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.     

RAPD and SCAR markers linked to the sex expression locus M in asparagus

Bulk segregant analysis (BSA), random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) methods were used to map molecular markers to the sex locus M of asparagus. Two parents, A19 (male, Mm) and MW25 (female, mm), and 63 progeny were used for the study. Two DNA bulks, one male and one female, were made by pooling equal amounts of DNA from 10 randomly selected progeny of each sex type. A total of 760 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Primer OPC15 produced two RAPD markers, OPC15-98 and OPC15-30, both of which were linked to the M locus at a distance of 1.6 cM. Subsequently, amplified RAPD fragment OPC15-98 was cloned and sequenced. The sequence was then used to design flanking 24-mer oligonucleotide SCAR primers SCC15-1 and SCC15-2. Both of these SCAR primers amplified a single 980 bp fragment; the same size as the cloned RAPD fragment. However, the SCAR marker was dominant as was the original OPC15-98 band from which it was derived. These RAPD and SCAR markers could be used for scoring male and female progeny in the mapping population, but were not found to be applicable to other asparagus germplasm studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

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.     

Development of molecular markers linked to the <Emphasis Type="Italic">Fom-1</Emphasis> locus for resistance to Fusarium race 2 in melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (F.o.m), is a worldwide soil-borne disease of melon (Cucumis melo L.). The most effective control measure available is the use of resistant varieties. Resistance to races 0 and 2 of this fungal pathogen is conditioned by the dominant gene Fom-1. An F₂ population derived from the ‘Charentais-Fom1’ × ‘TRG-1551’ cross was used in combination with bulked segregant analysis utilizing the random amplified polymorphic DNA (RAPD) markers, in order to develop molecular markers linked to the locus Fom-1. Four hundred decamer primers were screened to identify three RAPD markers (B17₆₄₉, V01₅₇₈, and V06₁₀₉₂) linked to Fom-1 locus. Fragments amplified by primers B17₆₄₉ and V01₅₇₈ were linked in coupling phase to Fom1, at 3.5 and 4 cM respectively, whereas V06₁₀₉₂ marker was linked in repulsion to the same dominant resistant allele at 15.1 cM from the Fom-1 locus. These RAPDs were cloned and sequenced in order to design primers that would amplify only the target fragment. The derived sequence characterized amplified region (SCAR) markers SB17₆₄₅ and SV01₅₇₄ (645 and 574 bp, respectively) were present only in the resistant parent. The SV06₁₀₉₂ marker amplified a band of 1092 bp only in the susceptible parent. These markers are more universal than the CAPS markers developed by Brotman et al. (Theor Appl Genet 10:337–345, 2005). The analysis of 24 melon accessions, representing several melon types, with these markers revealed that different melon types behaved differently with the developed markers supporting the theory of multiple, independent origins of resistance to races 0 and 2 of F.o.m.     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Development of a genetic marker linked to a new thermo-sensitive male sterile gene in rice (<Emphasis Type="Italic">Oryza sativa L</Emphasis>.)

Application of the thermo-sensitive genic male sterile (TGMS) system has a great potential to increase the efficiency of hybrid rice breeding. An indica rice TGMS mutant, 0A15-1, was crossed with a fertile indica line Guisi-8 to map the gene responsible to the TGMS. A RAPD (random amplified polymorphic DNA) maker, S187-770, linked to the TGMS gene at a distance of 1.3 cM in coupling phase was identified. The S187-770 was then cloned and sequenced to develop a dominant SCAR (sequence characterized amplified region) marker. Homology search against rice genome DNA sequence database indicated that S187-770 located on the short arm of chromosome 3 and close to centromere as a single copy sequence. This SCAR marker can be used in the marker-assisted transfer of this gene to different genetic background. As no other TGMS gene has been mapped on rice chromosome 3, the gene from 0A15-1 is a new TGMS gene and tentatively designated tms6(t).     

RAPD and SCAR markers for resistance to acochyta blight in lentil

Resistance to ascochyta blight of lentil (Lens culinaris Medikus),caused by the fungus Ascochyta lentis, is determined by a single recessive gene, ral ₂, in the lentil cultivar Indian head. Sixty F₂ individuals from a cross between Eston (susceptible) and Indian head (resistant) lentil were analyzed for the presence of random amplified polymorphic DNA (RAPD) markers linked to the ral ₂gene, using bulked segregant analysis (BSA). Out of 800 decanucleotide primers screened, two produced polymorphic markers that co-segregated with the resistance locus. These two RAPD markers, UBC227₁₂₉₀and OPD-10₈₇₀, flanked and were linked in repulsion phase to the gene ral ₂ at 12 cm and 16 cm, respectively. The RAPD fragments were converted to SCAR markers. The SCAR marker developed from UBC227₁₂₉₀ could not detect any polymorphism between the two parents or in the F₂. The SCAR marker developed from OPD-10₈₇₀ retained its polymorphism. The polymorphic RAPD marker UBC227₁₂₉₀ and the SCAR marker developed from OPD-10₈₇₀ can be used together in a marker assisted selection program for ascochyta blight resistance in lentil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a SCAR marker linked with a MYMV resistance gene in mungbean (Vigna radiata L. Wilczek)

