分子标记辅助选育萝卜雄性不育系

利用Ogura CMS不育基因orf138特异标记及细胞核育性恢复基因Rfo的KASP标记,在苗期对6个可育的中国水果萝卜育性基因型进行鉴定。结果表明:6个水果萝卜的细胞质均为正常可育胞质。细胞核育性恢复基因Rfo的基因型在不同单株间存在差异,所鉴定的60个单株中,基因型rfrf、Rfrf、Rf Rf分别占40.0%、33.3%和26.7%。以日本白萝卜品种献夏37为不育源,选择具有rfrf基因型的单株与不育源进行杂交与回交,经过5代的回交及父本自交,获得4个不育率100%、遗传稳定、整齐度高的绿皮绿肉水果萝卜雄性不育系及保持系。利用这些雄性不育系配制杂交组合,获得7个优良的杂交组合。分子标记辅助选育萝卜雄性不育系,避免配制大量杂交组合及田间育性鉴定,提高了选择效率与精准度。     

Ogura CMS育性恢复基因orf687在萝卜中的分布

以129个萝卜品种(包括5个栽培种和4个野生种)为试材,研究了Ogura CMS细胞核育性恢复基因orf687(Rfo)的分布。对orf687基因扩增的结果表明,除油用萝卜外,orf687目的片段广泛分布于东亚大长萝卜(83份,89.16%)、欧洲小萝卜(28份,85.71%)、黑萝卜(6份,100%)、荚用萝卜(1份,100%)和野生萝卜(10份,70%)中。东亚大长萝卜中的红萝卜品种95.45%含有orf687,高于白萝卜和绿萝卜。进一步利用PCR-RFLP对orf687的基因型进行鉴定发现,没有恢复功能的基因型(rfrf)在野生萝卜中所占比率最高,为57.14%,其次是东亚大长萝卜(47.13%)和欧洲小萝卜(37.50%)。在东亚大长萝卜中,红萝卜品种具有rfrf基因型的比率为76.19%,高于白萝卜(41.67%)和绿萝卜(23.53%)。在利用萝卜细胞质雄性不育材料育种的过程中,rfrf基因型的材料没有育性恢复功能,可作为保持系,用于雄性不育系的转育。上述结果可为萝卜雄性不育系的选育和利用提供指导。     

辣椒恢复基因连锁标记的适用性评价与应用

为了更准确、高效地利用分子标记辅助选择技术进行辣椒细胞质雄性不育育性基因的选择,本试验利用36份已知基因型(Rf Rf或rfrf)的辣椒自交系对11对已报道的辣椒恢复基因连锁标记进行适用性检验。结果表明,显性标记CRF3S1S和CRF-SCAR的适用性最广,其准确率分别为91.67%、88.89%;在6对共显性标记中Ca Rf-FL-M2的适用性最广,其准确率为80.56%。育种实际应用中可以利用标记CRF3S1S或CRF-SCAR对辣椒材料进行初次鉴定,对其中含有Rf基因的辣椒材料利用标记Ca Rf-FL-M2进行再次鉴定。     

KASP分子标记辅助选育甜椒核雄性不育两用系

为了进一步提高甜椒核雄性不育两用系育种效率,利用AB91核雄性不育候选基因Capana05g000747的突变位点设计KASP分子标记,以转育甜椒核雄性不育两用系的F2群体及育成的两用系为试验材料,对植株的育性进行基因型检测,并检验该标记的准确性。结果表明,该分子标记与甜椒核雄性不育的育性呈现共分离,在苗期对F2植株的基因型进行早期鉴别,分型准确率分别为98.91%和99.04%,可有效淘汰基因型为MSMS的可育株,筛选出基因型为MSms的可育株直接用于不育系的转育,转育出了黄色和红色甜椒核雄性不育两用系AB91-HTJ412和AB91-HLD163。在苗期对两用系群体的育性基因型进行早期鉴别,可有效淘汰基因型为MSms的可育株,选留基因型为msms的不育株直接用于杂交组合选配及杂交制种。     

萝卜细胞质雄性不育遗传机制研究

利用3对肉质根皮色分别为红、白、青的萝卜不育系及其保持系进行配组,对其后代进行不育率统计,结果表明同一保持系与不同不育系杂交的F1代育性表现不同,与同一不育系内不同单株的杂交F1代育性表现也不同;不同组合之间的育性表现呈连续变异的现象,说明萝卜的细胞质雄性不育(CMS)受多基因控制,并提出用核质不协调假说加以解释,还据此提出了创造、筛选和转育萝卜CMS不育源的方法.     

