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.     

Identification of RAPD markers linked to recessive genes conferring siliqua shatter resistance in Brassica rapa

Shattering of siliquae causes significant seed loss in canola (Brassica napus) production worldwide. There is little genetic variation for resistance to shatter in canola and, hence, the trait has been studied in B. rapa. Previous studies have shown two randomly segregating recessive genes to be responsible for shatter resistance. Three random amplified polymorphic DNA markers were identified as being linked to shatter resistance using bulked segregant analysis in a F₃B. rapa population. The population was derived from a cross between a shatter‐susceptible Canadian cultivar and a shatter‐resistant Indian line. Of the three markers, RAC‐3₉₀₀ and RX‐7₁₀₀₀ were linked to recessive sh1 and sh2 alleles, and SAC‐20₁₃₀₀ was linked to both dominant Sh1 and Sh2 alleles. The common marker for the dominant wild‐type allele for the two loci was explained to have resulted from duplication of an original locus and the associated markers through chromosome duplication and rearrangements in the process of evolution of the modern B. rapa from its progenitor that had a lower number of chromosomes. Segregation data from double heterozygous F₃ families, although limited, indicated the markers were not linked to each other and provided further evidence for the duplication hypothesis.     

Identification of RAPD markers closely linked to the mlo-locus in barley

Developing resistance to powdery mildew, Erysiphe graminis f.sp. hordei, is a major goal of many barley breeding programmes. Several resistance genes have been tagged or mapped with molecular markers. The mlo gene confers durable resistance towards all known isolates of the pathogen. In this study, RAPD markers and bulked segregant analysis were used to determine PCR-based markers linked to the mlo-locus. Sixty doubled haploid lines from a cross between an isogenic line of ‘Ingrid’ carrying the mlo11 allele and a susceptible cv. ‘Pokko’ were used as plant material. Seven linked RAPD markers were found, the closest lying 1.6 cM away from the resistance gene. When eight barley varieties were assayed for the presence of this band, F4-980, it was found in the resistant varieties but not in the susceptible ones. The linked marker bands could be amplified from DNA-samples prepared by using three different methods, including a quick squash technique. PCR-based markers linked to the resistance gene can be used as tools for selection in breeding programmes.     

Sclerotinia rot resistance in red clover: Identification of RAPD markers using bulked segregant analysis

Random amplified polymorphic DNA (RAPD) was used with the objective of identifying DNA markers linked to the sclerotinia crown and stem rot (SCSR) resistance of red clover. Bulked segregant analysis was used to detect polymorphism that should be linked to SCSR resistance. Two bulks were made by pooling previously extracted DNA. Each bulk (one resistant, and the other susceptible) consisted of eight genotypes from an F₂ population obtained from a cross between a susceptible and a resistant parent. A binomial model was used to select RAPD fragments with a low probability of no linkage with SCSR resistance. Four RAPD fragments were retained as candidate markers of SCSR resistance. Three are associated with resistance and one with susceptibility.     

RAPD and SCAR markers for powdery mildew resistance gene er in pea

In pea, a single recessive gene (er) on linkage group 6 confers resistance to powdery mildew caused by Erysiphe pisi. The present study aims to identify molecular markers linked to the er gene. Screening of the powdery mildew‐resistant cultivar ‘DMR11’ and its susceptible nearisogenic line for polymorphism revealed linkage of two RAPD primers (OPO‐02 and OPU‐17) to the er gene and a sequence characterized polymorphic region (SCAR) primer, ScOPD‐10₆₅₀ with er in a population of 83 F₂ plants in the order: OPU‐17 ‐ er ‐ ScOPD‐10₆₅₀ ‐ OPO‐02. The markers ScOPD‐10₆₅₀ and OPU‐17 being coupled with the allele causing resistance would substantially increase the efficiency of marker‐assisted selection in peabreeding for powdery mildew.     

RAPD markers linked to resistance to downy mildew disease in soybean

To determine and utilize RAPD markers linked to resistance to downymildew incited by Peronospora manshurica in soybean, a resistantcultivar `AGS129' was crossed to a susceptible cultivar `Nakhon Sawan 1'(NS1). F₂ and BC₁ populations were advanced from the F₁ and evaluatedfor resistance to the disease. ²-test demonstrated that the resistancewas controlled by a single dominant gene (Rpmx). Near-isogenic lines(NILs) and bulked segregant analysis (BSA) were used to identify RAPDmarkers linked to the gene. Six DNA bulks namely F₅(R), F₅(S),BC₆F₃(R), BC₆F₃(S), F₂(R) and F₂(S) were set up by pooling equalamount of DNA from 8 randomly selected plants of each disease responsetype. A total of 180 random sequence decamer oligonucleotide primerswere used for RAPD analysis. Primer OPH-02 (5 TCGGACGTGA 3 andOPP-10 (5 TCCCGCCTAC 3) generated OPH-02₁₂₅₀ and OPP-10₈₃₁fragments in donor parent and resistant bulks, but not in the recurrentparent and susceptible ones. Co-segregation analysis using 102 segregatingF₂ progenies confirmed that both markers were linked to the Rpmxgene controlling downy mildew disease resistance with a genetic distance of4.9 cm and 23.1 cm, respectively. Marker OPH-02₁₂₅₀ was presentin 13 of 16 resistant soybean cultivars and absent in susceptible cultivars,thus confirming a potential for MAS outside the mapping population.     