Yellow mosaic disease (YMD) caused by mungbean yellow mosaic virus (MYMV) is the most important disease of mungbean, causing great yield loss. The present investigation was carried out to study the inheritance and identify molecular markers linked with MYMV resistance gene by using F₁, F₂ and 167 F_2 : 8 recombinant inbred lines (RILs) developed from the cross ‘TM‐99‐37’ (resistant) × Mulmarada (susceptible). The F₁ was susceptible, F₂ segregated in 3S:1R phenotypic ratio and RILs segregated in 1S:1R ratio in the field screening indicating that the MYMV resistance gene is governed by a single recessive gene. Of the 140 RAPD primers, 45 primers showing polymorphism in parents were screened using bulked segregant analysis. Three primers amplified specific polymorphic fragments viz. OPB‐07_600, OPC‐06₁₇₅₀ and OPB‐12₈₂₀. The marker OPB‐07₆₀₀ was more closely linked (6.8 cM) with a MYMV resistance gene as compared to OPC‐06₁₇₅₀ (22.8 cM) and OPB‐12₈₂₀ (25.2 cM). The resistance‐specific fragment OPB‐07₆₀₀ was cloned, sequenced and converted into a sequence‐characterized amplified region (SCAR) marker and validated in twenty genotypes with different genetic backgrounds.     

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.     

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.     

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.     

SCAR and RAPD markers associated with 18-carbon fatty acids in rapeseed, Brassica napus

Breeding rapeseed for enhanced oil quality includes the development of varieties with low linolenic acid content. The breeder also aims to develop varieties with a high linoleic acid content because of its nutritional value. Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers have been developed for linolenic acid content, but they are not best suited for a direct application in marker-assisted selection. The RFLP technique is too complex and time-consuming and RAPD markers lack codominance, precluding the distinction of homozygous from heterozygous individuals. In this report the conversion of a RAPD marker to a codominant sequence characterized amplified region (SCAR) marker named L1L9 is described. One of the alleles consisting of an 899 bp fragment (allele A), is associated with low linolenic acid content. The other allele consists of an 641 bp fragment (allele B) and is associated with high linolenic acid content. This marker explains approximately 25% of the genetic variation for this trait. Linkage analysis in the mapping population indicates that the SCAR marker probably tags an ω-3 desaturase gene in B. napus. Two RAPD markers were found to be associated with oleic/linoleic acid content. Markers M14-350 and I06-650 explained approximately 10% and 7% of the genetic variation for linoleic acid content, respectively. These two markers were found linked at 12.3cM in the segregating B. napus F₂ progeny used for mapping. All the markers reported in this paper should be useful in breeding programmes for developing high linoleic and low linolenic acid rapeseed varieties.     

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.     

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.     

Molecular mapping of Aegilops speltoides derived leaf rust resistance gene Lr28 in wheat

In a segregating homozygous F₂ population of bread wheat involving a leaf rust resistance gene Lr28 derived from Aegilops speltoides, six randomly amplified polymorphic DNA (RAPD) markers, three each in coupling and repulsion phase were identified as linked to Lr28, mapped to a region spanning 32 cM including the locus. The F₂ and F₃ populations were studied in the phytotron challenged with the most virulent pathotype 77-5 of leaf rust. A coupling phase linked RAPD marker S464₇₂₁ and a repulsion phase linked RAPD marker S326₅₅₀ flanked the gene Lr28 by a distance of 2.4± 0.016 cM on either side. The flanking markers genetically worked as co-dominant markers when analyzed together after separate amplification in the F₂ population by distinguishing the homozygotes from the heterozygotes and increased the efficiency of marker assisted selection by reducing the false positives and negatives. One of the three RAPD markers, S421₆₄₀ was converted to locus specific SCAR marker SCS421₆₄₀ which was further truncated by designing primers internal from both ends of the original RAPD amplicon to eliminate a non-specific amplification of nearly same size. The truncated polymorphic sequence characterized amplified region marker (TPSCAR) SCS421₅₇₀ was 70 bp smaller, but resulted in a single band polymorphism specific to Lr28 resistance. The TPSCAR marker was validated for its specificity to the gene Lr28 in nine different genetic backgrounds and on 43 of the 50 Lr genes of both native and alien origin, suggesting the utility of the SCAR markers in pyramiding leaf rust resistance genes in wheat.     

