油菜抗咪唑啉酮类除草剂基因标记的开发与应用

草害已成为严重制约我国油菜生产的重要因素。种植抗除草剂品种和采用化学除草是防控草害的经济有效途径。为了开展分子标记辅助选择(marker-assisted selection,MAS)育种,加速抗除草剂品种培育,本研究针对已发现的抗咪唑啉酮类除草剂油菜M9中BnALS1R基因编码区第1913位点的SNP变异,开发高通量、低成本的竞争性等位基因特异PCR (kompetitive allele specific PCR, KASP)标记。采用筛选出的KBA1R19681913B标记在2个F_2群体中进行KASP反应。结果表明,该标记能有效检测群体中存在的BnALS1R 3种基因型,其分离比均为1︰2︰1,遵循单基因遗传规律,且基因型与表型完全吻合。将该标记用于BnALS1R抗性纯合基因的回交转育,获得200多个抗咪唑啉酮油菜恢复系。该标记还可在油菜苗期鉴定抗性杂交种的纯度。KASP标记KBA1R19681913B的获得为油菜抗除草剂MAS育种以及抗性新种质的培育提供了技术支撑。     

水稻抗咪唑啉酮类除草剂基因ALS功能标记的开发与应用

选育和利用抗除草剂水稻品种具有重要的生产实践意义。通过筛选水稻资源, 发现了抗除草剂材料金粳818, 其ALS基因编码区第1880位碱基存在一个由G到A的碱基变异, 导致丝氨酸突变为天冬酰胺, 从而具有除草剂抗性。本研究基于该位点的碱基变异, 设计了11条等位基因特异PCR (allelic-specific PCR, AS-PCR)引物, 经过优化筛选, 获得两个引物组合F1N (S1/S9)和F1M (S1/S10), 将该标记命名为AS-ALS。利用F₂群体及其亲本和杂交种, 结合AS-ALS标记检测和除草剂抗性分析, 结果表明感除草剂ALS-G等位基因型只能被F1N引物对有效扩增, 抗除草剂ALS-A等位基因型只能被F1M引物对有效扩增, 而杂合基因型能同时被两对引物F1N和F1M扩增, ALS-A纯合或杂合等位基因型都表现抗除草剂, ALS-G纯合基因型表现感除草剂。因此本研究开发的标记能有效区分除草剂抗性基因的3种基因型, 基因型与表型完全对应。该标记用于回交育种, 可以选择ALS-A杂合基因型单株, 剔除ALS-G纯合等位基因型, 在自交的F₂保留ALS-A纯合基因型单株, 连续自交, 能快速获得除草剂抗性稳定的水稻材料。该除草剂抗性基因的功能标记还可用于咪唑啉酮类除草剂抗性资源筛选。     

兼抗两种稻飞虱和稻瘟病多基因聚合系的创制

通过分子标记辅助选择创制兼抗稻飞虱和稻瘟病的水稻多基因聚合系,为选育持久多抗的水稻新品种提供材料来源。以携带稻瘟病抗性基因Pi1的材料‘CQ12261’为供体亲本,以兼抗白背飞虱和褐飞虱的优良恢复系‘桂恢1561’为轮回亲本,应用田间抗病虫性鉴定与分子标记辅助选择相结合的方法,并结合农艺性状的考察和分析。在BC₂F₄代中检测到6个株系的抗病基因Pi1已稳定遗传。至BC₂F₆代,GH-7、GH-8、GH-12这3个株系农艺性状与轮回亲本‘桂恢1561’相似,且丰产性好。通过常规的回交选育、分子标记辅助选择和田间抗性鉴定相结合的方法,筛选获得3份聚合了稻飞虱抗性基因Wbph9、Bph14、Bph15和稻瘟病抗性基因Pi1的水稻中间材料,为选育新的双抗病虫害水稻恢复系提供种质资源。     

