 h和48 h进行转录组分析,主要差异表达基因显著富集到光合作用、脂肪酸代谢、氨基酸代谢、植物激素信号转导、核糖体、昼夜节律及角质、木栓素和蜡质的生物合成等相关途径;将QTL与转录组测序相结合,鉴定到28个耐旱相关候选基因,主要编码FLC、bHLH105、TGA4、TEM1、ERF003、ACO3、CHLI1、LHCB6和PORC等,具有转录因子活性、乙烯产生和信号传导、叶绿素生物合成与结合、叶绿素氧化还原酶以及编码核糖体相关蛋白等功能。这些结果可为揭示甘蓝型油菜耐旱机理及分子标记辅助选育耐旱新品种奠定基础。

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.

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.

甘蓝型油菜RIL群体苗期根系耐铝性鉴定及评价方法研究

为减少铵肥的大量施用以及酸雨频降引发的耕地酸化,从而影响作物的生长,利用综合评价方法通过幼苗期根系表型筛选耐铝种质.以甘蓝型油菜重组自交系(RIL)亲本10D130和中双11为材料筛选苗期适宜的铝胁迫浓度,使用该浓度处理RIL内138个品种(系)幼苗,对其根长、根表面积、根平均直径、根体积、根交叉数、根尖数和根干质量7个根系指标的铝毒单项耐性系数进行变异系数分析、相关性分析、主成分分析、系统聚类分析和逐步回归分析,并采用隶属函数法综合评价不同油菜品种(系)根系的耐铝特性.结果表明:138份油菜品种(系)间各项根系指标差异有统计学意义(p<0.05);筛选出甘蓝型油菜幼苗期耐铝毒品种(系)27034,27036和27007等.根据灰色关联度及逐步回归分析结果,认为根据根长、根表面积、根体积、根交叉数等7个关键指标,可以初步判断甘蓝型油菜种质幼苗期的耐铝毒特性.通过这些方法综合评价油菜对铝的耐受性,可以避免单一指标的片面性和不稳定性,结果更为可靠.

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.