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.     

细胞质雄性不育辣椒育性恢复基因特异分子标记的筛选

利用集团分离分析法(Bulked segregant analysis BSA),以辣椒细胞质雄性不育系BU-12、恢复系RF-12为材料共筛选了336条RAPD引物,其中引物S418在恢复系中呈现特异性扩增,得到一条约3000bp的特异片段。回扩得到两条片段,测序表明大小为1515bp,1162bp。荧光原位杂交证实1515bp片段为恢复系特有,命名为S418_(1515)。序列分析表明S418_(1515)为一新发现的序列,Blastn序列比对同源性小于40%,tBlastx比对发现该序列与水稻2、4、7、10号染色体的几个BAC克隆上的序列高度同源。推测可能与其具有相似的编码功能,为进一步从分子水平研究辣椒育性恢复打下了坚实的基础。根据测序结果设计特异引物,将S418_(1515)转化成特异PCR标记,证明能用于候选材料的初筛。     

细胞质雄性不育辣椒育性恢复基因特异分子标记的筛选

利用集团分离分析法(Bulked segregant analysis BSA),以辣椒细胞质雄性不育系BU-12、恢复系RF-12为材料共筛选了336条RAPD引物,其中引物S418在恢复系中呈现特异性扩增,得到一条约3000bp的特异片段。回扩得到两条片段,测序表明大小为1515bp,1162bp。荧光原位杂交证实1515bp片段为恢复系特有,命名为S4181515。序列分析表明S4181515为一新发现的序列,Blastn序列比对同源性小于40％,tBlastx比对发现该序列与水稻2、4、7、10号染色体的几个BAC克隆上的序列高度同源。推测可能与其具有相似的编码功能,为进一步从分子水平研究辣椒育性恢复打下了坚实的基础。根据测序结果设计特异引物,将S4181515转化成特异PCR标记,证明能用于候选材料的初筛。     

Inheritance and fine mapping of fertility restoration for cytoplasmic male sterility in Gossypium hirsutum L

Genetics of CMS fertility restoration was presented through the analysis of classic genetics and molecular markers. Based on F(2) segregation of the crosses between CMS and the restoring lines, the testcrosses and F(1) x F(1) populations, together with RAPD and SSR mapping, one dominant gene was identified to control the CMS fertility restoration in cotton. The strategy of genotype representation analysis (GRA) was put forward to screen the markers linked with the Rf(1) locus. Using 1,025 random decamer primers and 282 pairs of SSR primers, two RAPD and three SSR markers were identified to be closely linked to the Rf(1) gene. Among the five markers, three were co-dominantly inherited. Additionally, based on the analysis of monosomic and telesomic lines with one SSR maker, the Rf(1) locus could be located on the long arm of chromosome 4. The molecular markers available here are helpful in the development of the elite restoring lines in cotton by marker-assisted selection.     

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     

Cytological and molecular characterization of a novel monogenic dominant GMS in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5′-CTATGCCGAC-3′) gave a repeatable 1500-bp DNA polymorphic segment S243₁₅₀₀ between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243₁₅₀₀ is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.     

Mitochondrial and total DNA RAPD patterns can distinguish restorers of CMS lines in sorghum

 Seven sorghum restorer lines that differentially restore (or maintain) the A1 and A2 cytoplasmic male-sterile (CMS) cytoplasms were studied by RFLP analyses of their mtDNAs and RAPD analyses of their mitochondrial DNA (mtDNA) and total DNA to understand nuclear mitochondrial combinations that are present in these lines. RFLP data from 11 mitochondrial gene probes were inadequate to classify these seven lines. However, the analysis of RAPD profiles of total DNA could distinguish these lines on the basis of their ability to restore completely or partially the fertility in the A1/A2 CMS cytoplasms. Interestingly, RAPD profiles of mtDNAs of these lines also followed the same pattern as that of the total DNA. These results indicate that the different restorer lines possess specific nuclear-cytoplasm combinations. Further, the results also show that the RAPD technique can be used to identify markers for different cytoplasms used in CMS. Received: 26 August 1997 / Accepted: 9 October 1997     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

