甘蓝型油菜隐性三系核不育上位基因Rf精细定位

甘蓝型油菜隐性三系核不育系因不育性稳定、不育基因易转育、恢复谱广、无胞质负效应、制种产量高等优点在生产上得到广泛应用.其不育性状受两对隐性重叠不育基因(ms1和ms2)与一对隐性上位抑制基因(rf)互作控制.ms1和ms2同时纯合(ms1ms1ms2ms2)表现不育,但隐性纯合rf(rfrf)对ms1ms1ms2ms2的表达起抑制作用,又使其表现可育(临保系,ms1ms1ms2ms2rfrf).本研究利用BSA群分法,通过AFLP分子标记和SRAP分子标记的筛选,利用Rf基因的BC1分离群体( ms1ms1ms2ms2Rfrf+ms1ms1ms2ms2rfrf)进行遗传连锁分析.结果表明,共筛选到4个与Rf基因均共分离的标记,这些标记在该基因不同遗传背景的BC1群体中均与Rf基因紧密连锁.标记测序序列BLASTn结果表明,其位于大白菜A7染色体Scaffold000017(3.3 Mb)上.根据该Scaffold序列信息,开发了一系列SSR引物,多态性SSR引物在Rf基因的一个BC1群体中进行连锁分析,最终将该基因限定在245kb的一个范围内.其中SSR标记A7-10、A7-24与Rf基因共分离,A7-24为共显性标记.     

甘蓝型油菜细胞核雄性不育性的遗传研究——Ⅰ.隐性核不育系9012A的遗传

研究结果表明:(1)甘蓝型油菜核不育系9012A不同于显性上位互作核不育,是一种新的基因互作核不育类型,其不育性受2对隐性重叠不育基因和1对隐性上位抑制基因互作控制,当隐性上位基因纯合时,对隐性不育基因起上位抑制作用。(2)在甘蓝型油菜中存在至少2套独立的不同隐性重叠不育基因,隐性上位基因与不同的隐性重叠不育基因之间的互作是非专一性的,这对于现有双基因隐性核不育杂种优势利用具有重要意义。 隐性上位互作核不育完全保持系的分离解决了传统隐性核不育杂种优势利用难点,为隐性核不育杂种优势利用开辟了新途径。这类核不育不仅在杂优育种实践中具有较高的开发利用价值,同时也是研究植物细胞核雄性不育机理的理想材料。     

甘蓝型油菜细胞核雄性不育性的遗传研究：I.隐性核不育系9012A的遗传

研究结果表明：（１）甘蓝型油菜核不育系９０１２Ａ不同显性上位互作核不育，是一种新的基因互作核不育类型，其不育性受２对隐性重叠不育基因和１对隐性上位抑制基因互作控制，当隐性上位基因纯合时，对隐性不育基因起上位抑制作用。（２）在甘蓝型油菜中存在至少２套独立的不同隐性重叠不育基因，隐性上位基因与不同的隐性重叠不育基因之间的互作是非专一性的，这对于现有双基因隐性核不育杂交优势利用具有重要意义。隐性上位互作     

甘蓝型油菜细胞核雄性不育材料118 A的遗传与应用研究

经遗传研究表明：核不育系118A的不育性受两对隐性重叠不育基因和一对隐性上位互作基因控制，不育基因与117AB、S45AB的不育基因为非等位基因，当两对基因隐性纯合，另一对上位互作基因为显性（RfRf或Rfrf）时，植株表现不育（aabbRfRf或aabbRfrf），其相应临保系的基因型为3对隐性纯合体（aabbrfrf）。纯合两型系内不育株与可育株（AabbRfRf或aaBbRfRf）杂交，F1为1：1的育性分离，系内可育株自交为3：1的育性分离。利用纯合两型系AabbRfRf或aaBbRfRf中不育株和可育株成对兄妹交可生产纯合两型系和不育系118A，不育系和临保系杂交生产全不育系118CA（aabbRfrf），全不育系与恢复系（AA-或-BB）杂交生产杂交种。     

