Independent Inheritance of Flower Colour and Male-Fertility Restorer Characters in Brassica napus L.

Available material of oilseed (Brassica napus L., AACC) comprises two yellow-flowered breeding lines and a white/pale-flowered line of resynthesized rape. The flower colour white/pale is dominant over yellow, and is controlled by a gene located in the C-genome. The yellow-flowered genotypes acted as restorer lines and the white/pale-flowered genotype as a maintainer line in a cytoplasmic male sterility system. The segregation pattern of flower colour and male fertility restorer characters were studied in F₂ generations of crosses between these lines, also in a three-way cross additionally including a yellow flowered B. campestris (AA) line. Evidense was obtained in support of the conclusion that the flower colour and male fertility restorer characters are monogenically controlled and independently inherited. Whether the male fertility restorer gene is located in the A or C genome remains to be determined.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

Identification of Maintainer Genes in Brassica napus L. for the Male-Sterility-Inducing Cytoplasm of Diplotaxis muralis L.

Male fertility of F¹ interspecific hybrid plants derived from crosses between cytoplasmic male-sterile Brassica campestris in Diplotaxis muralis cytoplasm and 147 B. napus cultivars was Investigated. F¹, plants obtained, from crosses with the B. napus cultivars‘Mangum’and‘Hinchu’were male-sterile while F¹ plants derived from all other crosses were male-fertile. This indicated that these two cultivars carried maintainer genes far the male-sterility-inducing cytoplasm of D. muralis. Sterility was stable In plants derived from backcrosses of male-sterile F; plants with‘Mangun and‘Hinchu’but the seed set of backcross plants was low. With restorer genes readily available in B. napus, these findings could lead to the development of a new cytoplasmic male sterility system for the breeding of B. napus hybrid cultivars.     

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

Cytoplasmic male sterility (CMS), a maternally transmitted failure in pollen formation, is an effective pollination control system in hybrid rapeseed (Brassica napus) breeding. However, CMS is not widely used in the related oilseed species Brassica rapa. In the past years, several male sterile plants have been isolated from the B. rapa landrace ‘0A193’, collected in Shaanxi, China, in 2011. It is noteworthy that the fertility expression of 0A193‐CMS was affected by temperature. In contrast to pol CMS, fertility tests with 18 B. rapa and 9 B. napus accessions suggest that a different system of maintaining and restoring is responsible for the observed phenotype. Further on, genetic investigation evidenced that fertility of 0A193‐CMS is controlled by both cytoplasmic and one pair of nuclear recessive genes. Interestingly, plants of the 0A193‐CMS type possess a highly specific fragment of the mitochondrial gene orf222, a crucial regulator of male sterility in nap CMS. Our study broadens the CMS resources in B. rapa and provides a highly applicable alternative to pol CMS and ogu CMS for hybrid breeding production.     

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.     

Haplotype analysis of CMS-associated DNA markers in sweet peppers

Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) is an economical and efficient system to produce F₁ hybrid seeds. Although the CMS/Rf system has been used to produce hybrid seeds of hot peppers, this system has never been used for sweet pepper seed production, presumably due to the inability to select stable restorer lines during the breeding process. To test the feasibility of the CMS/Rf system in sweet pepper breeding, we investigated the distribution of haplotypes of previously developed, CMS-associated markers (orf456, ψ atp6-2, CRF-SCAR, OPP13-CAPS, PR-CAPS, and PR-SNP) in 27 commercial sweet pepper F₁ hybrids and 12 breeding lines. When CMS-associated cytoplasmic markers orf456 and ψ atp6-2 were applied, male sterile cytoplasm was not detected in commercial sweet pepper cultivars. When nuclear haplotype markers linked to Rf were applied, all sweet pepper cultivars showed haplotype 3, haplotype 1, and the rf genotype for OPP13-CAPS, PR-CAPS, and CRF-SCAR, respectively. In contrast, we were able to detect male sterile cytoplasm in some breeding lines, and we were also able to detect polymorphisms for PR-CAPS between stable and unstable maintainer lines. The 17T7-SNP also showed polymorphisms between unstable and stable maintainer (or restorer) lines. In conclusion, we expect that it will be possible to develop stable A, B, and C sweet pepper lines using CMS-associated markers and that this will eventually lead to successful implementation of the CMS/Rf system to produce F₁ hybrid sweet pepper seeds.     

