SCAR and RAPD markers associated with 18-carbon fatty acids in rapeseed, Brassica napus

Breeding rapeseed for enhanced oil quality includes the development of varieties with low linolenic acid content. The breeder also aims to develop varieties with a high linoleic acid content because of its nutritional value. Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers have been developed for linolenic acid content, but they are not best suited for a direct application in marker-assisted selection. The RFLP technique is too complex and time-consuming and RAPD markers lack codominance, precluding the distinction of homozygous from heterozygous individuals. In this report the conversion of a RAPD marker to a codominant sequence characterized amplified region (SCAR) marker named L1L9 is described. One of the alleles consisting of an 899 bp fragment (allele A), is associated with low linolenic acid content. The other allele consists of an 641 bp fragment (allele B) and is associated with high linolenic acid content. This marker explains approximately 25% of the genetic variation for this trait. Linkage analysis in the mapping population indicates that the SCAR marker probably tags an ω-3 desaturase gene in B. napus. Two RAPD markers were found to be associated with oleic/linoleic acid content. Markers M14-350 and I06-650 explained approximately 10% and 7% of the genetic variation for linoleic acid content, respectively. These two markers were found linked at 12.3cM in the segregating B. napus F₂ progeny used for mapping. All the markers reported in this paper should be useful in breeding programmes for developing high linoleic and low linolenic acid rapeseed varieties.     

Identification of molecular markers associated with oleic and linolenic acid in spring oilseed rape (Brassica napus)

The quality of the oil derived from oilseed rape is determined by its fatty acid composition. Breeding oilseed rape for enhanced oil quality includes the development of cultivars with high oleic and low linolenic acid. Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) techniques were investigated for the development of molecular markers for genes controlling oleic and/or linolenic acid. Markers that were identified were converted to sequence characterized amplified region (SCAR) markers for use in breeding. Molecular markers associated with these two fatty acids were identified in a doubled haploid population derived from a cross between the oilseed rape lines TO99‐5318‐20, very high oleic (>79%) and very low linolenic acid (<2%) × DH12075, high oleic (68%) and higher linolenic acid (>7%). Eight RAPD markers were associated with oleic and linolenic acid contents. The RAPD marker UBC 2₈₃₀ accounted for 43% and 13% of the genetic variation for oleic and linolenic acid levels, respectively. The RAPD marker UBC 153₅₅₀ accounted for 19% of the genetic variation for linolenic acid. The UBC 2₈₃₀ fragment was converted to a SCAR marker. The markers identified in this study should be useful tools for the early generation selection of high oleic and low linolenic acid genotypes in oilseed rape breeding programmes.     

甘蓝型油菜遗传图谱的构建及芥酸含量的QTL分析

一个由甘蓝型油菜品种Quantum (黄花、低芥酸)和人工合成的甘蓝型油菜品系No.2127-17（白花、高芥酸）为亲本材料建立的DH群体中芥酸呈现单基因的遗传模式。为了发展与芥酸紧密连锁的分子标记对其实行有效的控制,随机选择121个结实正常的DH系为作图群体,利用SSR和RAPD标记构建了一张甘蓝型油菜的遗传连锁图谱。在亲本间检测     

甘蓝型油菜主要脂肪酸组成的QTL定位

应用RAPD、SSR和SRAP技术, 对甘蓝型油菜低芥酸品系APL01与高芥酸品系M083杂交组合的BC1F1群体进行检测, 获得251个分子标记, 构建了19个连锁群组成的分子标记遗传图谱; 应用WinQTLCart 2.0对油菜主要脂肪酸组成进行QTL扫描, 获得与棕榈酸含量相关的QTL 5个, 分别位于N3、N8、N10和N13连锁群, 其中效应值较大的主效QTL qPA8-1和qPA13分别可解释棕榈酸含量表型变异的11.31%和14.47%。获得与硬脂酸含量相关的QTL 3个, 分别位于N1、N8和N16连锁群, 其中效应值较大的主效QTL qST16可解释硬脂酸含量表型变异的12.22%。获得与油酸含量相关的QTL 2个, 位于N8和N13连锁群, 均为主效QTL, 其中qOL8位于N8连锁群的m11e37b~A0226Ba267区间, 可解释油酸含量表型变异的11.73%, qOL13位于N13连锁群的m18e46~m20e25a区间, 可解释表型变异的27.14%。获得与亚油酸含量相关的QTL 3个, 其中主效QTL qLI8-1位于N8连锁群, 可解释亚油酸含量表型变异的13.25%。获得与亚麻酸含量相关的QTL 3个, 效应值均较小, 属微效QTL。获得与廿碳烯酸含量相关的QTL 4个, 分别位于N8、N13和N15连锁群, 其中主效QTL qEI8-1、qEI8-2和qEI13分别可解释廿碳烯酸含量表型变异的12.20%、10.22%和11.14%。获得与芥酸含量相关的QTL 2个, 位于N8和N13连锁群, 均为主效QTL, 其中qER8位于N8连锁群的m11e37b~A0226Ba267区间, 可解释芥酸含量表型变异的16.74%; qER13位于N13连锁群的A0301Bb398~m18e46区间, 可解释芥酸含量表型变异的31.32%。在N8连锁群的分子标记m11e27b附近及N13连锁群的分子标记m18e46附近存在多个主要脂肪酸的主效QTL, 这些标记可用于油菜脂肪酸改良的分子标记辅助选择。     