芥菜型油菜多室基因Bjmc2的精细定位

芥菜型多室油菜的产量比普通两室油菜更高,定位乃至克隆多室基因可为油菜遗传改良及解释多室角果形成机制创造条件。本研究通过验证JD11-2家系衍生群体仅在BjMc2位点上存在差异,可用于BjMc2的定位。采用AFLP结合BSA法分析BC₅和BC₆群体,筛选到1个与BjMc2连锁的AFLP标记并转化为SCAR标记SC1。基于该AFLP标记序列信息,利用白菜同源序列设计SSR引物和SCAR引物,获得11对SSR标记和1对SCAR标记。通过在芥菜型油菜BAC文库中的挑选,获得2个覆盖目标区域的单克隆,由此开发1个SSR标记。将获得的SCAR和SSR标记扫描BC₇群体,构建了两室性状基因BjMc2的遗传连锁图,两侧最近标记ZX17和BACsr96与目标基因之间的遗传距离分别为0.048 cM和0.340 cM,并定位到白菜A7 scaffold000019的946~1014 kb之间,约68 kb物理距离。     

青海大黄油菜粒色性状分子标记的开发和图谱整合

利用青海大黄油菜和褐籽白菜型油菜09A-126构建BC₄和F₂分离群体, 结合AFLP与群体分离分析法(bulked segregant analysis, BSA)筛选引物, 获得5个与黄籽基因Brsc1紧密连锁的分子标记Y11~Y15。5个AFLP特异片段的序列, 均与白菜型油菜的A9染色体部分序列表现同源。将5个AFLP标记成功转化为5个SCAR标记(SC11~SC15)。利用目标基因所在染色体区段序列筛选到7个与目标基因紧密连锁的SSR标记(BrID10607、KS10760、B089L03-3和A1~A4)。利用SCAR和SSR标记扫描F₂群体中部分单株, 发现SC14和A1为共显性标记。用BC₄群体将Brsc1定位在标记Y06和A4之间1.7 Mb的区间内, 遗传距离分别为0.115 cM和0.98 cM。标记Y05和Y12与Brsc1共分离。本研究为黄籽油菜分子标记辅助选择育种体系的建立及目标基因的进一步精细定位和图位克隆奠定了基础。     

基于MAS技术对水稻保持系软华B稻瘟病抗性的分子改良

为选育优质抗稻瘟病保持系软华B,以携带稻瘟病抗性基因Pi46和Pi2的优质籼稻H281作为供体亲本、以软华B为轮回亲本,利用分子标记辅助选择(MAS)技术结合系谱选育法,聚合2个外源基因以改良保持系软华B。对性状稳定的改良株系进行稻瘟病抗性鉴定、稻米品质分析等。通过回交及多代自交,并结合分子标记检测,获得以软华B为遗传背景且含有2个纯合目标基因的BC₁F₆群体2个、BC₂F₅群体2个、BC₃F₄群体2个。田间自然诱发鉴定结果表明,不同回交世代改良材料在自然病圃均抗稻瘟病;育性鉴定结果显示,回交世代对不育系的不育度为52.7%～100.0%;农艺性状考查及米质分析表明,改良株系基本保留了软华B的主要农艺性状和稻米品质特性。SNP基因芯片分析结果显示,BC₁F₆的背景回复率为74.42%～77.77%,BC₂F₅的背景回复率为86.42%～87.75%,BC₃     

水稻高抗性淀粉含量基因sbe3-rs的KASP分子标记开发与利用

含有sbe3-rs基因型的高抗性淀粉含量水稻品种(系)具有调节餐后血糖、改善血脂和增强饱腹感等功效。以‘降糖稻1号’为sbe3-rs基因供体亲本通过杂交育种和回交转育方法培育优质高产高抗性淀粉水稻新品种具有重要意义。sbe3-rs基因第16个外显子的第105位处有T→C的碱基突变,基于该突变位点开发高通量KASP分子标记,利用该分子标记对杂交F₁后代50个单株和回交BC₁F₁后代44个单株共94份材料进行基因分型检测,结果显示50份F₁样品中有47个单株基因型为C/T杂合基因型,44份BC₁F₁群体材料中共有28个单株基因型为C/T杂合基因型。利用已开发的成熟的sbe3-rs的CAPS分子标记检测验证结果完全一致。同时利用开发的KASP分子标记对杂交F₂分离群体进行基因分型鉴定,对应高抗性淀粉基因型单株成熟期收获进行抗性淀粉含量测定,结果完全符合。结果表明针对sbe3-rs的突变位点开发KASP分子标记应用杂交回交群体,克服了前期开发...     