Identification of a molecular marker linked to an Agropyron elongatum-derived gene Lr19 for leaf rust resistance in wheat

The leaf rust resistance gene Lr19, transferred from Agropyron elongatum into wheat (Triticum aestivum L.) imparts resistance to all pathotypes of leaf rust (Puccinia recondita f.sp. tritici) in South‐east Asia. A segregating F₂ population from a cross between the leaf rust resistant parent ‘HW 2046’ carrying Lr19 and a susceptible parent ‘Agra Local’ was screened in the phytotron against a virulent pathotype 77‐5 of leaf rust with the objective of identifying the molecular markers linked to Lr19. The gene was first tagged with a randomly amplified polymorphic DNA (RAPD) marker S73₇₂₈. The RAPD marker linked to the gene Lr19 which mapped at 6.4 ± 0.035 cM distance, was converted to a sequence characterized amplified region (SCAR) marker. The SCAR marker (SCS73₇₁₉) was specific to Lr19 and was not amplified in the near‐isogenic lines (NILs) carrying other equally effective alien genes Lr9, Lr28 and Lr32 enabling breeders to pyramid Lr19 with these genes.     

Identification and characterization of a RAPD/SCAR marker linked to a resistance gene for soybean mosaic virus in soybean

Soybean line `ICGR95-5383' [Glycinemax (L.) Merr.] is a newly releasedgermplasm from China and is resistant (R)to soybean mosaic virus (SMV). ICGR95-5383was crossed to the susceptible (S)cultivars `HB1', `Tiefeng21', `Amsoy', and`Williams' to investigate the inheritanceof SMV resistance. The F<sub>1</sub> and F<sub>2</sub>plants were inoculated with SMV-3 (the mostvirulent) strain from Northeast China. Theresults showed that F<sub>1</sub> plants from thefour R × S crosses were necrotic (N) andall F<sub>2</sub> populations segregated in a3(R+N):1S ratio, indicating thatICGR95-5383 carries a single gene withincomplete dominance for resistance to SMV. In a bulked segregant analysis (BSA) of theF<sub>2</sub>population from ICGR95-5383 × HB1, a codominant RAPD marker,OPN11<sub>980/1070</sub>, was found to be linkedto the resistance gene in ICGR95-5383. The980-base pair (bp) fragment OPN11<sub>980</sub>was amplified in the R parent ICGR95-5383,R bulk, and resistant F<sub>2</sub> plants. Theother 1070-bp fragment OPN11<sub>1070</sub> wasamplified in the S parent HB1, S bulk, andsusceptible F<sub>2</sub>plants.OPN11<sub>980/1070</sub> was amplified in theF<sub>1</sub> plants and the necroticF<sub>2</sub> plants from the R×S cross.Segregation analysis of the RAPD marker inthe F<sub>2</sub> population revealed that themarker OPN11<sub>980/1070</sub> is closely linkedto the resistance gene with a map distanceof 3.03 cM. OPN11<sub>980/1070</sub> was clonedand sequenced, and specific PCR primerswere designed to convertOPN11<sub>980/1070</sub> into sequencecharacterized amplified region (SCAR) makerSCN11<sub>980/1070</sub>. SCAR analysis of theF<sub>2</sub> population confirmed thatOPN11<sub>980/1070</sub> and SCN11<sub>980/1070</sub> areat the same locus linked to the SMVresistance gene. The RAPD markerOPN11<sub>980</sub> was used as RFLP probefor southern hybridization to soybeangenomic DNA. Southern analysis showed thatsoybean genome contains low-copy sequenceof OPN11<sub>980</sub>. Using a recombinant inbredmapping population of `Kefeng No.1' (R) ×Nannong1138-2'(S), OPN11_980/1070 was mapped to thesoybean molecular linkage group (MLG) Fbetween the restriction fragment lengthpolymorphism (RFLP) markers B212 (0.7 cM) and K07 (6.7 cM) and 3.03 cM apart from theSMV resistance gene.     

对除草剂敏感致死水稻bel基因的RAPD和SCAR分子标记

以8077s与抗感的籼稻品种丰35亲本及杂交后自交所得的F2群体为材料，采用群分法（Bulked Segregant Analysis, BSA），从210个10mer随机引物，找到两个水稻苯达松敏感池和抗感池之间表现多态性的特异引物——S20和S316，分别产生的标记片段为S20-440和S316-590。它们与bel基因的连锁距离分别为12.132 cM和7.97 cM。对RAPD扩增标记的片段进行克隆、测序，根据测序结果合成两对特异性的SCAR引物，包含原有的RAPD序列。SC01引物在敏感单株中扩增出一条423 bp带；SC02引物在敏感单株中扩增出一条606 bp带，它们的SCAR标记与bel基因的连锁距离为10.66 cM和7.04 cM。应用SCAR标记对水稻恢复系进行了辅助选育。     

结球甘蓝迟抽薹基因RAPD标记转SCAR标记

本研究以与结球甘蓝迟抽薹基因连锁的N1750为引物,应用RAPD技术进行PCR扩增,检测到迟抽薹基因,对特异片段进行回收、克隆和测序,依据测序结果设计SCAR引物。在166株BC1群体(A21与P02杂交得到F1再与P02回交)中通过与RAPD标记的比较,SCAR扩增结果同RAPD扩增结果完全一致,从而证实了SCAR标记的准确性。实验结果表明,与甘蓝迟抽薹基因连锁的RAPD标记被成功转化为SCAR标记,为甘蓝分子标记辅助选择育种提供了基础。