油菜种子油脂分子标记及QTL定位的研究进展

阐述了油脂的合成途径,重点归纳了油菜种子含油量及重要脂肪酸（包括油酸、亚油酸、亚麻酸和芥酸）的分子标记及QTL定位。总结了分子标记技术在油菜遗传图谱构建、重要性状的基因定位、油菜遗传多样性与种质资源评价及油菜分子标记辅助选择中的应用。最后提出需要进一步研究和解决的问题,并做了展望。     

Allelic variation in linolenic acid content of high erucic acid Ethiopian mustard and incorporation of the low linolenic acid trait into zero erucic acid germplasm

Zero erucic acid germplasm of Ethiopian mustard is characterized by high levels of linolenic acid (about 21%). Two genetic sources of low linolenic acid (N2‐4961 and HF‐186, about 5%), have been developed in a high erucic acid background. The objectives of this research were to study the genetic relationship between the two low linolenic acid lines and to transfer the trait to zero erucic acid germplasm. F1 seed generations from crosses between both lines had higher average linolenic acid concentration than both parents. F2 seeds segregated for linolenic acid content following a continuous variation from 1.8 to 7.4%, exceeding the limits of the parental distribution ranges. Transgressive recombinants with very low linolenic acid concentration (0.7‐2.7%) were confirmed in the F3 seed generation. The results suggested that N2‐4961 and HF‐186 possess alleles for low linolenic acid at different loci. Transgressive low linolenic acid F3 plants were crossed with plants of a zero erucic acid line and a selection for zero erucic, low linolenic acid was conducted. As a result, a zero erucic acid F3:4 line containing 1.5 ± 0.7% linolenic acid was developed.     

Evaluation of cytoplasmic effects on agronomic and seed quality traits in two doubled haploid populations of Brassica napus L.

Cytoplasmic effects have been occasionally implicated in the inheritance of several traits in oilseed rape (Brassica napus L.), including linolenic acid concentration (18:3) in the oil. It is important that these be considered when choosing the direction of cross for producing new breeding populations. To study this phenomenon, a reciprocal cross was made between two genotypes of oilseed rape, Reston and LL09, which differed for their erucic and linolenic acid concentrations in the seed oil. Two DH populations, which were produced by microspore culture from reciprocal F₁ plants, were evaluated in the growth room for one generation and in the field at two locations in Southern Ontario in 1993and 1994. Field notes were taken on days to flower, days to maturity,plant lodging, plant height and, seed quality traits. In the growth room study, the phenotypic distribution of 18:3 differed significantly between the two reciprocal DH populations. In the field, significant reciprocal differences between the population means were detected for 18:3,flowering date and protein content in both years and for days to maturity and oil content in 1993 only. To further study the parental lines,chloroplast (cp) and mitochondrial (mt) DNA from parental lines were isolated and subjected to RFLP and RAPD analysis. Several random primers revealed reproducible DNA polymorphism (RAPD) between the parental mt DNA. It is concluded that the direction of cross should be taken into consideration by oilseed rape breeders relying solely on doubled haploids for developing genotypes with modified seed quality traits in Brassica napus L. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selection on pollen for linolenic acid content in rapeseed, Brassica napus L.

