大白菜的适宜AFLP-Pst I/Mse I引物组合

基于PCR的DNA指纹鉴定技术AFLP广泛应用于生物多样性、遗传图谱构建、基因定位、分子系统发育和品种鉴定等方面.为了筛选出大白菜适宜的AFLP引物组合,研究以抗根肿病大白菜双单倍体CR Shinki、感病自交系94SK及其后代F2构成的纯合抗病和纯合感病DNA混合池为材料,利用256对Pst I/Mse I引物组合进行了AFLP分析.结果表明:平均每个组合检测到55条带,共扩增13 079条带,平均每1 000条带检测到1.30个根肿病抗性基因连锁标记;多态性带数为3 167条,双亲间多态性频率约为0.24;扩增带数与多态性带数呈线性正相关,引物组合中的不同GC含量与扩增带数和多态性带数存在显著性差异,并表现出线性负相关.筛选出的适宜53对引物组合共扩增出4 381条带,占总扩增片断的33.50%,平均每对扩增出83条,其中9个组合扩增出与抗根肿病基因连锁标记.     

梨AFLP分子连锁图谱的构建与分析

遗传图谱是数量性状基因定位、基因图位克隆及分子辅助育种等研究的基础,而目前国内在梨树上这方面的研究尚属空白.试验以早美酥×八月红的杂交F1 92株实生苗为作图群体,采用了扩增稳定、多态性丰富的16对Mse I/EcoR I引物组合,对分离群体进行AFLP多态性检测,共获得208条多态性带,其中偏离孟德尔遗传的比率29.8%(P<0.01).应用Joinmap 3.0软件且结合"双假测交"策略,对208个AFLP分子标记进行了遗传连锁分析,构建了一张包含145个AFLP标记,分属20个连锁群的梨分子遗传连锁图谱,每个连锁群包含4～20个标记,平均为9.06个标记,图谱总长度覆盖梨基因组618.7 cM,标记间平均图距为6.58 cM.此图谱为梨树基因定位、比较基因组学和重要农艺性状的QTL定位等研究奠定了基础.     

高粱AFLP分子连锁图谱的构建

利用感蚜高粱品种千三与抗蚜品种河农16杂交获得的100个F2单株作为图谱构建群体,以AFLP标记构建高粱连锁图谱。通过对192对AFLP引物组合进行筛选,共得到75对多态性引物,利用筛选出的75对引物进行选择性扩增,得到了154个AFLP多态性位点。经Map maker/EXP 3.0软件处理,构建了包含12个连锁群,93个遗传标记的连锁图谱,该图谱覆盖686 cM,平均图距为7.38 cM。     

黄瓜远缘群体分子遗传连锁图谱的构建和分析

以野生黄瓜品种和普通栽培黄瓜品种作亲本获得的142个F2群体为材料,采用AFLP,SRAP,SSR等分子标记进行遗传分析,构建了包含10个连锁群,有159个标记组成的黄瓜遗传连锁图谱,其中包括112个AFLP标记,39个SRAP标记和8个SSR标记。该遗传图谱覆盖基因组长度743.11 cM,平均图距4.67 cM。     

大白菜遗传图谱的构建及与染色体关联分析

为构建大白菜遗传图谱,并与国际上公认的白菜参照图谱相关联。以大白菜晚抽薹DH系Y177-12和早抽薹DH系Y195-93为亲本建立的183个DH系为作图群体,利用SRAP、SSR、AFLP、STS、ESTP和CAPS等多种分子标记和形态标记构建大白菜遗传图谱,通过参照图上的锚定标记确定连锁群,并用最新的连锁群命名法进行命名。构建了包含10个连锁群、233个标记位点的分子遗传图谱,其中包括145个SRAP标记、62个SSR标记、12个AFLP标记、6个STS标记、4个ESTP标记、3个形态标记和1个CAPS标记。图谱总长度1 036 cM,标记间的平均图距为4.4cM。通过其中的38个SSR,4个ESTP和2个STS标记将各个连锁群与国际上认可A1-A10的参照连锁群相关联。该图谱的构建将为不同图谱的比较与整合及重要农艺性状的基因定位奠定基础。     

