Genetic mapping of nuclear fertility restorer genes for the ‘Polima’ cytoplasmic male sterility in canola (Brassica napus L.) using DNA markers

 Co-segregation of male fertility with DNA markers selected by targeted mapping approaches as being potentially linked to the Rfp1 restorer gene for the pol cytoplasmic male sterility (CMS) was analyzed using two canola (Brassica napus L.) backcross populations. Eleven DNA markers (10 RFLP markers and one RAPD marker) directly linked to the Rfp1 locus were identified. The linkage group containing the Rfp1 locus was found to correspond to B. napus linkage group 18 of Landry et al. (1991). A similar pattern of co-segregation between DNA markers and male fertility was observed in a backcross population segregating for the pol restorer gene Rfp2 from line ‘UM2383’; one RFLP marker, cRF1b, showed perfect linkage with both Rfp1 and Rfp2 and detected identical polymorphic fragments in both the Rfp1 and Rfp2 restorer lines. Our findings indicate that restoration of pol CMS is controlled by a single nuclear genetic locus on linkage group 18 and that Rfp1 and Rfp2 are likely allelic. Received: 2 October 1996 / Accepted: 20 December 1996     

甘蓝型油菜Pol CMS育性恢复基因的PCR标记

采用恢、保回交群体和集团混合分析法,筛选了１０４０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（Ｒｆｐ）连锁的两个ＲＡＰＤ标记Ｓ１０１９７２０和Ｓ１０３６８１０。它们位于Ｒｆｐ的一侧,与该基因的遗传图距分别为５．８ｃＭ和１２．３ｃＭ。随后,克隆并测序这２个多态性片段,根据其２端序列设计了２对２０～２４－ｍｅｒ的特异引物,它们在１３８株的回交群体中Ｐ     

Development of PCR-based markers linked to dominant genes for male-fertility restoration in Pampa CMS of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Cytoplasmatic male sterility (CMS) is the basis for commercial hybrid seed production of rye. Nuclear restorer genes are indispensable for a complete restoration of fertility of the CMS lines. The drawbacks of current European restorer lines require the utilisation of new genetic resources that have been recently detected in an Iranian primitive rye population (IRAN IX) and an Argentinean landrace (Pico Gentario). The introgression of these effective restorer genes (Rfp1 and Rfp2, respectively) into breeding material can be facilitated by marker-assisted selection. Using two F(2) populations based on crosses between the non-restorer inbred line Lo6 and the restorer IRAN IX, as well as Pico Gentario, RAPDs and AFLPs were screened and led to a closely linked marker set for each of these genes. The conversion of the closest markers into fragment-specific sequence-characterised amplified region (SCAR) markers resulted in flanking ranges of 2.9 cM (Rfp1) and 5.2 cM (Rfp2). The application of these markers in backcross programmes is discussed.     

The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats

A single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. A map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687-amino-acid protein with a predicted mitochondrial targeting pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis, although a smaller and related PPR gene is found about 40 kb from this site. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.     

Targeted mapping approaches to identify DNA markers linked to the Rfp1 restorer gene for the ‘Polima’ CMS of canola (Brassica napus L.)

 We have used two targeting approaches [pairs of nearly isogenic lines (NILs) and bulked segregant analysis] to identify DNA markers linked to the Rfp1 restorer gene for the pol CMS of canola (Brassica napus L.). We were able to target the Rfp1 locus as efficiently by comparing NILs as by bulked segregant analysis, and it was demonstrated in this instance that double-screening strategies could significantly improve the overall targeting efficiency. The chance occurrence of shared homozygosity at specific unlinked chromosomal regions in the bulks was found to limit the efficiency of bulked segregant analysis, while the efficiency of NIL comparison was limited by residual DNA from the donor cultivar at scattered sites throughout the genome of the NILs. Received: 6 June 1997 / Accepted: 12 February 1998     

Alloplasmic male sterile Brassica napus with Enarthrocarpus lyratus cytoplasm: introgression and molecular mapping of an E. lyratus chromosome segment carrying a fertility restoring gene.

A cytoplasmic male sterility (CMS) system for Brassica napus (2n = 38; AACC) was developed by backcross substitution of its nucleus into the cytoplasm of a wild crucifer, Enarthrocarpus lyratus. Male sterility was complete, stable, and expressed in small flowers with rudimentary anthers. Since the B. napus germplasm lines were complete or partial maintainers of male sterility, the required fertility restorer gene (Rfl) was introgressed from the cytoplasm donor species. Inheritance studies carried out on F1 and F2 populations derived from hybridizing cytoplasmic male sterile and male fertile near-isogenic (PNILs) lines of B. napus 'Westar', revealed a monogenic dominant control for fertility restoration. Bulked segregant analysis with 215 RAPD primers helped in the identification of putative primers associated with fertility restoration. Co-segregation analysis of eight such primers with Rfl gene revealed two markers, OPK 15700 and OPZ 061300, which flank the Rfl locus on either side at a distance of 8.2 and 2.5 cM, respectively. These DNA markers will be useful in marker-assisted selection for improving the commercial potential of this newly developed CMS-fertility-restorer system for hybrid seed production programs in rapeseed.     