油菜Ogura-CMS恢复系分子标记二重PCR体系研究

油菜Ogura-CMS是由细胞质中线粒体嵌合基因Orf138和1对隐性细胞核基因(rfrf)共同控制的一种最稳定的雄性不育材料,是油菜杂种优势利用的一种有效途径.本研究根据不育基因Orf138和恢复基因Orf687的基因序列,设计2对PCR特异引物,以自育的Ogura-CMS不育系、恢复系以及杂交F1代植株的DNA为模版,建立了同时扩增不育和可育恢复基因的的二重PCR体系,用于油菜Ogura-CMS恢复系的分子标记辅助选择.结果表明,以建立的二重PCR体系用于F2代群体的分子标记辅助选择,其结果与常规测交结果完全吻合,可以有效提高其选择效率.本研究所建立的油菜萝卜质雄性不育恢复系分子标记二重PCR体系检测结果稳定可靠,为油菜Ogura-CMS恢复系的选择提供了一种实用可靠的标记辅助选择技术体系.     

大豆质核互作雄性不育系NJCMS3A双亲雄性育性基因的SSR标记

利用大豆质核互作雄性不育系NJMCS3A的质、核供体亲本N21566和N21249构建F₂和BC₁F₁育性分离群体进行雄性育性的遗传分析与基因定位。结果表明, F₁正反交可育,F₂和BC₁F₁的可育株与不育株分离比例经χ²测验分别符合3∶1和1∶1,表明NJCMS3A供体亲本雄性育性由一对基因控制,可育等位基因为显性。该基因可能是NJCMS3A的一个恢复基因。选用793对SSR引物对F₂和BC₁F₁群体分别进行育性基因定位,发现该育性基因位于O连锁群上,在Satt331和Satt477标记之间,与Satt331、CSSR133和Satt477标记距离的次序一致,分别为8.1~10.4 cM、11.4~16.4 cM、13.3~19.2 cM。     

一种抗草甘膦转基因油菜的快速鉴定方法

为建立一种快速鉴定抗草甘膦转基因油菜的方法,以抗草甘膦转基因油菜品系及后代分离群体为研究材料,利用不同草甘膦浓度滤纸平板进行种子发芽,观察抗性材料和非抗性材料幼胚抗性反应表型,并通过PCR和苗期草甘膦处理进行抗性验证。结果表明,利用0.5~1 g/L的草甘膦溶液处理的抗性材料胚根根毛生长正常,而非抗性材料胚根生长迟缓且光滑无根毛;利用该浓度的处理BC₁和F₂抗性分离群体,幼胚根毛有无性状分离比符合1:1和3:1,幼胚个体的基因组PCR扩增结果与根毛有无呈共分离。通过观察在该浓度草甘膦发芽处理后的幼胚根毛有无,可有效区分抗草甘膦转基因油菜的抗性和非抗性材料。本研究建立的鉴定方法不仅能够对抗草甘膦油菜材料进行快速、准确鉴定,而且能保证材料成活,对抗草甘膦转基因油菜育种和种子纯度鉴定提供技术参考。     

油菜乙酰乳酸合酶突变体S638N的酶学特性及其对ALS类除草剂的抗性

在对油菜抗咪唑啉酮类除草剂基因Bn ALS1R克隆与功能验证基础上,为比较抗性基因编码的乙酰乳酸合酶突变体S638N酶学特性及其对ALS类除草剂抗性与野生型的差异,构建基因原核表达载体,在大肠杆菌中表达S638N和野生型的重组融合蛋白。SDS-PAGE和Western blot分析表明,S638N和野生型均能表达出约74 k D的特异性重组蛋白。纯化目的蛋白,在不同温度和pH条件下,测定S638N和野生型的酶活性。结果显示,温度和pH对突变酶活性的影响与野生型相同,表现为先升后降,在37℃、pH 7.0条件下催化活性均最高。同时,该突变酶的酶学动力学参数Km和Vmax与野生型没有显著差异,其对3个辅助因子的响应曲线也与野生型类似,缺少其中任何一个辅助因子均使突变酶S638N基本都没有活性。然而,突变酶S638N对IMI类除草剂抗性显著高于野生型,而对Su类除草剂敏感性和野生型相同。因此,突变酶S638N具有对IMI类除草剂的专一抗性,但未改变酶学反应特征。     

Identification of RAPD markers linked to the rust (<Emphasis Type="Italic">Uromyces fabae</Emphasis>) resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Summary Two RAPD markers linked to gene for resistance (assayed as pustule number cm⁻² leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC₁F₁ [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F₁, F₂, BC₁F₁ and BC₁F₂ generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC₁F₁ individuals. Two RAPD makers, viz., SC10-82₃₆₀ (primer, GCCGTGAAGT), and SCRI-71₁₀₀₀ (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.     