In order to enhance the economic value of edible rapeseed oil, an improvement of quality is necessary. Mutagenesis of rapeseed resulted in a low linolenic acid content and a low ‘linolenic acid (CIS: 3) level to linoleic acid (CIS: 2) level’ ratio, that is, the linoleic desaturation ratio (LDR), in the seeds of the Canadian variety ‘Stellar’. As an early breeding marker for low linolenic acid content, the pollen fatty acid composition was determined on 80 doubled haploid plants derived from a single F₁ hybrid obtained from a cross between ‘Stellar’ and a high CIS: 3 variety ‘Drakkar’. Fatty acid analysis on seed and pollen showed that the low CIS: 3 and the low LDR traits from the ‘Stellar’ variety were expressed in pollen and in seeds, and that a very close correlation (r = 0.88) existed between seed and pollen for these two traits. The inheritance of these traits is controlled by two major genes with additive effects, both in seed and pollen. However, minor genes also appeared to be expressed in pollen and seed. These genes may allow the production of plants with lower CIS: 3 levels than that of the low linolenic acid content parent. The efficiency of this new tool for early screening in breeding programmes is discussed.     

油菜种子中亚麻酸研究现状及改良思路

提高油菜种子（油）中亚麻酸含量对缓解目前亚麻酸资源日益紧缺的压力、提高国民健康水平均具有重要意义。本文从形成过程、含量差异、影响因素、杂种优势、遗传方式和控制基因等方面对油菜种子中亚麻酸研究现状进行了综述;在剖析油菜高亚麻酸育种的意义、可行性、难点的基础上,提出开展高亚麻酸种质资源创新利用,创建高亚麻酸育种体系,建立高效保优栽培技术体系,开展油菜籽加工技术和抗氧化研究,构建标准体系等5个方面促进油菜高亚麻酸产业发展的建议。     

Inheritance of reduced linolenic acid content in the Ethiopian mustard mutant N2-4961

The zero erucic acid Ethiopian mustard lines developed so far are characterized by an exceptionally high linolenic acid content in the seed oil. The mutant line N2‐4961, expressing low linolenic acid content in a high erucic acid background, was developed through chemical mutagenesis. The objective of this research was to study the inheritance of low linolenic acid content in this mutant. Line N2‐4961 was reciprocally crossed with its parent line C‐101 and the linolenic acid content of the reciprocal F₁, F₂ and BC₁ generations was studied. No maternal, cytoplasmic or dominance effects were detected in the analysis of F₁ seeds and F₁ plants from reciprocal crosses. Linolenic acid content segregated in 1: 2: 1 ratios in all the F₂ populations studied, suggesting monogenic inheritance. This was confirmed with the analysis of the reciprocal backcross generation. The simple inheritance of low linolenic acid content in N2‐4961 will facilitate the transference of this trait to zero erucic acid lines of Ethiopian mustard.     

Analysis of QTL for seed oil content in <Emphasis Type="Italic">Brassica napus</Emphasis> by association mapping and QTL mapping

Increasing seed oil content is one of the most important breeding targets for rapeseed (Brassica napus). In this study, we combined quantitative trait loci (QTL) mapping and marker-trait association analysis to dissect the genetic basis of seed oil content in rapeseed. A doubled haploid (DH) population with 261 lines was grown in two highly contrasting macro-environments, Germany with winter ecotype environment and China with semi-winter ecotype environment, to explore the effect of environment effect of on seed oil content. Notable macro-environment effect was found for seed oil content. 19 QTL for seed oil content were identified across the two macro-environments. For association analysis, a total of 142 rapeseed breeding lines with diverse oil contents were grow in China macro-environment. We identified 23 simple sequence repeat (SSR) markers that were significantly associated with the seed oil content. Comparative analysis revealed that five QTL identified in the DH population, located on chromosomes A03, A09, A10 and C09, were co-localized with 11 significantly associated SSR markers that were identified from the association mapping population. Of which, the QTL on chromosome A10 was found to be homeologous with the QTL on chromosome C09 by aligning QTL confidence intervals with the reference genomes B. napus. Those QTL associated with specific macro-environments provides valuable insight into the genetic regulation of seed oil content and will facilitate marker-assisted breeding of B. napus.     

Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.)

An essential quality improvement of rapeseed oil can be obtained by reduction of its linolenic acid (C18:3) content from about 10% to less than 3% of the total fatty acids. Genotypes low in C18:3 have been developed by mutagenesis. The initial summer rapeseed mutant had been low yielding and highly susceptible to various diseases. It has been debated whether the low C18:3 character can be successfully combined with high seed yield for physiological reasons. Therefore, the low linolenic character of mutant M48 was transferred into high-yielding genotypes by repeated backcrossing to well-adapted low erucic acid, low glucosinolate (00-) winter rapeseed cultivars. Lines with low C18:3 content were selected from BC3 and BC4 generations and examined in 1990–95. Positive selection response for seed yield was shown to continue over the years. Presently, the best lines are yielding as well as the control cultivars being equivalent also in oil and glucosinolate contents. In order to test the effect of a low C18:3 content on seed yield, plants with low and with high C18:3 content, respectively, were selected from 16 segregating BC5-F₂ populations and bulked to form 32 F₃ populations. These ‘isogenic’ bulk populations were tested for field performance at four locations in 1995. The results show that C18:3 content of the seed oil is not associated with seed yield, oil content, beginning of flowering, plant height and disease resistance. Means of relative seed yield for the high and the low linolenic F₃ bulk populations were not significantly different with 88.0% and 86.9% of the control cultivars, respectively. There was a significant interaction between genotypes with high or low C18:3 content and location. This shows that under specific environmental conditions a low C18:3 content may be either favourable or unfavourable. The results indicate that the low C18:3 character of the original mutants per se does not cause a decrease in seed yield, oil content or general field performance.     

