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.     

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.     

甘蓝型油菜显性细胞核雄性不育基因的AFLP标记

用甘蓝型油菜双基因显性细胞核雄性不育系Rs1046A和欧洲油菜品种Samourai构建了一个回交分离群体。在群分法（BSA）构建的不育池和可育池中共筛选了256对AFLP引物组合,找到了与不育基因紧密连锁的两个AFLP标记(EA03MC1599和EA07MC01235), 它们与不育基因的遗传图距分别是3.5 cM和5.5 cM,而且位于不育基因的同一侧,标记间相     

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.     

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.     

高产优质甘蓝型核不育杂交种双油杂1号的选育

双油杂1号是河南省农业科学院经济作物研究所选育的高产优质半冬性隐性核不育甘蓝型油菜两系杂交种。该品种农艺性状优良、抗寒、抗倒伏、较耐菌核病、抗病毒病。在2年河南省油菜区域试验中平均单产2731.2kg/hm2,比对照杂98009增产8.58%。在河南省油菜生产试验中,双油杂1号平均单产2489.1kg/hm2,比对照杂交种杂98009增产3.93％。品质优良,芥酸含量0.1％,硫甙含量18.85μmol/g饼,含油量40.96％,品质符合国家标准。     

Establishment of a random-mating population of 'Polima' CMS restorers in Brassica napus by use of dominant genie male sterility

Five restorers of ‘polima’ cytoplasmic male sterility (pol CMS) cannot restore the fertility in dominant genie male sterility (DGMS). A dominant male sterility gene from both, a DGMS line Rs l046AB and DGMS hybrid ‘Zhongza No. 3’, was successfully introduced into Polima cytoplasm. A random-mating population of pol CMS restorers was established by using many double-low pol CMS restorers as pollinators to cross continuously to the DGMS plants which had Polima cytoplasm.     

Efficiency of a novel gametocide amidosulfuron on rapeseed (Brassica napus)

The efficiency of a novel gametocide amidosulfuron, 1-(4, 6-dimethoxypyrimidin- 2-yl) -3-mesyl (methyl) sulfamoylurea, was evaluated on rapeseed ( Brassica napus L.). Double application of 0.09–0.12 g/ha amidosulfuron at the uni-nucleate stage of longest buds and 12 days later, made 97.4–100% plants male-sterile in seventeen out of 20 cultivars tested. The shrunken anthers could not release pollen or produced only dysfunctional pollen grains that could not be stained by aceto carmine. The treatment also caused 10–30% reduction on seed setting in comparison to the controls. The purity of hybrid seed from crosses of 'C161 × Huaye' and 'Zhongshuang No.2 × Huaye' based on amidosulfuron was 99.8% and 100.0%, respectively. The results suggested that amidosulfuron is an efficient gametocide for B. napus .     

甘蓝型油菜Pol CMS育性恢复基因的RAPD标记

采用恢、 保回交群体和集团分离分析(BSA), 筛选了860个10 mer随机引物, 找到了与甘蓝型油菜波里马细胞质雄性不育系(Pol cms)育性恢复基因(Rf)连锁的两个RAPD标记AH19690和AI16830。 它们位于Rf的一侧,与该基因的遗传图距分别为5.9 cM和13.6 cM,两标记间的遗传图距为7.8 cM。这两个RAPD标记的发掘,为进一步利用分子标记     

甘蓝型油菜隐性上位互作核不育基因(Ms1)精细定位

甘蓝型油菜细胞核雄性不育是杂种优势利用的重要途径.隐性上位互作核不育系9012A已经广泛用于杂交种子生产,其不育性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...     

