Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed

Chlorophyllase and peroxidase activities were measured in relation to seed maturation and degreening in canola ( Brassica napus cvs Westar and Alto) and mustard ( Brassica juncea cvs Cutlass and Lethbridge 22A). Samples of seed collected at the same moisture content were pooled, then divided and used for each assay. During maturation the green pigment (chlorophyll and related pigments) content of canola seed decreased linearly and was lower than that measured in mustard at all moisture contents studied, except for the highest and lowest moisture contents. Chlorophyllides and pheophorbides were detected in canola and were essentially absent in mustard. This difference in accumulation of dephytylated pigments infers differences in the pigment degradation pathways in Brassica species. Interspecific differences in the enzymology of degreening were found. Green pigment degradation was associated with increased chlorophyllase activity and low peroxidase activity in canola and low Chlorophyllase and high perosidase activity in mustard. The possible role of ethylene in seed degreening is discussed.     

甘蓝型油菜种子硫代葡萄糖苷含量的QTL定位及候选基因鉴定

【目的】为了解析油菜种子硫代葡萄糖苷性状的重要遗传位点及候选基因,【方法】本研究利用KN DH群体在冬性环境2015-2018连续4年的种子硫苷含量表型和KN 高密度SNP遗传连锁图谱,通过Wincart 2.5软件的符合区间作图法对甘蓝型油菜种子硫代葡萄糖苷含量进行QTL定位和潜在候选基因鉴定。【结果】共鉴定到47个硫苷含量QTL,单个QTL解释表型变异最大是qGC.16YL19-4(19.44%),解释表型变异最小的是qGC.15YL12-5(1.82%)。利用元分析的方法将初步鉴定的47个QTL整合为38个consensus QTL,其中7个consensus QTL(cqGC.A9-5、cqGC.A9-7、cqGC.A9-9、cqGC.C2-9、cqGC.C2-10、cqGC.C9-5和cqGC.C9-6)为环境稳定表达QTL,包括3个硫苷含量主效QTL(cqGC.A9-5、cqGC.C2-10和cqGC.C9-5)。在主效QTLcqGC.A9-5和cqGC.C9-5鉴定到3个候选基因BnaA09g05480D,BnaC09g05620D和BnaC09g05810D,其功能主要涉及了油菜硫苷生物合成途径中吲哚-3-乙醛肟(IAOx)的合成和将2-烷基-苹果酸异构化形成3-烷基-苹果酸酯,以及硫苷的转运与分配。【结论】本研究获得了油菜种子硫苷含量3个主效QTL及3个候选基因,该结果为硫苷含量相关基因的功能机械和优质油菜品种培育提供理论依据。     

Genome-wide association study for candidate genes controlling seed yield and its components in rapeseed (Brassica napus subsp. napus)

Genetic improvement of seed yield per plant (SY) is one of the major objectives in Brassica napus breeding programme. SY, being a complex quantitative trait is directly and indirectly influenced by yield-component traits such as siliqua length (SL), number of seeds per siliqua (NSS), and thousand seed weight (TSW). Therefore, concurrent improvement in SL, NSS and TSW can lead to higher SY in B. napus. This study was conducted to identify significant SNPs and putative candidate genes governing SY and its component traits (SL, NSS, TSW). All these traits were evaluated in a diverse set of 200 genotypes representing diversity from wide geographical locations. Of these, a set of 125 genotypes were chosen based on pedigree diversity and multi-location trait variation for genotyping by sequencing (GBS). Best linear unbiased predictors (BLUPs) of all the traits were used for genome-wide association study (GWAS) with 85,126 SNPs obtained from GBS. A total of 16, 18, 27 and 18 SNPs were found to be significantly associated for SL, NSS, TSW and SY respectively. Based on linkage disequilibrium decay analysis, 150 kb genomic region flanking the SNP was used for the identification of underlying candidate genes for each test trait. Important candidate genes involved in phytohormone signaling (WAT1, OSR1, ARR8, CKX1, REM7, REM9, BG1) and seed storage proteins (Cruciferin) were found to have significant influence on seed weight and yield. Genes involved in sexual reproduction and fertilization (PERK7, PERK13, PRK3, GATA15, NFD6) were found to determine the number of seeds per siliqua. Several genes found in this study namely ATS3A, CKX1, SPL2, SPL6, SPL9, WAT1 showed pleiotropic effect with yield component traits. Significant SNPs and putative candidate genes identified for SL, NSS, TSW and SY could be used in marker-assisted breeding for improvement of crop yield in B. napus. Genotypes identified with high SL, NSS, TSW and SY could serve as donors in crop improvement programs in B. napus.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01060-9.     