Molecular mapping of the <Emphasis Type="Italic">Rf1</Emphasis> gene restoring pollen fertility in PET1-based F<Subscript>1</Subscript> hybrids in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Up to now a single cytoplasmic male sterility (CMS) source, PET1, is used worldwide for hybrid breeding in sunflower. Introgression of the restorer gene Rf1, responsible for fertility restoration, into new breeding material requires tightly linked markers to perform an efficient marker-assisted selection. A survey of 520 decamer primers by bulked segregant analyses identified five RAPD markers linked to the restorer gene Rf1. In a F(2) population of 183 individuals one of the RAPD markers, OPK13_454, mapped 0.8 cM from Rf1, followed by OPY10_740 with 2 cM. Bulked segregant analyses using 48 AFLP primer combinations identified 17 polymorphisms, which could be mapped in the same linkage group as Rf1. E33M61_136, and E41M48_113 were mapped 0.3 cM and 1.6 cM from the gene, respectively. Conversion of E41M48_113 into a sequence-specific marker resulted in a monomorphic pattern. However, two of the RAPD markers, OPK13_454 and OPY10_740, were successfully converted into SCAR markers, HRG01 and HRG02, which are now available for marker-assisted selection. To investigate the utility of these SCAR markers in other cross-combinations they were tested in a set of 20 lines. Comparison of the patterns of 11 restorer and nine maintainer lines of PET1 demonstrated that the markers OPK13_454/HRG01 and HRG02 were absent in all maintainer lines but present in all restorer lines, apart from the high oleic line RHA348 and the dwarf line Gio55. In addition, restorer lines developed from the interspecific hybrids Helianthus annuus x Helianthus mollis and H. annuus x Helianthus rigidus gave the same characteristic amplification products.     

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).     

萝卜细胞质雄性不育恢复基因的RAPD标记

以萝卜恢复系9802和不育系9802A配制杂交组合,并以174株个体组成的F2分离群体作为恢复基因的标记群体.以分离群体的不育株和可育株分别建立不育池和恢复池,利用100个RAPD引物对两池间的多态性进行研究.分析表明引物OPC6在两池间扩增出稳定的多态性差异.经连锁分析,证明标记OPC61900与萝卜细胞质雄性不育恢复基因连锁,遗传距离为11.6cM(Centimorgan).这个标记可应用于对育性恢复基因的标记辅助选择.     

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.     

Genetics of resistance to anthracnose and identification of AFLP and RAPD markers linked to the resistance gene in PI 320937 germplasm of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medikus)

Anthracnose, caused by Colletotrichum truncatum, is a major disease problem and production constraint of lentil in North America. The research was conducted to examine the resistance to anthracnose in PI 320937 lentil and to identify molecular markers linked to the resistance gene in a recombinant inbred line (RIL) population developed from a cross of Eston lentil, the susceptible parent, and PI 320937, the resistant parent. A total of 147 F(5:6) RILs were evaluated for resistance to anthracnose in the greenhouse using isolate 95B36 of C. truncatum. Bulked segregant analysis (BSA) strategy was employed and two contrasting DNA bulks were constructed based on greenhouse inoculation of F(5)-derived F(6) RILs. DNA from the parents and bulks were screened with 700 RAPD primers and seven AFLP primer combinations. Analysis of segregation data indicated that a major dominant gene was responsible for resistance to anthracnose while variations in the resistance level among RILs could be the influences of minor genes. We designate the major gene as LCt-2. MapMaker analysis produced two flanking RAPD markers OPEO6(1250) and UBC-704(700) linked to LCt-2 locus in repulsion (6.4 cM) and in coupling (10.5 cM), respectively. Also, three AFLP markers, EMCTTACA(350) and EMCTTAGG(375) in coupling, and EMCTAAAG(175) in repulsion, were linked to the LCt-2 locus. These markers could be used to tag the LCt-2 locus and facilitate marker-assisted selection for resistance to anthracnose in segregating populations of lentil in which PI 320937 was used as the source of resistance. Also, a broader application of the linked RAPD markers was also demonstrated in Indianhead lentil, widely used as a source of resistance to anthracnose in the breeding program at the Crop Development Centre, University of Saskatchewan. Further selection within the few F(5:6) lines should be effective in pyramiding one or several of the minor genes into the working germplasm of lentil, resulting in a more durable and higher level of resistance.     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996     

Inheritance of seed colour and identification of RAPD and AFLP markers linked to the seed colour gene in rapeseed (Brassica napus L.)