花生栽培种SSR遗传图谱的构建

花生栽培种品种间分子多态性相对缺乏, 至今未构建出较完整的分子遗传图谱。本研究以粤油13和阜95-5为亲本, 通过杂交构建包含184个F6重组自交系的遗传作图群体。采用652对genomic-SSR引物和392对EST-SSR引物对亲本进行多态性检测, 从中筛选出121对多态性引物, 在亲本中共检测到123个多态性位点。利用作图群体对多态性SSR位点进行遗传连锁分析, 获得包含108个SSR标记(102个genomic-SSR标记和6个EST-SSR标记), 涉及20个连锁群, 总长568 cM, 平均图距为6.45 cM的花生栽培种遗传图谱。与前人构建的花生野生种(A. duranensis × A. stenosperma, AA genome)SSR遗传图谱比较, 初步确定本研究构建的遗传图谱中有11个连锁群与野生种遗传图谱的6个连锁群存在同源关系。     

基于SSR分子标记的高丹草遗传图谱构建及相关性状QTL定位

本试验以散穗高粱和红壳苏丹草为亲本,以其杂种F2代的170个分离单株为作图群体,利用SSR分子标记技术和Join Map 3.0作图软件构建高丹草遗传连锁图谱,并在此基础上对分蘖数、株高、氢氰酸含量等8个相关性状进行QTL定位。结果表明:从120对SSR引物中共筛选出50对多态性引物,利用这些引物对作图群体各单株进行PCR扩增,共获得226个多态性标记,平均扩增标记为4.5个/引物。构建出一张由10个连锁群组成的高丹草SSR分子遗传图谱,含181个SSR标记,图谱总长803.13 cM。各连锁群长度在5.8~152.3 cM之间,标记间平均距离4.38 cM,图谱密度较高。在构建图谱的基础上,对高丹草8个相关性状进行QTL定位,共获得17个QTLs,其中控制茎粗的QTL 4个,控制叶宽的QTL 3个,控制叶片数、叶长、分蘖数和穗长的QTL各2个;控制株高和氢氰酸含量的QTL各1个。这些QTL位点分布于高丹草SSR遗传连锁图谱的其中8个连锁群上,其遗传贡献率的范围为12.5%~25.6%。本研究可为进一步开展高丹草重要性状基因的精细定位、图位克隆、功能分析,以及分子标记辅助育种提供理论指导。     

中国白菜AFLP分子遗传图谱的构建

以白菜高抗TuMV白心株系91 112和高感TuMV桔红心株系T12 91为亲本建立的小孢子DH系作为图谱构建群体,以AFLP标记来构建白菜连锁图谱。通过对64对AFLP引物组合的筛选,利用20对引物得到了263个AFLP多态性位点。经Mapmaker/EXP3.0软件处理,构建了1张含255个标记位点,10个连锁群,覆盖长度为883.7cM的连锁图。255个AFLP位点中偏离孟德尔遗传的比率(P<0.05)为15 0%和27 5%(P<0.01)。     

甜菜遗传连锁图谱初步构建

以甜菜高产低糖型JV34-2和低产高糖型2B023两材料杂交, 构建了200个单株的F₂作图群体, 利用所筛选出的56对SRAP引物组合和20对SSR引物, 对F₂作图群体进行PCR扩增和遗传连锁分析, 初步构建了一张包含9个连锁群、141个(123个SRAP和18个SSR)标记位点的甜菜遗传连锁图谱。该图谱覆盖长度为1399.88 cM, 平均图距9.92 cM。未进入连锁群的有4个标记。9个连锁群包含3~26个标记不等, 连锁群遗传距离15.69~237.21 cM。连锁群上有20.56%的标记出现偏分离, 主要集中在Ch3连锁群上, 其余分散在Ch1、Ch2、Ch8和Ch9中。该图谱是我国甜菜领域利用SRAP和SSR相结合方法, 构建的第一个较精密的分子遗传图谱, 为重要性状的基因定位和优良基因的克隆奠定了基础。     