Identification and Inheritance of Fertility Restorer Genes for 'tour' CMS in Rapeseed (Brassica napus L.)

Eighteen genotypes of Brassica napus were crossed to a cytoplasmic male sterile (CMS) line of B. napus BO 15 carrying B. tournefortii cytoplasm (‘tour’ cytoplasm). Fourteen genotypes were found to be stable maintainers of the ‘tour’ CMS. Of the remaining four genotypes, GSL-1 and ‘Asahi-natane’ were found to be heterozygous and ‘Mangun’ and ‘Yudal’ were homozygous for the restorer gene. Analysis of the F₁ and F₂ progenies of (CMS) BO 15 בMangun’ and (CMS) BO 15 בYudal’ showed that fertility restoration is controlled by a single dominant gene. The availability of a number of stable maintainer lines and the simple inheritance pattern of fertility restorer gene makes ‘tour’ CMS a useful system for hybrid seed production in rapeseed.     

Conversion of the RAPD OPC021150 marker of the Rfo restorer gene into a SCAR marker for rapid selection of oilseed rape

The Rfo fertility restorer gene for the Ogura cytoplasmic male sterility (CMS) applied for oilseed rape hybrid seed production can be monitored with the use of the RAPD OPC02₁₁₅₀ marker while molecular breeding. The aim of this work was to convert the RAPD marker into a more suitable SCAR marker. Total DNA was isolated from a doubled haploid line derived from the line BO20 (INRA, France). A fragment of 1150‐bp linked to the Rfo gene was PCR amplified with the use of the RAPD OPC02 primer, cloned and sequenced. A pair of primers was designed and PCR amplification was performed to develop a SCAR marker for the Rfo gene. The new marker was applied for analysis of 220 oilseed rape lines comprising doubled haploid and inbred restorer lines, restored hybrids as well as F₁ and F₂ recombinant generations involving restorer lines. Simultaneously, the RAPD OPC02 marker was used and it revealed that the markers are equivalent to each other. However, the developed new SCAR marker has made the analysis more practical, rapid and efficient.     

Genetic relationships among resynthesized,semi-resynthesized and natural <Emphasis Type="Italic">Brassica napus</Emphasis> L. genotypes

The allopolyploidization event that created cultivated oilseed rape Brassica napus L, followed by intense breeding, reduced its genetic diversity. Resynthesized (RS) B. napus L. obtained by interspecific hybridization between genotypes of B. rapa L. and B. oleracea L. can be a valuable source for broadening genetic diversity in cultivated oilseed rape. In this study, we determined the extent of DNA polymorphism among natural accessions of oilseed rape, resynthesized B. napus, their parental species and double-low quality semi-RS lines carrying the Rfo gene. Using 10 selected primer combinations, 522 polymorphic AFLP markers were scored in the complete set of 100 Brassica sp. To detect relationships between these genotypes, a cluster analysis was performed using the Jaccard’s distance. Resynthesized allopolyploids clustered directly between their diploid parents. Cultivated accessions of oilseed rape created a compact group away from resynthesized allopolyploids as well as semi-RS lines. The natural oilseed rape group, which consists of 49 cultivars and breeding lines of oilseed rape, is characterized by lower genetic diversity than the group of 33 accessions of resynthesized oilseed rape, and the analysis showed that the double-low quality semi-RS lines represent a specific genetic variation of B. napus. The de novo resynthesized B. napus lines and the semi-RS lines of double-low quality generated from them, provide a significant opportunity for enrichment the gene pool of oilseed rape.     