In China Polima cytoplasmic male sterility (cms) is currently the most important hybrid system used for the breeding of hybrids. In an effort to develop yellow-seeded Polima cms restorer lines, we used yellow-seeded, doubled haploid (DH) line No.2127-17 as the gene source in crosses with two elite black-seeded Polima cms R lines, Hui5148-2 and 99Yu42, which originated from our breeding programme. The inheritance of seed colour was investigated in the F₂, BC₁ and F₁-derived DH progenies of the two crosses. Seed colour was found to be under the control of the maternal genotype and the yellow seed trait to be partially dominant over the black seed trait. Segregation analysis revealed a single gene locus for the partial dominance of yellow seed colour. Of 810 randomly amplified polymorphic DNA (RAPD) primers, 240 (29.6%) revealed polymorphisms between the parents. Of the 240 RAPD primers and 512 amplified fragment length polymorphism (AFLP) primer pairs, four RAPDs and 16 AFLP pairs showed polymorphisms between the bulks, with two RAPD and eight AFLP markers being identified in the vicinity of the seed-coat colour gene locus using a DH progeny population—derived from the cross Hui5148-2×No.2127-17—of 127 individuals in combination with the bulked segregant analysis strategy. Seven of these latter ten markers were linked to the allele for yellow seed, whereas the other three were linked to the allele for black seed. The seed-coat colour gene locus was bracketed by two tightly linked markers, EA02MG08 (2.4 cM) and S1129 (3.9 cM). The partial dominance and single gene control of the yellow seed-coat colour trait together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded hybrid varieties.     

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     

RAPD based DNA markers linked to anthracnose disease resistance in Sorghum bicolor ( L.) Moench

Anthracnose caused by Colletotrichum graminicola is one of the major diseases of sorghum. The locus for disease resistance in sorghum [Sorghum biocolor (L.) Moench] accession G73 was found to segregate as a simple recessive trait in a cross to susceptible cultivar HC136. In order to identify molecular markers linked to the locus for disease resistance, random amplified polymorphic DNA (RAPD) analysis was coupled with bulk segregant analysis. DNA from the parental cultivars and the bulks were, screened by PCR amplification with 114 RAPD primers. Three RAPD primers amplified a sequence that consegregated with the recessive resistance allele, while another three amplified a band linked to the susceptible allele. The six disease linked markers were screened with individual resistant and susceptible genotypes to observe degree of linkage of identified RAPD markers with the gene for resistance. Two primer sequences (OPI 16 and OPD 12) were found to be closely linked to the locus for disease resistance.     

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     

Mitochondrial DNA Polymorphisms Revealed by RAPD Assays Distinguish the Male-sterile and Male-fertile Cytoplasms in Pearl Millet

Diversification of cytoplamic male sterile (CMS) sources is of considerable significance in pearl millet, considering that almost all the commercially cultivated hybrids, particularly in India, are based on a single CMS source, A₁. We analyzed the mitochondrial DNA (mtDNA) polymorphisms among five pearl millet CMS sources (A₁ to A₅) and a male-fertile maintainer (B) line, all in an isonuclear background. Analysis using 21 random primers led to identification of polymorphic bands specific to the A₁, A₂, A3 and A₅ CMS lines. Two RAPD primers, OP-G12 and OP-G19, in combination were able to distinguish all the male-sterile and male-fertile cytoplasms. Highly effective for this purpose also were four RAPD markers, OP-B7, OP-D8, OP-F10 and OP-G12. Cluster analysis, followed by bootstrap analysis, of the mtDNA dataset revealed two distinct clusters: cluster-I comprising the A₁, A₂, A₃ CMS lines and the male-fertile line, and cluster-II comprising the A₄ and A₅ CMS lines.     

利用分子标记定位农垦58S的光敏核不育基因

对农垦５８Ｓ（Ｏｒｙｚａｓａｔｉｖａｓｐ．ｊａｐｏｎｉｃａ）／大黑矮生标记基因系ＦＬ２组合组建可育集团和不育集团,并以亲本为对照进行了ＲＦＬＰ、ＲＡＰＤ和双引物ＲＡＰＤ分析,结果第１２染色体上的一个单拷贝标记Ｇ２１４０与光敏核不育基因连锁遗传,二者间的遗传图距为１４．１ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）。在筛选过的１０４０个随机单引物和１９０个双引物中,仅引物ＯＰＡＵ１０扩增出与光敏核不育基因连锁的１．５ｋｂＤＮＡ片段,回收、克隆该ＤＮＡ片段并制备探针,将其转换成共显性的ＲＦＬＰ标记并命名为ＯＰＡＵ１０１５００。分离群体连锁分析表明该标记与标记Ｇ２１４０紧密连锁,将农垦５８Ｓ的一对光敏核不育基因定位于第１２染色体上。