基于EST-SSR的木薯分子标记遗传连锁图谱的构建

此实验以木薯推广品种‘KU50’为母本,‘SC124’为父本通过杂交得到包含240个单株的F1分离群体,利用300对EST-SSR引物,20对SSR和20对SRAP引物组合对亲本和部分群体株系进行多态性分子标记筛选,共获得具有多态性的引物110对。在此基础上,利用这110对多态性引物对该F1群体进行分子标记的多态性分析,共获得269个多态性标记。利用JoinMap 3.0软件对这269个多态性标记进行分组和遗传图谱构建,最后获得了一张包含140个标记的木薯分子遗传连锁图谱,其中EST-SSR标记111个,SSR标记22个,SRAP标记7个;共21个连锁群,其中连锁群1和3（LG1、LG3）上的标记位点最多（15个）,LG21标记位点最少（2个）。此遗传连锁图谱的总长度为1314.775 cM,单个连锁群最长为132.904 cM（LG3）,最短为0.431 cM（LG19）,标记间平均长度9.391 cM。     

抗根肿病甘蓝型油菜新品种华油杂62R的选育

我国油菜根肿病发病面积在66.7万公顷左右,约占总生产面积的10%,已严重威胁到油菜的安全生产。基于此,本研究以含有CRb抗根肿病位点的大白菜材料CR Shinki为供体亲本,以甘蓝型油菜国审杂交种华油杂62的父本Pol.CMS恢复系Bing409为受体亲本,通过杂交、回交及自交等育种程序,结合前景和遗传背景筛选,将CRb抗病位点导入到Bing409中。在BC3F2代获得了遗传背景高度接近Bing409且含CRb抗病位点的抗根肿病新恢复系Bing409R,进而成功选育了我国首个抗根肿病杂交油菜新品种华油杂62R。CRb抗病位点在甘蓝型油菜背景中表现为单基因显性遗传,根肿病抗性遗传改良的同时并未对Bing409R及由其配制的杂交种华油杂62R的产量、品质造成不良影响,Bing409R及华油杂62R对我国四川、湖北、安徽等地区根肿菌生理小种具有免疫抗性。本研究的开展为我国油菜抗根肿病育种提供了宝贵资源,为我国抵抗油菜根肿病的威胁提供了重要保障。     

芸薹根肿菌致病机理的研究进展

十字花科植物根肿病是由芸薹根肿菌的侵染而引起,由于该病原菌存活时间长,传染性强,传播速度快,传播途径多样,以及对根肿菌的生活史和致病成灾机制认识不足,使得近期根肿病在中国的发生越来越严重。笔者从近年来芸薹根肿菌致病机理的相关研究进展入手,综述了生长素、细胞分裂素、水杨酸、茉莉酸和脱落酸等植物激素,氧化酶、芥子油苷和类黄酮、多胺等次生代谢物质的变化与芸薹根肿菌致病的关系,分析了芸薹根肿菌致病过程和十字花科植物在应对根肿菌侵入过程中的主要生理生化反应、代谢改变过程,以期对进一步揭示芸薹根肿菌致病机理研究及根肿病有效防控起到一定的参考和借鉴作用。     

抗根肿病甘蓝型油菜新品种华油杂62R的选育

The rapeseed clubroot disease incidence in China is about 0.67 million hectare, accounting for 10% of the canola production area, which become a serious threat for the safety of Brassica napus industry. Based on this, we used CR Shinki, a Chinese cabbage material containing CRb clubroot disease resistance locus, as the donor parent, and Pol.CMS restorer line Bing409, the parent of Brassica napus national approved varieties Huayouza 62, as the recipient parent, and the CRb resistance locus was introduced into Bing409 by breeding programs such as crossing, backcrossing, self-cross with the foreground and genetic background selection. In the BC₃F₂ generation, a new restorer line Bing409R with a genetic background close to Bing409 containing CRb resistance locus was obtained, and Huayouza 62R, the first rapeseed hybrid resistant to clubroot disease in China was successfully developed. The results were as follows: CRb disease resistance locus appeared as a dominant single-gene inheritance in B. napus background, and the genetic improvement of resistance to clubroot disease did not at the expense of yield and quality losses for new restorer line Bing409R and its hybrid Huayouza 62R. Bing409R and Huayouza 62R were showed immune-resistance to physiological races of Plasmodiophora brassicae in Sichuan, Hubei, and Anhui provinces in China. This study will provide valuable resources for the breeding of rapeseed in China, and supplemented important support to overcome the threat of rapeseed clubroot disease.     