甘蓝型高油分隐性上位互作核不育系粱全1504A的选育研究

粱全1504A是贵州粱丰农业科技有限公司以引进甘蓝型油菜杂交种核优202杂交种后代可育株连续自交、测交、兄妹交育成纯合两型系(粱纯4A),与引进甘蓝型油菜杂交种核优202杂交种后代不育株和从贵州省油菜所引进隐性上位互作核不育黄籽临保系(S2375C-15)杂交转育的临保系(粱临4C)组配的甘蓝型油菜隐性上位互作核不育系,该不育系综合性状优良,株型紧凑,含油量较高,一般配合力较高,组配的隐性上位互作核不育杂交油菜组合丰产性好,抗性好,品质性状优良,有广泛的应用前景。     

高油分隐性上位互作核不育纯合两型系60AB及同型临保系60C的选育

甘蓝型油菜隐性上位互作核不育材料的选育中,临保系的选育是基础,纯合两型系的选育是关键,在实际育种过程中,由于甘蓝型油菜隐性上位核不育的育性受2对重叠基因和1对上位基因抑制[1],分离较为复杂,不育株率比例干扰较大,选育世代较长。本研究以贵州省油菜研究所选育的油分较低(含油量42.32%)的全不育系HF 1AC系为母本,通过在杂交后代的苗期或初花期提取油菜嫩叶DNA,标记上位基因Rf,结合生物学性状观察及品质分析,有针对性的进行测交、自交,成功选育了高油分隐性上位互作核不育纯合两型系60AB及同型临保系60C。60AB的品质为芥酸0.73%、硫甙24.44μmol/g(饼)、含油量48.73%、种子蛋白24.07%;60C的品质为芥酸0.45%、硫甙29.62μmol/g(饼)、含油量45.23%、种子蛋白27.13%;60AB与60C配制的全不育系60AC的品质为芥酸0.72%、硫甙29.63μmol/g(饼)、含油量45.38%、种子蛋白26.68%。分子标记技术在选育甘蓝型油菜隐性上位互作核不育纯合两型育系及同型临保系上的应用,提高了育种效率,加快了育种进程。     

甘蓝型油菜隐性核不育材料ZWA的遗传利用研究

通过遗传测交试验表明:甘蓝型油菜核不育系ZWA的不育性由两对隐性重叠基因和一对上位抑制基因共同控制.当两对重叠隐性基因呈双隐性纯合体(m1m1m2m2),而另一对抑制基因的基因型为RfRf或Rfrf时,植株表现为不育(m1m1m2m2RfRf或m1m1m2m2Rfrf,其相应临保系的基因型为三对隐性纯合体(m1m1m2m2rfrf.纯合两型系不育株(m1m1m 2m2RfRf与可育株(M1m1m2m2RfRf或m1m1M2m2RfRf兄妹交,后代可育株与不育株的分离比例为1:1;可育株自交,其可育株与不育株比例为3:1.利用纯合两型系中m1m1m2m2RfRf不育株与临保系(m1m1m2m2rfrf测交可获得基因型为(m1m1m2m2Rfrf全不育系,全不育系与相应恢复系(M1M1 或M2M2 )杂交可获得全可育杂交种,从而将隐型核不育系ZWA的三系杂交种应用于生产.     

甘蓝型油菜隐性核不育系20118A的育性遗传及分子标记辅助选择

以20118A不育系和临保系与22个油菜品种(系)杂交测交后代为材料, 采用经典遗传学和分子标记辅助选择方法, 验证该不育系统遗传控制体系及等位基因分布频率; 探索利用连锁共显性标记筛选两型系和临保系基因型的高效性和准确率。研究表明, 6个品种(系)与20118A测交产生的F₂世代, 所有组合可育株∶不育株均符合3∶1或13∶3分离规律, 而与20118A-TAM杂交产生的6个F₂中1个呈13∶3分离, 其余均为全可育, 育性符合1对隐性不育基因和1对隐性上位抑制基因互作控制的遗传模式; 进而采取反向验证方法, 从1 059个F₂分离单株中, 用新发展的Bnms₃/Bnrf连锁共显性标记跟踪选择, 直接获得临保系(ms₃ms₃rfrf)、纯合不育株(ms₃ms₃RfRf)和两型系可育株(Ms₃ms₃RfRf) 70、69和135株, 经测交或互交验证, 准确率均达95%以上; 根据20个测交品种的后代分离, BnRf位点上只出现Rf和rf两个等位基因, 推测第3个等位基因存在的频率很低, 基本可以根据两基因各2等位基因互作原理开展分子标记辅助育种。     