无芥酸甘蓝型油菜十八碳不饱和脂肪酸含量的QTL定位

用无芥酸的高油酸油菜品系HOP和低油酸油菜品种湘油15为父母本构建含189单株的F₂代作图群体。F₂代单粒种子播种前采用半粒取样,F₂代单株种子采用混合取样,进行脂肪酸含量的气相色谱分析。统计检测显示这两种方法测定结果极显著相关,各种脂肪酸含量之间大部分也呈显著相关。用该群体构建含342个SSR标记的遗传连锁图并对18碳不饱和脂肪酸含量进行了QTL定位。在A5和C5连锁群上各检测到1个油酸含量主效QTL,其中位于A5连锁群的QTL效应值较大,且与FAD2基因紧密连锁;位于C5连锁群的QTL为首次报道,与之紧密连锁的标记在A5 连锁群QTL区域有同源标记,说明可能与位于C5的FAD2基因有关。用两种方法测定性状值都能检测到这2个QTL,且效应值比较接近,共能解释60%~70%油酸含量变异。由于油酸含量与亚油酸之间高度相关,定位在A5和C5的油酸含量QTL也被确认为亚油酸含量主效QTL,但利用单株法测定的性状值能在A4连锁群上再发现1个LOD值较低的亚油酸含量QTL。两种测定法能比较一致地在A4、A5和C4连锁群上检测到3个亚麻酸含量主效QTL,共能解释72%~80%亚麻酸含量变异。用半粒法能在A4连锁群还能检测到1个解释变异度为12.42%的较小LOD值的亚麻酸含量QTL。     

Marker-assisted increase of genetic diversity in a double-low seed quality winter oilseed rape genetic background

Generation of novel genetic diversity for maximization of heterosis in hybrid production is a significant goal in winter oilseed rape breeding. Here, we demonstrate that doubled haploid (DH) production using microspore cultivation can simultaneously introgress favourable alleles for double‐low seed quality (low erucic acid and low‐glucosinolate content) into a genetically diverse Brassica napus genetic background. The DH lines were derived from a cross between a double‐low quality winter rapeseed variety and a genetically diverse semisynthetic B. napus line with high erucic acid and high glucosinolates (++ quality). Twenty‐three low‐glucosinolate lines were identified with a genome component of 50–67% derived from the ++ parent. Four of these lines, with a genome component of 50–55% derived from the ++ parent, also contained low erucic acid. Heterosis for seed yield was confirmed in test‐crosses using these genetically diverse lines as pollinator. The results demonstrate the potential of marker‐assisted identification of novel genetic pools for breeding of double‐low quality winter oilseed rape hybrids.     

Evaluation of microspore-derived embryos as models for studying lipid biosynthesis in seed of rapessed (Brassica napus L.)

Summary Microspore culture of rapeseed (Brassica napus L.) has provided a powerful tool not only for breeding but also in developmental studies. In this study, microspore-derived embryos (MDE) of B. napus were evaluated as a model in seed for studying accumulations of triacylglyceride (TAG) fatty acids in both a low and high erucic acid rapeseed line; and accumulations of TAG and free fatty acids (FFA) in a high erucic acid rapessed line. The accumulation patterns confirmed that MDE had a similar TAG fatty acid profile to seed during the embryo development within each genotype. The oil accumulation in MDE after 36 days in culture (DIC) approached levels similar to those in zygotic seed 25 days after flowering (DAF). Significant differences were detected in contents of both total free fatty acids and specific free fatty acids between MDE and seed. During the developmental period, total free fatty acids changed from 16% to 2.1% in MDE, but from 10.5% to 0.1% in seed. MDE had much higher percentage of free linolenic and erucic acids than seed, particularly during the late developmental stages. The current study indicated that MDE can be used as a model to study TAG and TAG fatty acids in seed but caution must be taken to study free fatty acid metabolism.     