The transfer of characters from Brassica campestris L. to Brassica napus L.: Production of clubroot-resistant oil-seed rape (B. napus ssp oleifera)

Summary Four oil-seed rape lines were crossed with a clubroot resistant Brassica campestris line from the European Clubroot Differential sct. The allotriploid hybrids were backcrossed to the rape lines to introgress clubroot resistance into oil-seed rape. Using a combination of screening for disease resistance and chromosome number, a high proportion of 38-chromosome, clubroot resistant selections were obtained.     

成都平原主栽大白菜品种根肿病抗性的鉴定

为明确成都平原地区主栽大白菜品种对根肿病的抗性情况,于2018年分别在成都市新都区、彭州市和崇州市3个试验田块对45个大白菜主栽品种采用田间抗鉴圃自然感染诱发法进行根肿病抗性鉴定,通过调查根部根肿病发病程度评价各品种的抗性。结果表明,在崇州自然病圃中表现为免疫的品种有4个,占总品种的8.9%;其余品种均表现为感病或高感,占比91.1%。彭州的结果显示,在所测定的45个品种中,免疫品种有6个;耐病品种有11个;剩余28个品种为感病或高感品种。而在新都发病田块中,只有2个品种表现为对根肿病免疫,16个品种表现为耐病,27个品种表现为感病或高感。部分品种在不同地区抗性结果表现不一致,试验鉴定到2个对根肿病免疫的优异品种,可为选育和应用抗性品种提供借鉴。     

Evaluation of F2 and F3 plants introgressed with QTLs for clubroot resistance in cabbage developed by using SCAR markers

Clubroot disease caused by Plasmodiophora brassicae is one of the major diseases of Brassica crops, often devastating to the cultivation of cruciferous crops in temperate regions. In a previous study (Moriguchi et al. 1999) identified three major quantitative trait loci (QTLs) for clubroot resistance, each in a separate linkage group, in a population derived from a cross between a clubroot‐susceptible inbred cabbage line, Y2A and a resistant inbred kale line, K269. In this study, the original random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were converted into sequence‐characterized amplified region (SCAR) markers to facilitate large‐scale marker‐assisted screening of clubroot resistance in cabbage breeding. Of 15 RAPD markers closely linked to the three QTLs, nine SCARs were developed as dominant markers after cloning and sequencing. In addition, two RAPD markers were converted into co‐dominant cleaved amplified polymorphic sequence (CAPS) markers, and one RFLP marker out of three tested was converted to a dominant SCAR marker. The effect of selection for resistance by the improved markers was evaluated in progeny plants in the F2 and F3. A total of 138 F2 plants were genotyped with nine SCARs and 121 well‐distributed makers consisting of 98 RAPD, 19 RFLP, two isozymes, and two morphological markers in order to estimate the level of resistance and the proportion of undesirable alleles from the kale in non‐target areas in each of the F2 populations. An F2 plant, YK118, had kale alleles at QTL1, QTL3 and QTL9. Three F2 plants, namely, YK107, YK25 and YK51 had kale alleles at only QTL1, QTL3 and QTL9, respectively. These F2 plants were selected for their low proportion of alleles derived from kale in non‐target regions. YK118, like the resistant kale parent, expressed very high resistance to three field isolates of Plasmodiophora brassicae, whereas the mean disease index in the F2 and F3 plants carrying only single QTLs was intermediate. The QTLs showed no differential response to the isolates. These plants with improved resistance will be useful as parental inbred lines for F1 hybrids.     