Development of IP and SCAR markers linked to the yellow seed color gene in Brassica juncea L.

Previous studies showed that the yellow seed color gene of a yellow mustard was located on the A09 chromosome. In this study, the sequences of the molecular markers linked to the yellow seed color gene were analyzed, the gene was primarily mapped to an interval of 23.304 to 29.402M. Twenty genes and eight markers’ sequences in this region were selected to design the IP and SCAR primers. These primers were used to screen a BC₈S₁ population consisting of 1256 individuals. As a result, five IP and five SCAR markers were successfully developed. IP4 and Y1 were located on either side of the yellow seed color gene at a distance of 0.1 and 0.3 cM, respectively. IP1, IP2 and IP3 derived from Bra036827, Bra036828, Bra036829 separately, co-segregated with the target gene. BLAST analysis indicated that the sequences of newly developed markers showed good collinearity with those of the A09 chromosome, and that the target gene might exist between 27.079 and 27.616M. In light of annotations of the genes in this region, only Bra036828 is associated with flavonoid biosynthesis. This gene has high similarity with the TRANSPARENT TESTA6 gene, Bra036828 was hence identified as being the gene possibly responsible for yellow seed color, in our research.     

Use of molecular markers to accelerate the breeding of common bean lines resistant to rust and anthracnose

The main goal of this work was to introduce resistance genes for rust, caused by Uromyces appendiculatus, and anthracnose, caused by Colletotrichum lindemuthianum, in an adapted common bean cultivar through marker-assisted backcrossing. DNA fingerprinting was used to select plants genetically closer to the recurrent parent which were also resistant to rust and to race 89 of C. lindemuthianum. DNA samples extracted from the resistant parent (cv. Ouro Negro), the recurrent parent (cv. Rudá), and from BC₁, BC₂ and BC₃ resistant plants were amplified by the RAPD technique. The relative genetic distances in relation to the recurrent parent varied between 9 and 59% for BC₁, 7 and 33% for BC₂, and 0 and 7% for BC₃ resistant plants. After only three backcrosses, five lines resistant to rust and anthracnose with, approximately, 0% genetic distance in relation to the recurrent parent were obtained. These lines underwent field yield tests in two consecutive growing seasons and three of them presented a good yield performance, surpassing in that sense their parents and most of the reference cultivars tested.     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Inheritance of resistance to angular leaf spot in common bean and validation of the utility of resistance linked markers for marker assisted selection out side the mapping population

Inheritance of resistance to angular leaf spot (ALS) disease caused by Phaeoisariopsis griseola (Sacc.) Ferr was investigated in two common bean cultivars, Mexico 54 and BAT 332. Both Andean and Mesoamerican backgrounds were used to determine the stability of the resistance gene in each of the two cultivars. Resistance to P. griseola was phenotypically evaluated by artificial inoculation with one of the most widely distributed pathotypes, 63–39. Evaluation of the parental genotypes, F₁, F₂ and backcross populations revealed that the resistance to angular leaf spot in the cultivars Mexico 54 and BAT 332 to pathotype 63–39 is controlled by a single dominant gene, when both the Andean and Mesoamerican backgrounds were used. Allelism test showed that ALS resistance in Mexico 54 and BAT 332 to pathotype 63–39 was conditioned by the same resistance locus. Resistant and susceptible segregating populations generated using Mexico 54 resistant parent were selected for DNA extraction and amplification to check for the presence /absence of the SCAR OPN02 and RAPD OPE04 markers linked to the Phg-2 resistance gene. The results indicated that the SCAR OPN02 was not polymorphic in the study populations and therefore of limited application in selecting resistant genotypes in such populations. On the other hand, the RAPD OPE04 marker was observed in all resistant individuals and was absent in those scored susceptible based on virulence data. Use of the RAPD OPE04 marker in marker-assisted selection is underway.