Diversity of root-associated bacteria associated with field-grown canola (Brassica napus L.) and wheat (Triticum aestivum L.)

Little is known about the composition and diversity of the bacterial community associated with plant roots. The purpose of this study was to investigate the diversity of bacteria associated with the roots of canola plants grown at three field locations in Saskatchewan, Canada. Over 300 rhizoplane and 220 endophytic bacteria were randomly selected from agar-solidified trypticase soy broth, and identified using fatty acid methyl ester (FAME) profiles. Based on FAME profiles, 18 bacterial genera were identified with a similarity index >0.3, but 73% of the identified isolates belonged to four genera: Bacillus (29%), Flavobacterium (12%), Micrococcus (20%) and Rathayibacter (12%). The endophytic community had a lower Shannon-Weaver diversity index (1.35) compared to the rhizoplane (2.15), and a higher proportion of Bacillus, Flavobacterium, Micrococcus and Rathayibacter genera compared to rhizoplane populations. Genera identified in the endophytic isolates were also found in the rhizoplane isolates. Furthermore, principal component analysis indicated three clusters of bacteria regardless of their site of origin, i.e., rhizoplane or endophytic. In addition, the rhizoplane communities of canola and wheat grown at the same site differed significantly. These results indicate that diverse groups of bacteria are associated with field-grown plants and that endophytes are a subset of the rhizoplane community.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase activity.     

Mapping QTL controlling yield and yield components in a spring barley (Hordeum vulgare L.) cross using marker regression

An RFLP map constructed from 99 doubled haploid lines of a cross between two spring barley varieties (Blenheim × Kym) was used to localize quantitative trait loci (QTL) controlling grain yield and yield components by marker regression and single-marker analysis. Trials were conducted over three years. Genotype-by-year interaction was detected for plant grain weight and ear grain weight so they were analysed separately for each year. None was detected for thousand-grain weight and ear grain number so data were pooled over years. A total of eleven QTL were detected for plant grain weight over two years and fourteen for ear grain weight over three years. Seven QTL were detected for plot yield. The locus with the largest effect was on chromosome 2(2H)L and accounted for 19% of the variation in the progeny. Eight QTL were detected for thousand-grain weight and five for ear grain number. Many of the QTL detected were in comparable positions in each year. Yield and yield components were only partly correlated. Comparisons based on common RFLP markers showed that some QTL were found in positions similar to those identified in other studies. For a number of QTL the identification of linked markers provided suitable opportunities for marker-assisted selection and improvement of barley and reference markers with which to analyse the homoeologous chromosome regions of wheat and other cereals.     

甘蓝型油菜千粒重性状的QTL初步定位研究

千粒重是油菜重要的产量相关性状之一,构建油菜遗传连锁图谱是研究其产量性状基因的前提。本研究利用小孢子培养技术,选育出了甘蓝型油菜大粒品系(G-42)和小粒品系(7-9)的纯合DH系DH-G-42和DH-7-9,其千粒重分别为6.24 g和2.42 g,二者比值达2.58。以DH-G-42为母本、DH-7-9为父本,构建了含190个单株的F2遗传作图群体,利用SSR和SRAP标记技术绘制遗传连锁图谱,该图谱共包含20个连锁群,涉及128个SSR标记和100个SRAP标记,图谱总长1546.6cM,标记间平均图距为6.78cM。本研究共检测到3个与千粒重性状相关的QTL,分别位于A9和C1连锁群,其中qSW-A9-1和qSW-A9-2贡献率分别达到10.98%和27.45%,均可视为控制粒重的主效QTL。本研究为后续进行油菜千粒重性状QTL的精细定位分析、分子标记辅助选择育种及新基因的克隆等奠定了基础。     

Peroxidase-mediated chlorophyll bleaching in degreening canola (Brassica napus) seeds and its inhibition by sublethal freezing

Peroxidase activity in isolated thylakoids from degreening canola ( Brassica napus cv. Westar) seeds was demonstrated. The enzyme catalyzes the degradation of thylakoid-bound pigments in the presence of H₂O₂ and 2,4-dichlorophenol. Peroxidase activity is related to degreening, with periods of rapid degreening associated with high enzyme activity. Both de novo synthesis and substrate availability appear to control enzyme activity. Peroxidase is initially inhibited and then stimulated by sublethal freezing. Therefore, inhibition of peroxidase activity following sublethal freezing may be responsible, in part, for a failure of the seed to degreen.     