油菜抗根肿病资源创新与利用的研究进展与展望

根肿病是原生动物界根肿菌门根肿菌属芸薹根肿菌( Plasmodiophora brassicae Wor.)侵染引起, 并专性危害十字花科作物的一种传染性强的土传病害。近些年来, 加拿大和我国油菜的安全生产均受到根肿病的严重威胁。本文简要介绍了根肿菌的生物学特性、分类及主要防治途径, 重点总结了在根肿病抗性资源发掘、抗病基因遗传定位和克隆以及抗病育种等方面的进展, 分析了目前我国油菜根肿病综合防控过程中存在的主要问题, 并探讨与展望了相应的对策, 旨在为油菜抗根肿病遗传育种的高效顺利开展提供技术支持, 进而保障油菜产业的可持续发展。     

油菜感抗品种混播对根肿病防控效果的影响

近年来,油菜根肿病危害在我国呈蔓延趋势,主要原因是化学防治成本高且效果差。实际生产中,种植抗性品种是最经济、最有效的措施。不同抗感品种混播,在保证产量稳定的前提下可减轻对病原菌小种的选择压,从而稳定病原菌种群结构,延长抗性品种使用寿命。本试验在安徽绩溪、云南临翔2个油菜根肿病严重发生区域,选择2组互为近等基因系的感病、抗病的油菜品种,按不同比例混播(感病、抗病比例分别为10:0、1:9、2:8、3:7和0:10),在根肿病明显发生后调查发病率等指标。结果表明:不同比例感病、抗病油菜品种混播均降低了根肿病发病率和病情指数,并稳定油菜产量和品质;且油菜发病株率随抗病品种的比例下降而升高,但均低于理论发病率;不同抗性组合间混播的相对防效(REM)均低于1,且随着作物生育期的推迟呈现下降趋势。以上结果表明,油菜感抗品种混播对根肿病有较好的防控效果。本研究主要在大田条件下明确了2组不同抗感近等基因组合混播对油菜根肿病的防治效果,可为油菜根肿病防治提供思路和对策。     

Introgression of clubroot resistance into an elite pak choi inbred line through marker‐assisted introgression breeding

Clubroot is a soilborne disease that severely infects cruciferous species. Pak choi (Brassica rapa subsp. chinensis) is an economically important cruciferous crop cultivated throughout the world. However, no clubroot‐resistant germplasms have been identified in pak choi to date. To improve disease resistance, we used marker‐assisted selection (MAS) to introgress the clubroot resistance (CR) trait from the ‘CCR13685’ Chinese cabbage (B. rapa subsp. pekinensis) inbred line into an elite pak choi inbred line, ‘GHQ11021’. Genetic analysis of F₂ and BC₁ progeny showed that CR of ‘CCR13685’ was controlled by a single dominant gene. We designed nine candidate sequence‐characterized amplified region markers, K‐1 to K‐9, based on two molecular markers linked to the CR gene. We found that K‐3 co‐segregated with CR and an inoculation test confirmed that K‐3 could be used for MAS. Two introgression lines, BC₃‐1‐4 and BC₃‐2‐18, were developed using K‐3 for foreground selection. These lines displayed the same phenotypic properties as ‘GHQ11021’, but were highly resistant to clubroot, indicating that the CR gene of ‘CCR13685’ had been successfully introduced into pak choi.     

小白菜根肿病的接种方法及条件研究

十字花科作物根肿病是一种重要的世界性植物土传病害,该病引起的损失占世界十字花科作物总量的10%以上。探索病害的最佳接种方法是开展相关或同类研究的重要基础。试验比较了5种根肿病的人工接种方法,并探索了种子预浸、病原菌浓度和土壤pH等接种条件对接种效果的影响。结果表明,密封菌土法优于菌土法,而浸种加密封菌土法的接种效果最为显著。对比不同接种条件的试验结果得知,无菌水预浸种1天破胸后再进行接种处理会降低接种效果;用于接种处理的根肿病菌浓度越高,发病的效果越明显;而土壤条件为酸性时更利于根肿病的发生。