Identification of AFLP and SCAR markers linked to the male fertility restorer gene of <Emphasis Type="Italic">pol</Emphasis> CMS (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The pol cytoplasmic male-sterility system has been widely used as a component for utilization of heterosis in Brassica napus and offers an attractive system for study on nuclear–mitochondrial interactions in plants. Genetic analyses have indicated that one dominant gene, Rfp, was required to achieve complete fertility restoration. As a first step toward cloning of this restorer gene, we attempted molecular mapping of the Rfp locus using the amplified fragment length polymorphism (AFLP) technique combined with bulked segregant analysis (BSA) method. A BC₁ population segregating for Rfp gene was used for tagging. From the survey of 1,024 AFLP primer combinations, 13 linked AFLP markers were obtained and five of them were successfully converted into sequence characterized amplified region (SCAR) markers. A population of 193 plants was screened using these markers and the closest AFLP markers flanking Rfp were at the distances of 2.0 and 5.3 cM away, respectively. Further the AFLP or SCAR markers linked to the Rfp gene were integrated to one doubled-haploid (DH) population derived from the cross Quantum × No.2127-17 available in our laboratory, and Rfp gene was mapped on N18, which was the same as the previous report. These molecular markers will facilitate the marker-assisted selection (MAS) of pol CMS restorer lines.     

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.     

Sequence characterized amplified region markers tightly linked to the dwarf mosaic resistance gene <Emphasis Type="Italic">mdm1 (t)</Emphasis> in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Maize dwarf mosaic is one of the devastating and wide spread viral diseases in the world. The present investigation was carried out to develop DNA markers closely linked to the resistance gene mdm1 (t). Linkage between the markers and phenotypes was confirmed by analyzing an F₂ population obtained from a cross between a resistant parent ‘Huangzaosi’ and a susceptible parent ‘Mo17(478)’. Four AFLP markers were found in the maize dwarf mosaic resistant plants. By using (BSA) bulked segregant analysis, two of the four AFLP markers were transformed into Sequence-characterized amplified regions markers (SCARs), nominated Rsun-1 and Rsun-2. The two amplified fragment length polymorphism (AFLP) markers, RHC-1and RHC-2, from the amplification products of primer combination E-AGC/M-CAA and E-AGC/M-GAA, showed linkage with the mdm1 (t) gene in a genetic distance 1.6 and 2.0 cM, respectively. The results indicate that the new SCAR markers will be valuable to distinguish resistant plants from susceptible plants in plantlets growing in seedbeds. The markers developed in this study are suitable for marker-assisted selection for maize dwarf mosaic resistance.     

Identification of two SCAR markers co-segregated with the dominant Ms and recessive ms alleles in onion (Allium cepa L.)

Cytoplasmic genetic male-sterility is used to produce hybrid onion (Allium cepa L.) seeds worldwide. In this paper, we present the results of research aimed toward identifying PCR-based markers linked to the Ms locus through amplified fragment length polymorphism (AFLP). After screening 512 AFLP primer combinations, only one AFLP fragment was identified as being flanking linked to the dominant Ms allele. Subsequently, the AFLP marker was converted into a sequence-characterized amplified region (SCAR) marker, designated as DNF-566, co-segregated with the dominant Ms allele in first backcross (BC₁) segregated populations. Furthermore, we designed another molecular marker (RNS-357) co-segregated with the ms allele to identify different genotypes (i.e., MsMs, Msms, or msms). Both markers could be used for evaluating onion lines with different genetic backgrounds (including male-sterile lines, maintainer lines, male-fertile lines, and commercial based F₁ hybrid cultivars). The results of this study indicate that maintainer plants could be directly selected by using these 2 SCAR markers in the onion breeding process, and this may contribute significantly toward breeding onion F₁ hybrid cultivars.     