The Effect of A Genome Substitution on the Resistance of Brassica napus to Infection by Leptosphaeria maculans

Six accessions belonging to four subspecies of Brassica rapa, including three accessions of B. rapa subsp. sylvestris, were crossed with B. oleracea subsp. alboglabra in order to develop a series of synthetic B. napus lines with a common C genome but contrasting A genomes. Different A genomes had significant effects on the efficiency of B. napus resynthesis and the sexual compatibility of the synthetic lines with oilseed rape cultivars. The synthetic lines were used to investigate the effect of A genome substitution on the resistance of B. napus to infection by Leptosphaeria maculans, and to explore the potential for the use of wild forms of B. rapa in oilseed rape breeding programmes. Synthetic lines derived from two wild accessions of B. rapa, and their F₁ hybrids with oilseed rape cultivars, expressed high levels of resistance to L. maculans in glasshouse experiments. One of these lines also expressed high levels of resistance in field experiments in England and Australia when exposed to a genetically diverse pathogen population. All other synthetic lines and cultivars were highly susceptible in both glasshouse and field experiments. F₁ hybrids between oilseed rape cultivars and synthetic lines derived from B. rapa subsp. chinensis were significantly more susceptible than either parent.     

Inheritance and mapping of a restorer gene for the rapeseed cytoplasmic male sterile line 681A

A novel cytoplasmic male sterility‐fertility restoration system has been developed in rapeseed (Brassica napus). The cytoplasmic male sterile line 681A was derived from a spontaneous male sterile mutant in a newly released double‐low rapeseed cultivar ‘Xiangyou 13′. The restorer line 714R was identified in the interspecific progeny from a B. napus×B. juncea‐cross. Genetic analysis showed that fertility restoration for 681A cytoplasmic male sterility was controlled by a single dominant nuclear gene which might originate from B. juncea. The RAPD marker S1039_‐520 was found to be linked to the restorer gene in F₂ progeny of 681A × 714R with a recombination frequency of 5.45%.     

油菜野芥NSa细胞质雄性不育系的特异性分子鉴定

常规细胞质类型鉴定方法花费时间长、工作量大、鉴定效率低、应用性较差。基于基因组重复序列的PCR技术(rep-PCR)在重复序列丰富的线粒体和原核生物的基因组鉴定中效果较好, 本研究对8份油菜资源的细胞质进行分子鉴定,结果表明rep-PCR可以明确区分甘蓝型油菜中常见的6种雄性不育细胞质。同时对甘蓝型油菜和野芥(Sinapis arvensis)体细胞杂交创建的、育性较为稳定的NSa野芥雄性不育胞质进行了分子特异性鉴定,获得了两条NSa不育胞质的特征DNA条带。但波里马和萝卜质不育细胞质的特异性引物在NSa中不能扩增出特异性片段,说明NSa不属于这两种细胞质类型。本研究结果为该不育胞质系统的利用和知识产权保护提供了理论依据和技术支撑。     

野生稻（O.rufipogon）新胞质改良不育系稻米品质的研究

通过核置换回交，获得一种普通野生稻（O. rufipogon）雄性不育细胞质，称为FA细胞质，育成新质源不育系金农1A。研究表明，FA细胞质不育系与WA和HL不育系的恢保关系不同，野败型的保持系和恢复系、红莲型的保持系和恢复系都可以作为FA细胞质的保持系，而不能成为FA细胞质的恢复系。这是一种新发掘的水稻雄性不育细胞质源。FA细胞质突破了WA和HL细胞质恢保关系的遗传局限，扩大了保持系育种范围，可以用品质优良的栽培品种培育成优质稻米不育系。新质源不育系金农1A不育度高，不育性稳定，农艺性状优良，抗稻瘟病和白叶枯病，开花时间早，稻米12项品质指标全部达到农业部颁发的优质米一级或二级标准，实质性地提高了不育系的稻米品质和综合水平，为培育优质米杂交稻奠定良好的遗传基础。     

Identification of a Stable Maintainer Line for 'Polima' Cytoplasmic Male Sterility in Rapeseed (B. napus L.)

‘Polima’ cytoplasmic male sterility (CMS) was transferred to three different genotypes of B. napus i.e. GSL-1, ISN706 and HNS-8 by repeated backcrossing and was found to be completely stable in the nuclear background of synthetic B. napus genotype, ISN706, derived from a cross B. campestris ssp. oleifera var. brown sarson ×B. oleracea var. botrytis cv. Tusa Katki'. The BC₅ and BC₆ generation lines of ISN706, GSL-1 and HNS-8 with ‘Polima’ CMS were grown under a range of temperature and photo-period conditions to test their stability. No breakdown in sterility was observed in ‘Polima’ ISN706 and, therefore, this genotype can be used as a maintainer for hybrid seed production.     

