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.     

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.     

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.     

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.     

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.     

Development of dominant nuclear male-sterile lines with a blue seed marker in durum and common wheat

In order to develop genie male‐sterile lines with a blue seed marker, male‐sterile plants, controlled by a dominant nuclear gene Ms2, were used as female parents against a 4E disomic addition line ‘Xiaoyan Lanli’(2n= 44, AABBDD+4EII) as the male parent to produce monosomic addition lines with blue seed. Male‐sterile plants from the monosomic addition lines were pollinated with durum wheat for several generations and in 1989 a male‐sterile line with the blue grain gene and the male‐sterile gene Ms2 on the same additional chromosome was detected and named line 89‐2343. Using this line, the blue seed marker was successfully added to a short male‐sterile line containing Ms2 and Rht10. The segregation ratios of male sterility and seed colour as well as the chromosome figurations of different plants indicated that the blue grain genes, Ms2 and Rht10 were located on the same additional chromosome. Cytological analysis showed that the blue marker male‐sterile lines in durum wheat and common wheat were monosomic with an additional chromosome 4E. The inheritance ratio for blue seed male‐sterile plants and white seed male‐fertile plants was 19.7% and 80.3%, respectively, in common wheat. The potential for using blue marker sterile lines in population improvement and hybrid production is discussed.     

A unique introgression from Moricandia arvensis confers male fertility upon two different cytoplasmic male-sterile lines of Brassica juncea

A Brassica juncea line carrying an introgression from Moricandia arvensis restored male fertility to two cytoplasmic male‐sterile (CMS) B. juncea lines carrying either M. arvensis or Diplotaxis catholica cytoplasm. Genetics of fertility restoration was studied in the F₁, F₂, F₃ and backcross generations of the cross between CMS and fertility‐restorer lines. No male‐sterile plants were found in F1‐F₃ generations of the cross between CMS [M. arvensis] B. juncea and the restorer. However, a 1: 1 segregation for male sterility and fertility was observed when the F1 was pollinated with non‐restorer pollen from a euplasmic line. These results clearly show that restoration is mono‐genic and gametophytic. In CMS lines carrying D. catholica cytoplasm, the restorer conferred male fertility to the F1 and showed 3: 1 and 1: 1 segregations for male fertility and sterility in F₂ and BC1 generations, respectively, indicating a monogenic, sporophytic mode of fertility restoration. The results were also supported by pollen stainability in the F1 which was about 65% in M. arvensis‐based CMS and >90% in D. catholica‐based CMS. The above results are discussed in the light of previous molecular studies which showed association between CMS and atpA in both systems.     

Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

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.     

核不育复等位基因向娃娃菜中转育的研究

根据"复等位基因遗传假说",以甲型两用系AB12为不育源(直筒生态型),采用杂交、自交、兄妹交的方法,将核不育基因向娃娃菜可育品系06006中转育,获得了不育株率100%的新核基因雄性不育系,扩大了原有不育系的遗传基础,拓宽了该优良雄性不育基因的应用范围.     

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...     

Conversion of the genic male sterility (GMS) system of bell pepper (Capsicum annuum L.) to cytoplasmic male sterility (CMS)

Non‐pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F₁ hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non‐pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non‐pungent bell pepper line GC3 was conducted by introgression of S‐type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC₁F₁ containing S‐type cytoplasm and four lines from BC₂F₂ containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non‐pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC₁F₁ and BC₁F₂ populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.     

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。     

利用分子标记辅助选育大白菜核基因雄性不育系

本研究以含不育基因Ms的大白菜细胞核复等位基因型雄性不育两用系"AB01"的可育株(MsfMs)为供体亲本,以高代自交系‘a20’(msms)为轮回亲本,采用杂交和连续回交转育方法,利用与不育基因Ms连锁的SCAR标记syau_scr01辅助不育基因Ms选择,成功地将不育基因转育到可育品系‘a20’中,育成了不育度和不育株率均为100%,植物学性状与自交系‘a20’相近的新核不育系GMS4。选择结果表明syau_scr01选择的准确率为100%,验证了该标记可以用于大白菜核不育系转育辅助选择。     