油菜硫甙性状QTL 区间的加密和整合

目的:加密油菜控制硫甙性状QTL区间,并进行QTL整合预测候选基因。方法:利用生物信息学方法根据已知测序的白菜BAC序列信息设计引物,在油菜TN DH群体中进行多态性扩增和定位,并根据加密后构建的遗传连锁图重新检测QTL,进行QTL整合。结果:将根据白菜BAC设计的3对多态性标记成功定位到油菜控制硫甙性状QTL区间,进行QTL整合后将QTL置信区间进一步缩小,并判定了初步的候选基因。结论:充分利用白菜已测序的BAC或者基因组信息,将能加快油菜基础研究的步伐。     

甘蓝型黄籽油菜种皮色泽QTL作图

甘蓝型黄籽油菜具有低纤维、高蛋白及高含油量的优点,因而己成为广大油菜育种工作者研究的重点之一。利用甘蓝型黑籽品系油研2号作父本,计蓝型黄籽品系GH06为母本,获得132个单株的F2群体;以AFLP和SSR为主要分析方法,构建了包括164个标记的甘蓝型油菜遗传连锁图谱,其中包括125个AFLP标记、37个SSR标记及一个RAPD和一个SCAR标记,分布在19个连锁群上,覆盖油菜基因组2549．8cM,标记间平均距离15．55cM。利用多区间作图法,对种皮色泽QTL进行分析,在第5及第19连锁群上各检测到一个QTL位点,分别解释表型变异46％及30．9％。     

Effects of sowing time and plant age on critical nitrogen concentrations in canola (Brassica napus L.)

Critical concentrations of NO₃-N in fresh petiole tissue and total N in the dried lamina were determined for the youngest mature leaf (YML) of field-grown canola. For dry matter yield of canola sown on 4 May, critical NO₃-N concentration in the YML petiole at the rosette stage (RS) was 1.46 mg/g fresh wt. At the flower-buds-visible stage (BV) it was 0.45 mg/g fresh wt. For seed yield the values were 1.72 and 0.53 mg/g fresh wt. Critical total N concentration in the YML lamina for dry matter yield were 69 mg/g dry wt. at RS and 57 at BV. For seed yield they were 71 and 59 mg/g dry wt. Critical NO₃-N concentrations in the YML petiole of canola sown on 30 May were reduced by 50%; critical total-N concentrations in the YML lamina were not reduced to the same extent. Despite the reductions in critical N concentrations in the YML, critical N fertilizer rates for vegetative growth and seed yield were unaffected by sowing date or plant growth stage.     

Triacontanol modulates photosynthesis and osmoprotectants in canola (Brassica napus L.) under saline stress

Abstract

An experiment was conducted to assess the effect of pre-sowing seed treatment with triacontanol (TRIA) in canola (Brassica napus L.) cultivar (RBN-3060) under saline stress. Canola seeds were soaked in three levels of TRIA (0, 0.5, and 1 mg L^?1) for 12 hours. Three levels of salt stress (0, 100, and 150 mM NaCl) in full strength Hoagland's nutrient solution were applied to 56-days-old plants. Salt stress caused a significant reduction in growth, gas exchange, photochemical quenching (qP), and shoot and root K⁺ contents, while increased leaf glycine betaine, free proline, and shoot Na⁺ contents. Pre-sowing seed treatment with TRIA increased shoot fresh weight, number of seeds per plant, photosynthetic rate, transpiration rate, ratio of chlorophyll a/b, qP, electron transport rate, shoot and root K⁺ contents, and free proline and glycine betaine contents of canola plants at various TRIA levels under nonsaline or saline conditions.     