特早熟甘蓝型油菜异交率的测定

利用SSR分子标记技术构建3个特早熟甘蓝型油菜恢复系的指纹图谱,筛选出这3个恢复系的共显性SSR标记,测定了这3个恢复系的异交率。结果表明,恢复系材料4395、3509、4152的异交率分别为46.02%、33.32%、18.12%,在P<0.01时呈极显著差异,说明这3个恢复系的异交差异明显,为确定这3个恢复系在综合杂交种中的比例提供依据。     

Molecular markers linked to the Rf2 fertility restorer gene in cotton.

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.     

Development of conserved ortholog set markers linked to the restorer gene Rfp1 in rye

Restoration of male fertility is a prerequisite for hybrid rye breeding and currently the most straightforward approach to minimize ergot infection in hybrid rye varieties. Molecular markers are important tools for the efficient introgression and management of restorer genes like Rfp1 originating from unadapted genetic resources. Furthermore, closely linked markers flanking Rfp1 are indispensible for identifying and selecting individuals with haplotypes showing recombination between Rfp1 and other gene(s) that reside in close proximity and have a negative influence on yield. We identified orthologous gene sets in rice, Brachypodium, and Sorghum and used these gene models as templates to establish conserved ortholog set (COS) markers for the restorer gene Rfp1 on the long arm of rye chromosome 4R. The novel co-dominant markers delimit Rfp1 within a 0.7-cM interval and allow prediction of Rfp1 genotypes with a precision not feasible before. The COS markers enabled an alignment of the improved genetic map of rye chromosome 4R with wheat and barley maps and allowed identification of regions orthologous to Rfp1 in wheat and barley on the short arms of chromosomes 6D and 6H, respectively. Results obtained in this study revealed that micro-collinearity around the Rfp1 locus in rye is affected by rearrangements relative to other grass genomes. The impact of the novel COS markers for practical hybrid rye breeding is discussed.     

新疆野生油菜胞质雄性不育系1193A对应恢复基因的SSR标记分析

以油菜细胞质雄性不育系1193A和恢复系1193R2为亲本构建F2分离群体,并运用BSA法构建了可育和不育基因池。利用1521对SSR引物进行了多态性分析,结果表明有36对引物在亲本和基因池间都表现多态性,用F2单株验证表明有11对引物与恢复基因连锁,离恢复基因较近的2个标记CB10316和Bn GMS171分布在恢复基因Rf的两侧,遗传距离分别为3.9 c M和5.7 c M,可作为恢复系标记辅助育种的候选标记。     

甘蓝型油菜pol CMS育性恢复基因对orf224/atp6的转录调控

用 10个线粒体基因探针对波里马细胞质雄性不育 (polimaCMS)三系 1141A(pol) ,1141B(nap)和 1141R(pol)的花蕾线粒体RNA进行了Northern检测。结果表明 ,只有 3个探针atp6、orf2 2 4和orf2 2 2检测到转录本的差异。atp6在可育的 1141B中只转录产生一个丰度很高的 1 1kb转录本 ,在雄性不育的 1141A和pol胞质恢复系 1141R中 ,这个转录本的丰度明显减少并出现了分子量较大的 2个转录本 2 2kb、1 9kb转录本。与 1141A相比 ,恢复系1141R的 2 2kb和 1 9kb转录本丰度明显减少 ,并伴随着两个新的转录本 1 4kb和 1 3kb。表明orf2 2 4 atp6的表达与polCMS有关 ,并且其转录受到恢复基因Rfp的调控。同时通过对杂种F1 ( 1141A× 1141R)与另一个恢复系RS35 (pol)的比较证实 ,Rfp对orf2 2 4 atp6的调控与Rfp纯合与否无关。orf2 2 4 atp6在 1141A的苗期叶片中还转录产生育性恢复特异的 1 4kb转录本 ,这可能与细胞核基因型和相对低温条件有关。     

Identification of AFLP markers linked to fertility restorer genes for tournefortii cytoplasmic male-sterility system in Brassica napus

The tournefortii cytoplasmic male-sterility system is being used as a method of pollination control to develop hybrids in Brassica napus. Genetic analyses have indicated that two dominant genes, one major ( Rft1) and another minor ( Rft2), were required to achieve complete fertility restoration. Though the major gene ( Rft1) can cause complete fertility restoration on its own, its expression was significantly enhanced in the presence of the minor gene ( Rft2). In the absence of Rft1, Rft2 caused only partial fertility restoration. We used a pair of near-isogenic lines (NILs), differing for the presence/absence of Rf genes, to identify AFLP markers linked to fertility restorer genes. A total of 64 EcoRI/ MseI primer combinations were surveyed which produced 3,225 bands, of which 19 (0.006%) were polymorphic between parental NILs. Primer combinations which led to the identification of polymorphic bands present in fertile parental NILs were used for assaying a mapping population of 70 F(2) plants for determining the segregation pattern of markers. Initial screening resulted in the identification of five AFLP markers. The recombination analyses of these AFLP markers revealed that at least two (EACC/MCTT(105), EAAG/MCTC(80)) were present in the same linkage group along with the Rf loci. Marker EACC/MCTT(105) was separated from the major gene ( Rft1) by a distance of 18.1 cM, while it was 33.2 cM away from the minor fertility restorer gene ( Rft2). Another marker EAAG/MCTC(80) was also located adjacent to Rft1 at a distance of 18.1 cM, but on other side. Identification of flanking markers (EACC/MCTT(105), EAAG/MCTC(80)) for the major fertility restorer gene ( Rft1) provides a crucial component for marker-assisted selection and map-based cloning of the restorer genes, and can hence be used to construct elite restorer genotypes.     