Development of cytoplasmic-genic male sterility in safflower

An interspecific cross was made between Carthamaus oxyacantha and the cultivated species C. tinctorius to develop a cytoplasmic‐genic male sterility (CMS) system in safflower. C. oxyacantha was the donor of sterile cytoplasm. The 3: 1 segregation pattern observed in BC₁F₂ suggested single gene control with dominance of male‐fertility over male‐sterility. The information obtained from crossing male sterile X male fertile plants in BC₁F₃ and BC₁F₄ generations showed statistically significant single gene (1: 1) segregation for male sterility vs. male fertility. The results demonstrated that C. tinctorius possesses a nuclear fertility restorer gene and that a single dominant allele restored fertility (Rf) in progeny carrying CMS cytoplasm of C. oxyacantha. Male sterility occurred with the homozygous recessive condition (rfrf) in a sterile C. oxyacantha cytoplasm background and not in the normal cytoplasm of C. tinctorius. The genetic background of different restorer lines of C. tinctorius having normal cytoplasm did not effect fertility restoration. The absence of male sterile plants in C. tinctorius populations ruled out the possibility of genetic male sterility. Normal meiosis in F₁ and BC₁F₂ ruled out a cytogenetic basis for the occurrence of male sterility.     

Three AFLP markers tightly linked to the genic male sterility <Emphasis Type="Italic">ms</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript> gene in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and conversion to a CAPS marker

Genic male sterility (GMS) has long been used as a tool for hybrid seed production in chili pepper (Capsicum annuum L.). We developed DNA markers linked to the GMS ms ₃ gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 512 primer combinations. Three AFLP markers (Eagg/Mccc₂₇₆, Eagc/Mctt₁₇₈, and Ecag/Mtgc₂₀₄) were identified as tightly linked to the ms ₃ locus. Among them, we converted the AFLP marker Ecag/Mtgc₂₀₄ to the cleavage amplified polymorphic sequence (CAPS) marker, named GMS3-CAPS, based on sequencing analysis of internal and flanking regions for the markers between male-fertile and sterile plants. This marker will be useful for pepper breeding using the GMS system.     

谷子显性雄性不育基因Msch的AFLP标记

利用雄性不育是实现谷子杂种优势利用最经济、有效的途径之一.为了寻找与不育基因Msch紧密连锁的分子标记,提高不育系的选育效率,本研究构建了Msch不育/可育近等基因系(NILs),通过对400对AFLP引物组合进行筛选,找到了与不育基因紧密连锁的两个AFLP标记(P17/M37224和P35/M52208),与不育基因的遗传距离分别是2.1 cM和1.4 cM,而且位于不育基因的同一侧,标记间相距0.7 cM.这两个AFLP标记可有效用于分子标记辅助选择育种.     

Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice

The wild abortive cytoplasmic male sterility (CMS‐WA) system, an ideal type of sporophytic CMS in indica rice, is used for the large‐scale commercial production of hybrid rice. Searching for restorer genes is a good approach when phenotyping is very time‐consuming and requires the determination of spikelet sterility in testcross progeny. To establish more precisely the genetical and physical maps of the Rf4 gene, high‐resolution mapping of this locus was carried out using simple sequence repeat (SSR) markers and newly designed markers in a F₂ population. The genetic linkage analysis indicated that five SSR markers (RM6737, RM304, RM171, RM5841 and RM228) on the long arm of chromosome 10 were linked with the Rf4 gene. Rf4 was flanked by two SSR markers RM171 and RM6737 at distances of 3.2 and 1.6 cM, respectively. Also, within the region between Rf4 gene and RM171, a newly designed primer pair, AB443, produced two sterile‐specific markers, AB443‐400 and AB443‐500, 0.5 and 1.03 cM from the gene. The flanking markers identified give promise for their application in molecular marker‐assisted selection (MAS) and they are also suitable for starting chromosome walking to clone Rf4 gene in the near future.     

SSR and SCAR markers linked to the fertility-restoring gene for a D2-type cytoplasmic male-sterile line in wheat

‘Yi 4060’ is an elite restorer line of a non‐photoperiod‐sensitive D²‐type cytoplasmic male‐sterile (CMS) line of wheat. Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were employed to map one major fertility‐restoring gene (D²Rf₁) in ‘Yi 4060′. The sterile and fertile DNA pools were established from individuals in BC₆, based on bulked segregant analysis. One RAPD marker E09, linked to D²Rf₁, was converted to a SCAR marker and designated as E09‐SCAR₈₆₅. The genetic distance between E09‐SCAR₈₆₅ and D²Rf₁ is 9.5 cM. Two SSR markers, Xgwm11 and Xgwm18, were also linked to a D²Rf₁ and co‐segregated with E09‐SCAR₈₆₅. The three molecular markers are useful in marker‐assisted breeding of the elite restorer lines for D² ‐type CMS lines in wheat.