Development of SCAR and CAPS markers linked to a recessive male sterility gene in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Genetic male sterility (GMS) has been a useful system for the production of hybrid varieties in self-pollinated plants. We obtained a GMS line developed from a spontaneous mutation in lettuce (Lactuca sativa L.). Genetic analysis in our previous study revealed that the sterility was controlled by a recessive gene which was named ms-S. For simple and quick screening of individuals showing male sterility, we attempted molecular mapping of the ms-S locus using an amplified fragment length polymorphism (AFLP) technique. From the examination of 4,096 AFLP primer combinations, 63 AFLP markers were found to be linked to the gene and nine of them were successfully converted into sequence characterized amplified region (SCAR) markers and cleaved amplified polymorphic sequence (CAPS) markers. Linkage analysis indicated that these nine markers were closely linked to the ms-S gene and all were located on the same side of the gene. The minimum genetic distance between the ms-S gene and a marker was 3.1 cM. These results provide additional information for map-based cloning of the ms-S gene and will be of great help for lettuce breeding using GMS to produce F₁ hybrids.     

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.     

High-resolution mapping of the nud locus controlling the naked caryopsis in barley

The hulled or naked caryopsis character of barley is an important agronomic trait because of the direct link to its use. A single recessive gene, nud, located on the long arm of chromosome 7H, controls the naked caryopsis character. Previously, linked amplified fragment length polymorphism (AFLP) bands from bulked segregant analysis were screened, and the nud gene was mapped in a population of 151 F₂ plants. In the present study, the aim was to construct a high‐resolution map of the nud gene towards its positional cloning. Two AFLP bands were converted into sequence‐characterized amplified region (SCAR) markers (sKT5 and sKT9), and a previously reported SCAR marker sKT3 was improved for more reliable detection of polymorphism. A total of 2380 segregants derived from five cross‐combinations were analysed, and the nud gene was flanked by sKT3 and sKT9, at the 0.6‐cM proximal and the 0.06‐cM distal side, respectively. The SCAR markers developed in this study should be useful for marker‐assisted selection in naked barley breeding employing crosses between naked and hulled accessions.     

Identification of SCAR markers linked to <Emphasis Type="Italic">or</Emphasis>, a gene inducing beta-carotene accumulation in Chinese cabbage

The or mutation in Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a recessive, single-locus mutation that causes the head leaves of the plant to accumulate carotenoids and turn orange. In China, considerable attention has been focused in recent years on breeding the variety with orange head leaves. In this study, sequence-characterized amplified region (SCAR) markers linked to the or gene were identified based on random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) by performing a bulked segregant analysis (BSA) using a doubled haploid (DH) population derived from the F₁ cross between 91-112 (white head leaves) and T12-19 (orange head leaves) via microspore culture. Two RAPD markers—OPB01-845 and OPAX18-656—and 1 AFLP marker, namely, P67M54-172, were identified to be linked to the or gene, and they were successfully converted into the SCAR markers SCR-845, SCOR204, and SCOR127, respectively. In a linkage analysis, these 3 SCAR markers and 2 previously published simple sequence repeat markers, namely, BRMS-51 and Ni4D09 (located on R9 linkage group), were mapped to the same linkage group with the or gene at a LOD score of 6.0, indicating that the or gene should be located on the linkage group R9 of the A genome. In addition, accuracies of 92%, 90%, and 89.1% were obtained when 110 different inbred breeding lines of Chinese cabbage were used for investigation with these 3 SCAR markers, indicating that these makers could be used in marker-assisted selection in orange head leaf breeding programs for Chinese cabbage.     

多重PCR技术鉴定番茄Ty-1和Mi基因

利用同一PCR反应体系,对分别与番茄(Lycopersion esculentum)抗黄化曲叶病毒(Tomato YellowLeaf Curl virus)病的Ty-1基因和番茄抗根结线虫(root-knot nematode)的Mi基因紧密连锁的SCAR标记进行同时扩增筛选,扩增的特异性片段与单引物扩增片段完全吻合.与Ty-1基因紧密连锁的SCAR1标记为共显性标记,抗感材料均产生398 bp的特异片段,纯合和杂合抗病基因型存在TaqⅠ酶切位点,酶切后分别产生了303 bp和95 bp以及398 bp、303 bp和95 bp的特异性片段,而感病基因型无此酶切位点.与Mi基因紧密连锁的SCAR2标记也为共显性标记,抗感材料均产生750 bp的特异片段,纯合和杂合抗病基因型存在TaqⅠ酶切位点,酶切后分别产生了570bp和180bp以及750bp、570bp和180bp的特异性片段,而感病基因型无此酶切位点.酶切结果仍为750 bp的产物.经反复验证,结果准确可靠,可以用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定.     