与甘蓝型油菜重要经济性状有关的DNA克隆在拟南芥遗传图谱中的整合

拟南芥与油菜同属十字花科植物芸寡族,亲缘关系很近,基因组间的同源性很高,在用拟南芥EST克隆和油菜DNA克隆作探针定位了甘蓝型油菜一系列重要性状的基础上,对25个与油菜雄性不育恢复基因,硼高效利用基因,抗菌核病QTL及油菜种间杂种营养优势相关联的克隆进行了测序,在拟南芥基因组数据库中寻找到与这25个克隆高度同源的序列,根据这些高度同源序列在拟南芥染色体上的相位位置,将油菜DNA克隆整合到了拟南芥遗传图谱上,其中油菜硼高效基因BE1两侧的标记克隆整合在拟南芥第一染色体长臂一个较小的区段内,以该目标区段内的拟南芥EST克隆PA24为探针对甘蓝型油菜基因组比较作图,将该克隆定位在油菜连锁图BE1两侧标记之间,表明了利用基因组间的相互比较作图来精细定位芸薹属作物重要基因的可能性。     

Fine mapping of the restorer gene <Emphasis Type="Italic">Rfp3</Emphasis> from an Iranian primitive rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Key message

A comparative genetics approach allowed to precisely determine the map position of the restorer gene Rfp3 in rye and revealed that Rfp3 and the restorer gene Rfm1 in barley reside at different positions in a syntenic 4RL/6HS segment.

Abstract

Cytoplasmic male sterility (CMS) is a reliable and striking genetic mechanism for hybrid seed production. Breeding of CMS-based hybrids in cereals requires the use of effective restorer genes as an indispensable pre-requisite. We report on the fine mapping of a restorer gene for the Pampa cytoplasm in winter rye that has been tapped from the Iranian primitive rye population Altevogt 14160. For this purpose, we have mapped 41 gene-derived markers to a 38.8 cM segment in the distal part of the long arm of chromosome 4R, which carries the restorer gene. Male fertility restoration was comprehensively analyzed in progenies of crosses between a male-sterile tester genotype and 21 recombinant as well as six non-recombinant BC₄S₂ lines. This approach allowed us to validate the position of this restorer gene, which we have designated Rfp3, on chromosome 4RL. Rfp3 was mapped within a 2.5 cM interval and cosegregated with the EST-derived marker c28385. The gene-derived conserved ortholog set (COS) markers enabled us to investigate the orthology of restorer genes originating from different genetic resources of rye as well as barley. The observed localization of Rfp3 and Rfm1 in a syntenic 4RL/6HS segment asks for further efforts towards cloning of both restorer genes as an option to study the mechanisms of male sterility and fertility restoration in cereals.

     

Generation and mapping of SCAR and CAPS markers linked to the seed coat color gene in Brassica napus using a genome-walking technique.

The yellow seed coat trait in No. 2127-17, a resynthesized purely yellow Brassica napus line, is controlled by a single partially dominant gene, Y. A double-haploid population derived from the F1 of No. 2127-17 x 'ZY821' was used to map the seed coat color phenotype. A combination of AFLP analysis and bulked segregant analysis identified 18 AFLP markers linked to the seed coat color trait. The 18 AFLP markers were mapped to a chromosomal region of 37.0 cM with an average of 2.0 cM between adjacent markers. Two markers, AFLP-K and AFLP-H, bracketed the Y locus in an interval of 1.0 cM, such that each was 0.5 cM away from the Y locus. Two other markers, AFLP-A and AFLP-B, co-segregated with the seed color gene. For ease of use in breeding programs, these 4 most tightly linked AFLP markers were converted into reliable PCR-based markers. SCAR-K, which was derived from AFLP-K, was assigned to linkage group 9 (N9) of a B. napus reference map consisting of 150 commonly used SSR (simple sequence repeat) markers. Furthermore, 2 SSR markers (Na14-E08 and Na10-B07) linked to SCAR-K on the reference map were reversely mapped to the linkage map constructed in this study, and also showed linkage to the Y locus. These linked markers would be useful for the transfer of the dominant allele Y from No. 2127-17 to elite cultivars using a marker-assisted selection strategy and would accelerate the cloning of the seed coat color gene.