甘蓝型油菜与蔊菜的原生质体融合与植株再生

以甘蓝型油菜下胚轴和蔊菜叶片为外植体提取原生质体, 采用PEG-高pH、高Ca²⁺附加DMSO的原生质体融合方法, 用液体浅层静置培养融合体, 获得了10株融合杂种, 观察了杂种形态学和细胞学。结果表明：1%纤维素酶+0.2%离析酶+3 mmol/L MES 酶解14 h 可获得较高产率的油菜原生质体, 0.25% 纤维素酶+0.5%离析酶+5 mmol/L MES酶解12 h可获得较高产率的蔊菜原生质体; 30% PEG + 0.3 mol/L葡萄糖+50 mmol/L CaCl₂•2H₂O +15%DMSO的融合条件下, 获得了10.4%的融合率; 实验所获的原生质体融合材料可作为新种质。     

Identification of QTL involved in field resistance to light leaf spot (Pyrenopeziza brassicae) and blackleg resistance (Leptosphaeria maculans) in winter rapeseed (Brassica napus L.)

 Quantitative trait loci (QTL), involved in the polygenic field resistance of rapeseed (Brassica napus L.) to light leaf spot disease, were mapped using 288 DNA markers on 152 doubled-haploid (DH) lines derived from the cross ‘Darmor-bzh’בYudal’. Over two years (1995 and 1996), the DH population was evaluated for light leaf spot resistance on leaves (L) and stems (S), and for blackleg disease resistance in same field trials. For the L resistance criterion, a total of five and seven QTL were detected in 1995 and in 1996 respectively, accounting for 53% and 57% of the genotypic variation. For the S criterion, three and five QTL were identified in 1995 and in 1996 respectively, explaining 29% and 43% of the genotypic variation. The locations of the QTL detected were quite consistent over the two years (4- and 2-year common QTL for L and S, respectively). Three genomic regions, located on the DY5, DY10 and DY11 groups, were common to the resistance on leaves and stems. In comparison with the QTL for blackleg resistance described by Pilet et al. (1998), two regions on the DY6 and DY10 groups, were associated with the two disease resistances. These ‘multiple disease resistance’ (‘MDR’) QTL may correspond to genes involved in common resistance mechanisms towards the two pathogens or else to clusters of resistance genes. Received: 21 November 1997 / Accepted: 3 March 1998     

蛋白质组学研究揭示的甘蓝型油菜非生物胁迫应答机制

油菜(Brassica napus L.)是我国的主要油料作物之一,在生长发育过程中经常受到干旱、高温、高盐和营养缺乏等非生物胁迫。这些胁迫通常会阻碍油菜的生长发育,导致品质和产量下降。近年来,快速发展的高通量蛋白质组学技术为揭示油菜胁迫响应分子机制提供了新线索。本文综合分析了油菜不同组织/器官(如:叶片、根、下胚轴和种子)在响应盐、高温、干旱、草酸和缺素(磷、硫和硼)等逆境过程中675种蛋白质的丰度变化特征,揭示了其胁迫应答机制,主要包括:(1)通过G蛋白介导的信号通路感知与传递胁迫信号;(2)通过改变参与糖类与能量代谢相关酶的丰度调节代谢水平;(3)通过叶绿素合成的变化调节光合作用;(4)调节转录因子、蛋白质合成与命运相关蛋白质的丰度,从而在转录、翻译以及翻译后修饰等水平上应答逆境;(5)通过调节膜联蛋白、V型H+-ATP酶等质膜蛋白质,促进细胞内物质吸收与转运;(6)通过细胞骨架动态重塑保持正常细胞结构;(7)利用调节抗氧化酶系统清除活性氧,并通过合成多种防御物质减轻细胞受到的伤害。本综述为解析油菜逆境应答网络体系中的关键调控及代谢通路的变化提供了重要信息。     

Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves

The glucosinolate content of oilseed rape {Brassica napus) leaves was monitored over the growth period 30–70 days after planting, and a comparison made between a single-low cultivar (low in erucic acid), Bienvenu, and a double-low cultivar (low in erucic acid and glucosinolate), Cobra. In older, fully-expanded leaves the glucosinolate concentration was very low (< 0.3 μmol/ml tissue water) and did not alter during the course of the experiment. In developing sixth leaves glucosinolate content increased rapidly and reached a maximum concentration (4–5 μmol/ml tissue water) 40 days after planting (6 days after leaf emergence). The concentration then declined, to about 1 μmol/ml after 60 days although the total glucosinolate content in leaves continued to increase until 50 days; much of the reduction in concentration was simply a result of leaf expansion. No major differences were seen between the two varieties in total glucosinolate content or in the individual compounds present. Cv. Cobra developed more quickly than cv. Bienvenu so direct comparison between leaves of the two cultivars was complex. When comparing the glucosinolate content of oilseed rape leaves, between cultivars or between treatments, it is vital to ensure that carefully matched leaves of comparable developmental age are selected.     

Cytokinins in a genic male sterile line of Brassica napus

Endogenous cytokinin levels were analyzed in four different organs viz., root, stem, leaf and mature flower, of the wild type (cv. Westar) and a genic male sterile (GMS) line of Brassica napus . Various cytokinins viz., bases (Z, DZ), ribosides (ZR, DZR), glucosides (OGZ + OGZR, OGDZ + OGDZR) and nucleotides (ZNT, DZNT) were quantified using Amaranthus betacyanin bioassay and ELISA. The major cytokinin in all the organs was DZ. In general, leaves had the highest levels of cytokinins as compared to the other organs. Root, stem and mature flowers of the wild type plants had higher levels of cytokinins and their metabolites than the GMS line. However. levaves of the GMS line had greater amount of various cytokinins as compared to the wild type. The major cytokinin quantitatively different between the two lines was DZ. The results suggest the possible involvement of cytokinins in the expression of genic male sterility in Brassica napus .     

甘蓝型油菜遗传图谱的构建及单株产量构成因素的QTL分析

采用常规品系04-1139与高产多角果品系05-1054构建的F2代群体为作图群体, 运用SSR(Simple sequence repeat)和SRAP(Sequence-related amplified polymorphism)构建分子标记遗传图谱并对甘蓝型油菜单株产量构成因素进行QTL分析。遗传图谱包含200个分子标记, 分布于19个连锁群上, 总长度1 700.23 cM, 标记间的平均距离8.50 cM。采用复合区间作图法(Composite interval mapping, CIM)对单株产量构成因素(单株有效角果数、每果粒数和千粒重)进行QTL分析, 共检测到12个QTL: 其中单株有效角果数4个QTL, 分别解释表型变异为35.64%、12.96%、28.71%和34.02%; 每果粒数获得5个QTL, 分别解释表型变异为8.41%、7.87%、24.37%、8.57%和14.31%; 千粒重获得３个QTL, 分别解释表型变异为2.33%、1.81%和1.86%。结果表明: 同一性状的等位基因增效作用可以同时来自高值亲本和低值亲本; 文章中与主效QTL连锁的标记可用于油菜产量性状的分子标记辅助选择和聚合育种。     

Gene transfer between canola (Brassica napus L. and B. campestris L.) and related weed species

Brassica species are particularly receptive to gene transformation techniques. There now exists canola genotypes with transgenic herbicide resistance for glyphosate, imidazolinone, sulfonylurea and glufosinate herbicides. The main concern of introducing such herbicide resistance into commercial agriculture is the introgression of the engineered gene to related weed species. The potential of gene transfer between canola (Brassica napus and B. campestris) and related weed species was determined by hand pollination under controlled greenhouse conditions. Canola was used as both male and female parent in crosses to the related weed species collected in the Inland Northwest region of the United States. Weed species used included: field mustard (B. rapa), wild mustard (S. arvensis) and black mustard (B. nigra). Biological and cytological aspects necessary for successful hybrid seed production were investigated including: pollen germination on the stigma; pollen tube growth down the style; attraction of pollen tubes to the ovule; ovule fertilisation; embryo and endosperm developmental stages. Pollen germination was observed in all 25 hybrid combinations. Pollen tubes were found in the ovary of over 80% of combinations. About 30% of the hybrid combinations developed to the heart stage of embryo development or further. In an additional study involving transgenic glufosinate herbicide resistant B. napus and field mustard it was found that hybrids occurred with relatively high frequency, hybrids exhibited glufosinate herbicide resistance and a small proportion of hybrids produced self fertile seeds. These fertile plants were found to backcross to either canola or weed parent.