大白菜Ogura胞质雄性不育恢复材料的创制与检测

针对白菜类蔬菜(Brassica rapa L.)中缺乏Ogura胞质雄性不育育性恢复材料的问题,利用远缘杂交技术将恢复基因Rfo从甘蓝型油菜导入大白菜中,利用种间杂交种不断与不育系进行回交,已分别获得BC₁、BC₂和BC₃代育性恢复材料。同时,利用形态学、细胞遗传学、分子生物学手段对所创制的材料进行检测,利用恢复基因Rfo特异性引物扩增,最后筛选获得了一株育性稳定、形态特征接近大白菜、染色体数目为20的BC₃单株。利用所创制BC₃单株进行Ogura雄性不育大白菜商品杂交种恢复测试,结合表型及恢复基因Rfo、不育基因orf138特异性引物检测,结果表明,所创制恢复系恢复基因有效。     

春夏及秋冬萝卜核-质互作型雄性不育系转育的研究

根据不同萝卜可育品系与不育系测交一代的育性表现,确定了萝卜雄性不育基因ms在萝卜品种中的分布频率,同时也明确了萝卜可育父本的基因型.可将萝卜可育父本的基因型分为三种:(1)保持型:与不育系杂交后代表现全不育;(2)部分保持(或恢复)型:与不育系杂交后代表现育性分离;(3)恢复型:与不育系杂交后代表现全可育.父本基因型不同,转育模式不同.本文提出了不育系的两种转育方案,四种不同的转育模式.同时,为选育经济性状优良的雄性不育系,对各种性状的转化速度进行了测定,结果表明,性状转化速度较快,可在较短时间内选育出经济性状符合要求的雄性不育系.     

辣椒雄性不育分子生物学研究进展

遗传定位、基因标记与克隆、标记辅助选择(MAS)等分子生物学技术在作物品种改良中发挥了重要作用。近年来,辣椒分子生物学研究取得了一定进展:(1)构建了一批分布广泛、覆盖整个基因组的种间或种内遗传图谱。(2)对C.annuum及其野生种C.annuum var.glabriusculum 和C.baccatum的全基因组进行测序,明确了辣椒基因组的大小(3.48 Gb)为番茄(Solanum lycopersicum L.)基因组(900 Mb)的4倍。(3)开发了一批连锁标记,奠定了利用基因克隆和MAS技术培育优良品种的基础。(4)发现20个独立遗传的核雄性不育(NMS)基因,开发了与ms1、ms3、ms8、ms10、ms_k、msc-1和一个未命名基因的连锁标记,但除ms1、ms3、ms8和ms10外,其他的NMS基因尚未定位。(5)开发了与辣椒胞质雄性不育系(CMS)不育性相关的线粒体基因atp6的标记,鉴定了与育性恢复(Rf)相关基因,但Rf没能在遗传图谱中定位;鉴定和标记了影响CMS系育性恢复的相关核基因(pr)。综述了辣椒分子生物学研究进展,这些信息将对辣椒种质资源评价、利用MAS培育NMS系和提高NMS系选育效率、利用rf和Rf基因选育CMS的保持系和恢复系,以及杂交种子纯度鉴定等具有一定参考价值。     

Ogura CMS青花菜育性恢复材料创制及其遗传背景研究

利用含有育性恢复基因Rfo的芥蓝中间材料(14Y12),通过杂交转育途径将该基因导入6份优良Ogura CMS青花菜中,并从农艺性状、细胞水平和遗传多样性方面揭示其变化特征及遗传背景.分子鉴定结果表明,Ogura CMS青花菜Rfo杂交后代(F1代)中,14个株系含有Rfo基因,Rfo基因传递效率约为40％.在农艺性状...     