Identification and characterization of important sterile and maintainer lines from various genotypes for advanced breeding programmes of onion (Allium cepa)

Onion is one of the major vegetable crops in terms of production as well as consumption. In the current research, available onion genetic stock was evaluated to identify male-sterile lines and produce high-yielding F₁ hybrids for future breeding programmes. A mitochondrial DNA-based marker was mapped and correlated with phenotypic traits to isolate male-sterile plants. Based on the floral and pollen structure, nine putative male-sterile lines were identified. On the other hand, for nuclear marker identification at Ms locus, two sets of primers were used, one for Ms dominant allele and another for sterile and maintainer plants. Results revealed that 70% of open pollinated varieties (OPVs) possess plants with sterile cytoplasm coupled with genetic sterility at Ms locus, called sterile “A” line. Approximately 20% of plants in some genotypes were identified with normal (N) cytoplasm having recessive fertility gene at Ms locus, called maintainer “B” line. Based on the present findings, “A”, “B” and “R” (restorer line), future F₁ hybrid seed production systems in onion is discussed.     

Marker-assisted increase of genetic diversity in a double-low seed quality winter oilseed rape genetic background

Generation of novel genetic diversity for maximization of heterosis in hybrid production is a significant goal in winter oilseed rape breeding. Here, we demonstrate that doubled haploid (DH) production using microspore cultivation can simultaneously introgress favourable alleles for double‐low seed quality (low erucic acid and low‐glucosinolate content) into a genetically diverse Brassica napus genetic background. The DH lines were derived from a cross between a double‐low quality winter rapeseed variety and a genetically diverse semisynthetic B. napus line with high erucic acid and high glucosinolates (++ quality). Twenty‐three low‐glucosinolate lines were identified with a genome component of 50–67% derived from the ++ parent. Four of these lines, with a genome component of 50–55% derived from the ++ parent, also contained low erucic acid. Heterosis for seed yield was confirmed in test‐crosses using these genetically diverse lines as pollinator. The results demonstrate the potential of marker‐assisted identification of novel genetic pools for breeding of double‐low quality winter oilseed rape hybrids.     

Detection and characterization of two new CMS systems in faba bean (Vicia faba) *

Wide crosses were made to identify new cytoplasmic male sterility (CMS) systems in faba beans, based on the interaction of cytoplasm with restorer and maintainer alleles. A total of 330 F₁ hybrids were produced in both reciprocal forms. Male sterile segregates were observed in one reciprocal version in the F₂ generation of six crosses. Two of these crosses with female parents originating from Afghanistan and Egypt expressed stable male sterility in subsequent backcross generations. Based on the female parents of the two crosses, these two CMS systems were designated CMS 199 and CMS 297. CMS 199 was more stable than CMS 297 during backcross generations and across different environments. Maintainer and restorer lines for both CMS systems were identified. Lower expression of male sterility occurred in CMS 297 in the greenhouse during the winter generations than in isolation cages during the summer generations, which may be utilized to maintain male sterile lines by selfing. Regarding practical applications, the CMS 199 shows great promise for hybrid breeding in faba beans.     

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.     

Characterization of Brassica nigra chromosomes and of blackleg resistance in B. napus–B. nigra addition lines

Brassica napus-B. nigra addition lines were previously created using the variety ‘Darmor’ as the oilseed rape genetic background. Two isozyme loci and 46 RAPD markers were added on five different B. nigra chromosomes. The oilseed rape variety used was highly susceptible to blackleg at the cotyledon stage and only the addition of chromosome 4 gave the same level of blackleg resistance as B. nigra. This resistance was efficient whatever the isolates used. A significant effect on the development of stem canker under field conditions was observed only for the line carrying chromosome 4 which was more resistant than the susceptible control. The potential effects of two other chromosomes have to be confirmed. F1 hybrids obtained by crosses between two highly susceptible lines and the monosomic addition line carrying chromosome 4 were examined under field conditions. No effect of the oilseed rape genetic background on the expression of resistance was detected. The introduction of this resistance and mapping of the gene(s) into oilseed rape varieties are discussed.