工业专用型高芥酸油菜新品种选育

高芥酸油菜品种是在工业上具有广泛用途的专用型品种。本研究通过两个常规芥酸品种杂交,采用以单株和单粒筛选相结合对芥酸含量正向选择为核心的技术,育成了芥酸含量达60%的甘蓝型高芥酸油菜新品种高芥1号。同时阐明了在高芥酸含量背景下各种脂肪酸间的相关性,为相关育种提供了—些理论依据。     

Estimating the fatty acid composition of the oil in intact-seed rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy

The objective of this work was to evaluate the potential of near-infrared reflectance spectroscopy (NIRS) as a rapid method to estimate the fatty acid composition of the oil in intact-seed samples of rapeseed. A total of 549 samples (3 g intact seed) from selected mutant and breeding lines were scanned by NIRS, and 220 of them were selected and scanned again by using two different adapters, which reduced the sample size to 300 and 60 mg, respectively. Selected samples were analysed by gas liquid chromatography and calibration equations for individual fatty acids were developed. Calibrations for oleic, linoleic, linolenic, and erucic acid were highly accurate, with values of r² in cross validation from 0.95 to 0.98 (samples of 3 g), from 0.93 to 0.97 (300 mg), and from 0.84 to 0.96 (60 mg). Calibrations for palmitic and stearic acid were less accurate, with values of r² in cross validation always lower than 0.8, probably because of the narrow range available for these fatty acids. The accuracy of the calibration equations for eicosenoic acid was very low (r² = 0.69 in 3 g samples), although improved equations were developed (r² from 0.78 to 0.91) when the relationship between erucic and eicosenoic acid was taken into account. We conclude that NIRS is a powerful technique to estimate the fatty acid composition of the oil in rapeseed, provided that samples covering a wide range of fatty acid levels are available, with the advantage that such estimation is possible with few additional costs when NIRS is used for the determination of other seed quality traits. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of RAPD markers linked to linolenic acid genes in rapessed

Summary The inheritance of the low linolenic acid content (derivated from mutant lines) in rapeseed was investigated. Molecular techniques of gene mapping through RAPD markers were applied on a microspore-derived progeny from a high × low linolenic acid F₁ hybrid. Bulked segregant analysis made it possible to test rapidly number of RAPD primers. Two linkage groups of 6 markers (72.7 cM and 75.6 cM) were determined. Each corresponded to a major QTL which explained 24% and 30.7% of the total phenotypic variation of the linolenic acid content. It was confirmed that two independant mutations were implied in the low linonenic acid content.     

近10年我国冬油菜区试品种品质及产量性状的演变分析

油菜是我国最重要的食用油料作物。本文综合分析了2000年～2009年10个年度国家冬油菜区域试验参试品种的品质及产量性状。分析表明：2002年是我国油菜品种品质（低芥酸、低硫苷）和产量的转折点,此后双低油菜品种比例在80%以上,产量也逐步提高,到目前基本达到双高品种的产量水平。分析表明双低油菜产量的提高,主要归因于单株结角数和千粒重的增加;此外,双低油菜品种含油量有了较大的突破,尤其是近3年含油量有了显著提高。这表明在我国油菜品种双低化过程中,品质和产量得到了协同发展。目前在双低品质达标的情况下,提高产量和含油量仍是我国油菜育种的主攻目标。     

甘蓝型油菜遗传图谱构建与无花瓣性状QTL定位

以无花瓣油菜APL01与正常有花瓣品种M083杂交的BC₁F₁为基础群体,利用RAPD、SSR和SRAP技术获得251个分子标记,包括219个SRAP、25个SSR和7个RAPD标记,构建了由19个连锁群组成的分子标记遗传图谱,根据共同的分子标记,建立该图谱与甘蓝型油菜高密度图谱的对应关系。利用WinQTLCart 2.0软件对无花瓣性状进行QTL扫描,获得4个与无花瓣性状相关的QTL,QAP5位于N5连锁群的A0226Bb152~m31e40b区间,解释花瓣度表型变异的3.71%;QAP6位于N6连锁群的m25e7~OPY9区间,解释花瓣度表型变异的3.02%;QAP8位于N8连锁群的A0226Gb468~m29e20区间,解释花瓣度表型变异的30.94%;QAP15位于N15连锁群的m21e4b~A0225Bb201区间,解释花瓣度表型变异的21.96%。QAP8和QAP15为2个主效QTL,可用于无花瓣性状的标记辅助选择,QAP5和QAP6为修饰基因位点。