辣椒恢复基因SSR标记定位及分子标记辅助选择育种

利用175对均匀分布在辣椒染色体上的SSR引物对辣椒不育系113A、恢复系139及其F2代群体进行筛选,发现AF208834引物和Rf基因连锁,遗传距离为20.8cM。用CRF-SCAR标记对36份恢复系材料进行分子标记辅助选择验证,结果表明,30份恢复系有特异条带,正确率为83.33%,并从108份未知育性材料中,检测到41份有恢复基因的材料;同时将SSR标记和SCAR标记结合起来,建立了与育性基因有关的多重PCR技术,进行辣椒恢复基因及其等位基因纯合状态的选择,显著提高了辣椒雄性不育三系的选择效率。     

黑萝卜胞质类型的分子鉴定与育种利用

萝卜胞质类型鉴定是杂种优势利用的前提.以NWB/DCGMS CMS及Ogura CMS两个萝卜细胞质雄性不育特异基因设计引物,对3份黑萝卜材料进行胞质类型鉴定.结果表明,3份可育黑萝卜资源均含有NWB/DC-GMS CMS不育主控基因orf463,利用3份黑萝卜为母本与其他萝卜构建的F2代遗传群体表现出育性分离现象,根...     

辣椒细胞质雄性不育基因对不育系及杂交一代农艺性状和生化特性的影响

 研究了两个不育系与它们各自的保持系间, 及其分别与恢复系的杂种一代间农艺性状和花蕾、叶片中生化物质含量间的差异。结果表明, 不育系杂种和保持系杂种在株高上的差异达到了显著水平。花蕾和叶片中过氧化物酶活性不育系高于保持系, 不育系杂种显著或极显著高于保持系杂种。花蕾中游离脯氨酸含量不育系小于保持系, 不育系杂种极显著小于保持系杂种。花蕾和叶片中IAA 含量不育系低于保持系, 不育系ABA 含量在叶片中高于保持系, 在花蕾中低于保持系。细胞质雄性不育基因对杂种一代内源激素IAA、ABA 和ZRs 含量影响的规律不明显。     

Genetics and distribution of fertility restoration associated RAPD markers in inbreds of pepper (Capsicum annuum L.)

Experiments were conducted to study genetics of fertility restoration and to examine distribution of RAPD markers (OPW19₈₀₀ and OPP13₁₄₀₀) linked with fertility restoration gene (Rf) in pepper (Capsicum annuum L.) inbreds. Forty-two hot and five sweet pepper inbreds were crossed on a cytoplasmic male sterile (cms) line CCA-4261 and F₁s were evaluated for fertility restoration under open field conditions. DNA of 5 plants of CCA-4261 and individual plants of 47 inbreds was isolated and PCR reaction was performed using OPW19 and OPP13 primers. The results revealed that most of the hot pepper lines posses Rf gene. The Rf gene associated two markers, viz., OPW19₈₀₀ and OPP13₁₄₀₀ were not frequently distributed in the restorer inbred lines because presence of marker bands often does not coincide with the presence of Rf gene identified in many restorer inbreds. The case specific applications of both the RAPD markers have been described.     

Inheritance of fertility restoration of male-sterility conferred by cytotype Y and identification of instability of male fertility phenotypes in onion (Allium cepa L.)

A novel onion (Allium cepa L.) cytoplasm, cytotype Y, was found in a previous study. Cytotype Y contained unique stoichiometry of coxI and orf725, a candidate gene responsible for male-sterility induction in onions. A S₁ segregating population was produced from a single plant selected from PI273626. Although male-fertility segregated in this population, the ratio significantly deviated from single-gene inheritance. However, genotypes of RF31446 marker perfectly linked to Ms locus-controlling fertility restoration completely matched with male-fertility phenotypes, indicating that male-fertility restoration of male-sterility conferred by cytotype Y might be determined by the Ms locus. One plant derived from the S₁ population showed discrepancy between male-fertility phenotype and RF31446 genotype. Although the RF31446 genotype was homozygous recessive, reduced amount of pollen grains were observed in anthers. Many pollen grains of the unstable male-sterile plant were deformed. Analysis of 13 molecular markers flanking the Ms locus showed no crossover between the Ms locus and the RF31446 marker. Ten more unstable male-sterile plants were identified from open-pollinated progenies of the unstable male-sterile plant. Viable seeds were successfully produced from unstable male-sterile plants, indicating that pollen grains of the unstable male-sterile plants were partially viable. In addition, an umbel containing unstable male-fertile flowers was identified from one of maintainer lines, although both male and female organs might be sterile in these flowers.     

Identification of allelic variation in the atp6 gene and its use in developing molecular markers for distinguishing two cytoplasmic types of bunching onion (Allium fistulosum L.)