Inducing male sterility in Brassica napus L. by a sulphonylurea herbicide, tribenuron-methyl

A sulphonylurea herbicide, tribenuron‐methyl, methyl 2‐[[[[(4‐methoxy‐6‐methyl‐1,3,5‐triazin‐2‐yl) methylamino] carbonyl] amino] sulphonyl] benzoate, was used to induce male sterility in rapeseed. Application of 0.2 μg tribenuron‐methyl per plant at the bolting stage with the longest floral bud <2 mm and repeated 15 days afterwards, resulted in 94.5–100% plants being male sterile in six different breeding lines, but combined with low phytotoxicity. However, excessive double application of the chemical (>0.2 μg per plant) did have some significant impact on rapeseed, including stunting, fading leaves and petals, reduction in the size of floral parts and a shortened duration of flowering. The percentage of hybrid seeds from ‘84004‘ treated with 0.2 μg × 2 tribenuron‐methyl per plant and pollinated by a male parent ‘Huaye’ was 92.7%, which met the hybridity requirement in China. The results suggest that tribenuron‐methyl could be used as an efficient chemical hybridizing agent.     

Identification and Genetic Studies of the Inhibition of Dominant Male Sterility in Brassica napus

By transferring dominant male sterility (DMS), caused by the gene Ms, to genotypes with various types of cytoplasm 12 DMS lines were developed and a number of crosses made between the DMS lines and other genotypes of Brassica napus. During the course of this population improvement programme, 16 genotypes were identified as having the capacity to restore the fertility of F₁ plants with the Ms gene. According to pedigree analysis, the inhibitory gene in those lines probably originated from a few genotypes from Australia and Germany. In further studies the inheritance of the sterility inhibition was determined, providing definite evidence that dominant male sterility and its inhibition in B. napus are controlled by two dominant interacting genes rather than by multiple alleles.     

Molecular markers for genic male sterility in Chinese cabbage

The gms gene of Chinese cabbage (Brassica campestris ssp. chinensis) conferring a recessivegenic male sterility was mapped with AFLPmarkers. Four markers were found to betightly linked to the gene with a distanceof <1 cM using a mapping population of327 individuals. The AFLPs were cloned andsequenced. Sequence derived primers yieldedclearly distinguishable PCR fragmentsrepresenting sequences tightly linked tothe gms gene. The sequence taggedsite (STS) markers can be used for markerassisted selection of male sterile plantsamong segregating populations.     

Development and primary genetic analysis of a fertility temperature-sensitive polima cytoplasmic male sterility restorer in Brassica napus

Over the past decade, the polima cytoplasmic male sterility ( pol CMS) three-line and two-line systems have been developed for the production of hybrid seed in Brassica napus oilseed rape in China. The discovery of the novel pol CMS restorer line FL-204 is described here. It restores male fertility of hybrid plants in the pol CMS system, but hybrid seed production can only be carried out under autumn sowing in Wuhan in south China under moderate temperatures at flowering. The restorer cannot be used as a male for hybrid seed production in northwestern China (Gansu) under spring sowing conditions, because there it is more or less male sterile due to high temperatures at flowering. Because of this behaviour, it is referred to as a fertility temperature-sensitive restorer (FTSR) in this paper. F₂, BC₁ as well as double haploid populations were constructed to determine the inheritance of fertility restoration of FL-204 in the autumn at Wuhan and under spring sowing conditions at Gansu, respectively. Deviations from Mendelian genetics were observed. It was hypothesized that the change of fertility was the result of the interaction between nuclear genes [restoring gene ( Rf ) and temperature-sensitive genes ( ts )] and the cytoplasm. The Rf gene in FL-204 was incapable of restoring male fertility of pol CMS lines under spring sowing conditions at Gansu where it is inactivated by the recessive ts gene present in FL-204. However, the ts gene(s) could be non-functional under moderate temperature conditions at flowering at Wuhan which allows full expression of male fertility in FL-204. The recessive ts gene(s) can only be expressed in plants containing the pol sterile cytoplasm. A method for the utilization of the FTSR pol CMS restorer FL-204 for the production of hybrid seed in B. napus oilseed rape is proposed.     

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.     