甘蓝型油菜隐性核不育的遗传与应用研究

雄性不育是植物杂种优势利用的重要途径，利用杂种优势成功地解决了油菜品质育种过程中高产与优质的矛盾，从而使双低杂交油菜的种植面积迅速扩大。目前我国油菜雄性不育的研究呈现多类型并用的局面。以Po1A、陕2A和MICMS为代表的细胞质雄性不育系已大量应用于生产中。细胞核雄性不育已应用的有三类：一是李树林(1985)报道的两对显性基因互作控制的双显性核不育；第二类是由两对隐性重叠基因控制的双隐性核不育；第三类是陈凤祥(1993)报道的二对隐性重叠不育基因与一对隐性上位抑制基因控制的三隐性核不育。     

油菜隐性细胞核雄性不育的研究进展

油菜细胞核雄性不育是油菜杂种优势利用的重要途径。近年来,油菜隐性细胞核雄性不育研究取得了一些进展并育成了一批核不育杂交种。为了促进核不育研究的深入及油菜杂种优势利用水平,对双隐性核不育和隐性上位互作核不育2种类型的核不育系统的遗传模式、相关基因定位以及其不育分子机理方面的研究进行综述,并对其研究成果在将来油菜杂交育种中的利用进行展望。     

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 AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Identification of AFLP fragments linked to one recessive genic male sterility (RGMS) in rapeseed (Brassica napus L.) and conversion to SCAR markers for marker-aided selection

117AB is a recessive genic male sterility (RGMS) line in which the sterility is controlled by a duplicate recessive gene named ms, located at two separate loci. In the RGMS line, the genotype of the sterile plant (117A) is msmsmsms, and that of the fertile plant (117B) is Msmsmsms. The present study was aimed to identify DNA markers linked to the ms locus by amplified fragment length polymorphism (AFLP). From the survey of 512 AFLP primer combinations, 6 AFLP fragments (y1, k1, k2, k3, k4, k5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among which two fragments, designated as k2 and k3, co-segregated with the target gene in the tested population. Fragment k2 was successfully converted into a sequence characterized amplified region (SCAR) marker. The markers detected could be valuable in marker-assisted breeding of RGMS in Brassica napus.     

Molecular mapping of a dominant genic male sterility gene Ms in rapeseed (Brassica napus)

Rs1046AB is a genic male sterile two‐type line in rapeseed that has great potential for hybrid seed production. The sterility of this line is conditioned by the interaction of two genes, i.e. the dominant genic male sterility gene (Ms) and the suppressor gene (Rf). The present study was undertaken to identify DNA markers for the Ms locus in a BC₁ population developed from a cross between a male‐sterile plant in Rs1046AB and the fertile canola‐type cultivar ‘Samourai’. Bulked segregant analysis was performed using the amplified fragment length polymorphism (AFLP) methodology. From the survey of 480 AFLP primer combinations, five AFLP markers (P10M13₃₅₀, P13M8₄₀₀, P6M6₄₁₀, E7M1₂₃₀ and E3M15₁₀₀) tightly linked to the target gene were identified. Two of them, E3M15₁₀₀ and P6M6₄₁₀, located the closest, at either side of Ms at a distance of 3.7 and 5.9 cM, respectively. The Ms locus was subsequently mapped on linkage group LG10 in the map developed in this laboratory, adding two additional markers weakly linked to it. This suite of markers will be valuable in designing a marker‐assisted genic male sterility three‐line breeding programme.     

A codominant SCAR marker linked to the genic male sterility gene (ms1) in chili pepper (Capsicum annuum)

As one of the genic male sterility (GMS) materials in chili pepper ( Capsicum annuum L.), GMS1 has been used for commercial F₁ hybrid seed production. The male sterility of GMS1 is controlled by a recessive nuclear gene, named ms ₁ . In this study, we developed DNA markers linked to the ms ₁ locus using a combination of bulked segregant analysis and amplified fragment length polymorphism (AFLP) in a segregating sibling population. From the screening of 1024 AFLP primer combinations, the AFLP marker E-AGC/M-GTG (514 bp) was identified as being linked to the ms ₁ locus at a distance of about 3 cM. Based on internal sequencing analysis of the E-AGC/M-GTG marker between male fertile and sterile plants, we identified three small deletions with a size of altogether 42 bp in the male-fertile plant and developed a codominant sequence characterized amplified region (SCAR) marker. This SCAR marker may be valuable for marker-assisted breeding in the hybrid seed production system of chili pepper using the GMS1 line.     