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.     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

Clustering of amplified fragment length polymorphism markers in a linkage map of rye

Amplified fragment length polymorphisms (AFLPs) are now widely used in DNA fingerprinting and genetic diversity studies, the construction of dense genetic maps and in fine mapping of agronomically important traits. The AFLP markers have been chosen as a source to extend and saturate a linkage map of rye, which has previously been generated by means of restriction fragment length polymorphism, random amplified polymorphic DNA, simple sequence repeat and isozyme markers. Gaps between linkage groups, which were known to be part of chromosome 2R, have been closed, thus allowing the determination of their correct order. Eighteen EcoRI‐MseI primer combinations were screened for polymorphism and yielded 148 polymorphic bands out of a total of 1180. The level of polymorphism among the different primer combinations varied from 5.7% to 33.3%. Eight primer combinations, which revealed most polymorphisms, were further analysed in all individuals of the F₂ mapping population. Seventy‐one out of 80 polymorphic loci could be integrated into the linkage map, thereby increasing the total number of markers to 182. However, 46% of the mapped AFLP markers constituted four major clusters located on chromosomes 2R, 5R and 7R, predominantly in proximity to the centromere. The integration of AFLP markers caused an increase of 215 cM, which resulted in a total map length of almost 1100 cM.     

An AFLP-based linkage map of Zoysiagrass (Zoysia japonica)

To construct an amplified‐fragment length polymorphism (AFLP)‐based molecular linkage map of zoysiagrass, the selfed progenies of a clone consisting of 78 individuals were analysed using 471 AFLP markers derived from 126 PstI/MseI primer combinations. Of these markers, 364 were grouped into 26 linkage groups. The maps covered a total length of 932.5 cM, with an average spacing of 2.6 cM between markers. This information proves useful for gene targeting, quantitative trait loci mapping, and marker‐assisted selection in zoysiagrass.     

An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata)

Similar to SCAR, an extended random primer amplified region (ERPAR) marker is a PCR amplified genomic DNA fragment at a single genetically defined locus. However, ERPAR uses specific primer pairs derived from RAPD primers by adding bases sequentially to their 3′-ends. As an example, an ERPAR marker was derived from a RAPD marker (OT11₉₀₀) linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). After two cycles of base adding and primer pair screening, a primer pair (5′-TTCCCCGCGACT-3′and 5′-TTCCCCGCGAGA-3′) amplified a single intense band with the same size as OT11₉₀₀. The identity of the new marker and OT11₉₀₀ was verified by segregation analysis. The new marker amplified by this extended primer pair was named as EPT11₉₀₀. The development of ERPAR exploits the importance of 3′-end bases of primers in PCR ERPAR shares advantages of SCAR, but eliminates the need for cloning and sequencing. It is a fast and universal way of converting RAPD markers into stable markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.     

Identification of a sequence-tagged site (STS) marker linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L.) by non-radioactive AFLP analysis

To identify DNA markers linked to a fertility restorer (Rf) genefor Ogura cytoplasmic male sterility in radish (Raphanus sativus L.),a non-radioactive, amplified fragment length polymorphism (AFLP) analysiswas performed on bulked DNA samples from male-sterile and male-fertileradishes. Ten male-fertile and 10 male-sterile plants selected arbitrarilyfrom an F₂ population made by selfing of F₁ plant from a crossbetween a male-sterile (`MS-Gensuke') plant and a restorer (`Comet') plantwere used as material. Using 32 AFLP primer pairs, one AFLP fragment(AFLP190) which is specific to the bulked DNA samples from male-fertileF₂ plants was identified. AFLP190 was characterized by molecularcloning and nucleotide sequencing, and was converted to a sequence-taggedsite (STS) marker, STS190. A linkage analysis performed in 126individuals of two independent F₂ populations showed tight linkageof STS190 to the Rf gene. The rate of recombination between themarker and Rf was estimated to be less than 1%, making STS1901.2 cM from the gene.     

Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences

Efficiencies of SCAR, CAPS and PCR-RF-SSCP marker production were investigated using two combinations of breeding lines in Brassica oleracea. Published EST sequences of B. oleracea, Brassica rapa, Brassica napus, and Arabidopsis thaliana and newly determined nucleotide sequences of anther cDNA clones from B. oleracea were used for designing primer pairs to amplify genes. The percentage of primer pairs yielding DNA amplification of a single gene was higher in primer pairs of B. oleracea (91%) than those of B. rapa (56%) and A. thaliana (17%). Single DNA fragments amplified by 9% of the primer pairs showed polymorphism as SCAR markers between a broccoli line and a Chinese kale line by agarose-gel electrophoresis. CAPS analysis showed different band patterns in 32% of the same-sized DNA fragments, and PCR-RF-SSCP analysis revealed DNA polymorphism in 52% of those showing no DNA polymorphism by CAPS. In total, 71% of the single DNA fragments were converted to DNA markers. The frequency of DNA polymorphism between parental lines of a cabbage F₁ hybrid was lower, 5% by SCAR and 12% by CAPS. However PCR-RF-SSCP analysis revealed DNA polymorphism in 21% of the DNA fragments showing no polymorphism by CAPS. These results suggest that PCR-RF-SSCP analysis enables highly efficient DNA marker production for mapping of genes in Brassica using progeny, even progeny of closely related parents. Analysis of selfed seeds of broccoli F₁ cultivars using PCR-RF-SSCP markers indicated that PCR-RF-SSCP analysis is also applicable to seed purity tests.     

Genetic diversity among selected Argentinean garlic clones (Allium sativum L.) using AFLP (Amplified Fragment Length Polymorphism)

Genetic diversity of eight selected Argentinean garlic clones (Allium sativum L.) were investigated at the DNA level with the amplified fragment length polymorphism DNA (AFLP) procedure. A total of 405 unambiguous bands were identified by six primer combinations of EcoR I +3 and Mse I +3, of those, 398 showed a clear polymorphism, representing 98% of the total bands. A presence/absence matrix was constructed with the polymorphic bands, and a dendrogram was obtained from it with the UPGMA method. The accessions showed different levels of similarity ranging between 0.24 and 0.97, using the coefficient of Jaccard. The dendrogram showed six arbitrary groups. Accessions typically considered as different clones show similarities between 0.97 and 0.495. The garlic clones were clustered according to the physiological group and bulb color. We could detect an association between AFLP and the geographical origin of the clones. The potential use of AFLP could allow not only the differentiation among species, but also between botanical varieties and well-defined ecotype groups. This is the first report of the use of AFLP to characterize Argentinean garlic clones. This revised version was published online in August 2006 with corrections to the Cover Date.     

青海大黄油菜粒色性状分子标记的开发和图谱整合

利用青海大黄油菜和褐籽白菜型油菜09A-126构建BC₄和F₂分离群体, 结合AFLP与群体分离分析法(bulked segregant analysis, BSA)筛选引物, 获得5个与黄籽基因Brsc1紧密连锁的分子标记Y11~Y15。5个AFLP特异片段的序列, 均与白菜型油菜的A9染色体部分序列表现同源。将5个AFLP标记成功转化为5个SCAR标记(SC11~SC15)。利用目标基因所在染色体区段序列筛选到7个与目标基因紧密连锁的SSR标记(BrID10607、KS10760、B089L03-3和A1~A4)。利用SCAR和SSR标记扫描F₂群体中部分单株, 发现SC14和A1为共显性标记。用BC₄群体将Brsc1定位在标记Y06和A4之间1.7 Mb的区间内, 遗传距离分别为0.115 cM和0.98 cM。标记Y05和Y12与Brsc1共分离。本研究为黄籽油菜分子标记辅助选择育种体系的建立及目标基因的进一步精细定位和图位克隆奠定了基础。