A male sterile line with dominant gene (Ms) in cabbage (Brassica oleracea var. capitata) and its utilization for hybrid seed production

A male sterile plant, 79-399-3, was identified from a spring cabbage line 79-399 in 1979. Light-microscopic studies with paraffin section indicated that meiotic division stopped at the tetrad stage. The ratio of male sterile plants to fertile plants in the progenies of test crosses was 1:1 in five experiments conducted in 1982 and 1991–1994. However, some male sterile plants were sensitive and developed a very low number of viable pollen grains. When sensitive male sterile plants were selfed, the progenies segregated into male sterile and fertile plants at a ratio of 3:1. The male sterile plants from selfing of sensitive male sterile plants were propagated by tissue culture and crossed with different inbred lines. From the progenies of the crosses, populations with 100% male sterile plants were observed. The results indicated that homozygous dominant male sterile (MsMs) plants segregated from the selfed progenies. Populations with 100% male sterile plants and stable male sterility were developed as male sterile lines. Several favorable combinations were also selected by crossing the male sterile lines with inbred lines with high combining ability for desirable horticultural characters. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tournefortii male sterility system in Brassica napus. Identification, expression and genetic characterization of male fertility restorers

A germplasm collection of 152 diverse rapeseed accessions from Canada, China, France, India, Poland and South Korea was assayed for identifying new fertility restorers and sterility maintainers for a Tournefortii (tour) cytoplasmic male sterility (CMS) system in rape‐seed. Only 16 (10.5%) genotypes showed complete fertility restoration following hybridization with tour CMS line NE 409A. Notable among these were GSL 8851, GSL 8953, Mokpo # 9, Mali, Buk‐wuk‐13, Kuju‐27 and Mokpo # 84. As many as 78 (51.3%) genotypes were perfect maintainers of sterility, the remaining 58 (38.2%) genotypes were classified as partial maintainers. To study the inheritance of fertility restoration, 20 CMS (tour) rapeseed lines were crossed with the four best fertility restorers, namely GSL 8851, GSL 8953, Kuju‐27 and Mokpo # 9, to obtain F₂ and test cross populations. Segregation data indicated that fertility restoration for tour CMS was governed by two genes, of which, one is stronger than the other (χ²_12:3:1). Differences in gene interactions were also observed (χ²_9:3:4) which could be explained on the basis of influence of female parent genotypes/or modified expression of the restorer gene(s) in different genetic backgrounds. Tests of allelism indicated that the restorer genes present in the four restorers evaluated were allelic.     

甘蓝型油菜雄性不育完全保持的途径

甘蓝型油菜雄性完全不育系在杂种优势利用中具有重要作用。本文综述了甘蓝型油菜雄性完全不育保持的途径和方法,并对此研究领域的发展前景和探索方向进行了展望。     

甘蓝型油菜轮回选择研究

以同时携带显性核不育基因Ms和显性不育抑制基因Rf的甘蓝型油菜为父本,含有相应隐性等位基因的同类型油菜为母本,经一代杂交成功地把显性核不育基因导入具不同细胞质来源的基因型中.90%以上的杂种一代\回交和复交组合均表现为1可育:1不育的分离.在第一批转育所用的15个亲本中,有3个对由Ms基因控制的雄性不育性有抑制作用,它们     

Molecular characterization of novel resynthesized rapeseed (Brassica napus) lines and analysis of their genetic diversity in comparison with spring rapeseed cultivars*

Resynthesized (RS) rapeseed generated from interspecific hybridization between suitable forms of Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and B. oleracea L. (CC, 2n = 18) represents a potentially important resource to expand genetic diversity in the narrow gene pool of oilseed rape (B. napus L., AACC, 2n = 38). In this study 165 RS rapeseed lines originating from crosses between an Indian Yellow Sarson (B. rapa ssp. trilocularis) and five different cauliflower (B. oleracea convar. botrytis) cultivars were studied using amplified fragment length polymorphism (AFLP) markers and their genetic diversity was compared in relationship to an assortment of 40 diverse spring oilseed and fodder rape varieties. Using three AFLP primer combinations, a total of 467 polymorphic bands were scored. Cluster analysis allowed differentiation among the different RS lines, which, as expected, were genetically highly divergent from the cultivars. The genetic diversity of the material is discussed in relation to its morphological variability with a view to the implementation of RS lines in oilseed rape breeding.