Development and characterization of SCAR markers associated with a dominant genic male sterility in rapeseed

Rs1046AB is a dominant genic male sterility (DGMS) line in rapeseed, in which the sterility has always been thought to be conditioned by the interaction of a male sterility gene ( Ms ) and its non-allelic restorer gene ( Rf ). This system provides not only a tool for assisting in recurrent selection but also a promising system for hybrid production. Based on previous studies, two amplified fragment length polymorphism markers linked with the Ms gene were converted into a dominant and a co-dominant sequence characterized amplified region (SCAR) marker, respectively. The putative linear order relationship of three dominant SCAR markers with the same genetic distance from the Rf gene, was also determined by an examination of whether the homologues of these markers are present or not in different lines carrying Rf . A bigger fragment generated by the closest marker linked to the Rf gene was observed in all lines carrying the recessive allele rf , suggesting that this marker is a co-dominant marker, which was further confirmed by nucleotide sequence comparison of these fragments. SCAR markers specific for Ms and Rf will be especially valuable in marker-assisted DGMS three-line breeding.     

Molecular markers linked to <Emphasis Type="Italic">Bn</Emphasis>;<Emphasis Type="Italic">rf</Emphasis>: a recessive epistatic inhibitor gene of recessive genic male sterility in <Emphasis Type="Italic">Brassica napus </Emphasis>L.

7–7365AB is a recessive genic male sterile (RGMS) two-type line, which can be applied in a three-line system with the interim-maintainer, 7–7365C. Fertility of this system is controlled by two duplicate dominant epistatic genes (Bn;Ms3 and Bn;Ms4) and one recessive epistatic inhibitor gene (Bn;rf). Therefore an individual with the genotype of Bn;ms3ms3ms4ms4Rf_ exhibits male sterility, whereas, plant with Bn;ms3ms3ms4ms4rfrf shows fertility because homozygosity at the Bn;rf locus (Bn;rfrf) can inhibit the expression of two recessive male sterile genes in homozygous Bn;ms3ms3ms4ms4 plant. A cross of 7–7365A (Bn;ms3ms3ms4ms4RfRf) and 7–7365C (Bn;ms3ms3ms4ms4rfrf) can generate a complete male sterile population served as a mother line with restorer in alternative strips for the multiplication of hybrid seeds. In the present study, molecular mapping of the Bn;Rf gene was performed in a BC₁ population from the cross between 7–7365A and 7–7365C. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) technique was used to identify molecular markers linked to the gene of interest. From a survey of 768 primer combinations, seven AFLP markers were identified. The closest marker, XM5, was co-segregated with the Bn;Rf locus and successfully converted into a sequence characterized amplified region (SCAR) marker, designated as XSC5. Two flanking markers, XM3 and XM2, were 0.6 cM and 2.6 cM away from the target gene, respectively. XM1 was subsequently mapped on linkage group N7 using a doubled-haploid (DH) mapping population derived from the cross Tapidor × Ningyou7, available at IMSORB, UK. To further confirm the location of the Bn;Rf gene, additional simple sequence repeat (SSR) markers in linkage group N7 from the reference maps were screened in the BC₁ population. Two SSR markers, CB10594 and BRMS018, showed polymorphisms in our mapping population. The molecular markers found in the present study will facilitate the selection of interim-maintainer.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

EST辅助的甘蓝型油菜显性核不育AFLP标记转化

甘蓝型油菜显性核不育广泛应用于轮回选择和杂种优势利用,不育基因标记的开发与应用对于基因克隆和育种实践具有重要意义。基于AFLP标记SA12MG14的序列信息,从拟南芥整合数据库中,检索与标记序列同源的甘蓝型油菜EST,结合标记和EST序列设计特异引物,转化成新的SCAR标记。获得的SCAR标记S6B3,具有很高的检测稳定性,在回交群体Popu2上分析验证,结果与AFLP标记完全一致。该标记与不育基因相距0.3 cM,将其用于临保系同源的纯合型不育系选育,可有效提高育种工作效率。