The atp6 is a significant candidate gene that may be related to cytoplasmic male sterility (CMS) in higher plants. In this study, atp6 gene fragments were cloned from the CMS line and its maintainer line in bunching onion. The primers were designed according to sequences obtained from GenBank accession no. JQ283733. An important variation between the CMS and maintainer line was observed at the 171T and 171A position, respectively. The atp6 genes obtained from the CMS line had 171T mutations designated as T-atp6 (GenBank accession no. KR973431), and the maintainer line had 171A mutations designated as A-atp6 (GenBank accession no. KR973430). Furthermore, some plants in the maintainer line possessed both 171A and 171T mutations designated as heteroplasmic genotype A/T-atp6. To confirm this point mutation site, the derived cleaved amplified polymorphic sequence (dCAPS) method was used. Consequently, we developed a competitive allele-specific PCR (KASP) marker based on the observed single nucleotide polymorphisms (SNPs) in the atp6 genes. This marker will allow breeders to identify male-sterile or fertile cytoplasmic types on a large scale and may significantly contribute to the breeding of bunching onion F₁ hybrid cultivars.     

大葱细胞质雄性不育基因的SCAR 标记开发

 以大葱（Allium fistulosum L.）雄性不育系和保持系为试材,开发了一个能够鉴别细胞质雄性不育类型的SCAR 标记。此标记在大葱雄性不育系（S 型细胞质）中扩增出１条607 bp 片段,而在保持系（N 型细胞质）中没有扩增出此片段,将此片段命名为S607。对5 组不同遗传背景的不育系和相应保持系,以及4 份杂交组合进行验证,SCAR 标记S607 鉴定结果与实际细胞质类型完全相符。     

洋葱T型细胞质雄性不育与花蕾ATP含量的关系

以洋葱T型细胞质雄性不育系8-43A、保持系8-43B和恢复系8-43-57C为试材,采用萤火虫荧光素酶检测技术,分析花蕾不同发育时期三磷酸腺苷(ATP)的含量变化,研究洋葱T型细胞质雄性不育与花蕾ATP含量的关系。结果表明:在整个花药发育过程中的各个阶段,保持系和恢复系的花蕾之间ATP含量均无显著差异,不育系花蕾ATP含量均极显著低于保持系和恢复系,尤其在花粉粒成熟早期,仅为保持系和恢复系的1%;花粉粒成熟早期,不育系花蕾ATP含量下降到不足四分体时期的1/10,说明花药发育通过单核期后耗费了组织内大量的ATP,更重要的是ATP的产生可能出现了障碍;洋葱T型细胞质雄性不育系花蕾ATP含量极显著低于各时期的可育株,可能是导致其雄性不育的原因。     

大白菜pol CMS育性恢复基因的表达分析

 为进一步探明pol CMS育性恢复基因作用的分子机理,利用数字基因表达谱技术,选用不育系与恢复系杂交的F₂代分离群体,对大白菜polCMS育性恢复相关基因的差异表达进行了分析,并选取部分基因进行了实时荧光定量PCR验证。共有2 826个基因差异表达,其中441个上调表达,2 385个下调表达。GO功能注释表明,差异表达基因显著富集的细胞位置为细胞质、细胞器及大分子复合物等位置,细胞器包括线粒体、叶绿体及质体等,分子功能主要为核酸外切酶的活性,参与的生物过程是花粉壁的形成和组装。与pol CMS显著相关的通路主要是核糖体、糖和氨基酸代谢、核苷酸切除和修复、RNA降解等通路。表达谱和RT-PCR结果表明恢复基因主要通过下调表达调控育性恢复,有4个差异表达基因与育性恢复密切相关。     

几种类型甘蓝雄性不育的研究与显性不育系的利用

隐性不育材料83121ms，不育株率仅50％。黑芥胞质不育材料CMSN78091不育花不能完全开放，蜜腺小。萝卜胞质不育材料CMSR1409等及其转育后代开花结实性状不良，且低温下叶色黄化，以上3类不育材料在甘蓝实际育种中应用困难。改良的萝卜胞质不育材料CMSR29551等及其转育后代低温下叶色不黄化，在其转育后代中筛选出几份开花结实性状较好的不育系，但多代回交后不育系配制的F1杂种优势弱，应用有局限性。改良的萝卜胞质不育系CMSR3625等，植株性状、开花结实特性及配合力均较好，有较好的应用前景。显性雄性不育系不育性及经济性状优良，已配制出中甘16号和中甘17号两个优良杂种一代，并通过全国农作物品种审定委员会审定。