Identification of resistant sources and DNA markers linked to genomic region conferring dry root rot resistance in chickpea (Cicer arietinum L.)

A set of 520 chickpea germplasm lines was screened under laboratory conditions using blotter paper technique for reaction to dry root rot caused by Rhizoctonia bataticola (Taub.) Butler. The lines PG06102, BG2094 and IC552137 were identified as resistant for dry root rot. Phenotyping the mapping population consisting of 129 F_2:3 progeny derived from the cross L550 × PG06102 during 2013 winter indicated monogenic inheritance of dry root rot resistance. Fifty‐two of 381 simple sequence repeat (SSR) primers polymorphic between the two parents were used to genotype F₂ resistant and susceptible bulks prepared on the basis of reaction of F_2:3 progeny. Four markers differentiated the resistant and susceptible bulks. All the four polymorphic markers were then assayed on the entire F₂ population. Linkage analysis using 129 F₂ plants revealed that two markers ICCM0299 and ICCM0120b were co‐segregating with resistance to dry root rot. These two markers appeared to have additive effects on resistance and could be potentially utilized in dry root resistance breeding programme.     

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     

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 of a molecular marker linked to the closed capsule mutant trait in sesame using AFLP

The identification of an amplified fragment length polymorphism (AFLP) marker linked to an agronomically useful trait in sesame is reported. A bulked segregant analysis (BSA) approach was adopted on segregating progenies of a cross between the closed capsule mutant line ‘cc3’, and the Turkish variety ‘Muganli‐57′. A total of 72 primer combinations were screened for linkage to the trait, but only one closely linked amplified fragment length polymorphism (AFLP) marker was identified. The linkage was confirmed by analysing the AFLP profile from single plants. The marker has the potential to accelerate breeding programmes aimed at modifying unwanted side‐effects of the closed capsule mutation by marker‐assisted selection.     

Searching for molecular markers linked to male sterility and self-compatibility in apricot

In apricot, there are two traits related to flower biology that can be a limitation for the breeder: self‐incompatibility and male sterility. Fully mature trees are needed to determine both phenotypes. Molecular biology techniques may provide a potential tool for increasing selection efficiency by means of marker‐assisted selection. In this study, randomly amplified polymorphic DNA markers combined with bulked segregant analysis have been used to search for markers linked to these traits. A screening of 228 primers resulted in a marker linked to male‐fertility (M4‐650). No markers linked to S alleles were obtained. A second approach consisted of the screening of primers in a subset of seedlings and resulted in three markers linked to male‐fertility and two markers linked to the Sc allele. These markers may allow an earlier determination of the phenotype in progenies segregating for these traits.     

Molecular mapping of the leaf rust resistance gene Rph7 in barley

Leaf rust of barley, caused by Puccinia hordei Otth, is an important foliar disease in most temperate regions of the world. Sixteen major leaf rust resistance (Rph) genes have been described from barley, but only a few have been mapped. The leaf rust resistance gene Rph7 was first described from the cultivar ‘Cebada Capa’ and has proven effective in Europe. Previously mapped restriction fragment length polymorphism (RFLP) markers have been used to determine the precise location of this gene in the barley genome. From the genetic analysis of a ‘Bow‐man’/‘Cebada Capa’ cross, Rph7 was mapped to the end of chromosome 3HS, 1.3 recombination units distal to the RFLP marker cMWG691. A codominant cleaved amplified polymorphic site (CAPS) marker was developed by exploiting allele‐specific sequence information of the cMWG691 site and adjacent fragments of genomic DNA. Based on the large amount of polymorphism present in this region, the CAPS marker may be useful for the marker‐assisted selection of Rph7 in most diverse genetic backgrounds.     

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.     

Two RAPD markers linked to a major fertility restorer gene in pepper

Both major and minor genes control the restoring of fertility in the cytoplasmic male-sterility system in pepper (Capsicum annuum L.). Bulked segregant analysis (BSA) was applied to identify molecular markers linked to a major restorer gene (Rf) using the F₂ population of NiujiaojiaoNo.21 (rfrf)/Xiangtanwan (RfRf). Two random amplified polymorphic DNA (RAPD) markers linked to this allele were detected with 520 decamer primers with arbitrary sequences. OP13₁₄₀₀ is a tightly linked marker with a genetic distance of0.37 cm. OW19₈₀₀ is on the opposite side with a distance of 8.12 cm. Both markers were repeatable and easy to score. A panel of genotypes, including 13elite inbred lines with different fertility restoring ability, were assayed for the presence ofOP13₁₄₀₀ and OW19₈₀₀. The markers are absent in all sweet pepper lines, indicating that they will be most helpful for transferring Rf into sweet pepper lines. With the aid of these markers, the size of the backcross population for testcrosses can be minimized. Furthermore, these markers will be useful in genetic analysis of the minor genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD markers linked to a rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

Groundnut rust (Puccinia arachidis Speg.) is an important air borne pathogen, which causes substantial losses in groundnut yield and quality. Although large numbers of accessions were identified as rust resistant in wild, interspecific derivative and cultivated groundnut species, transfer of resistance to well-adapted cultivars is limited due to linkage drag, which worsens yield potential and market acceptance. A F₂ mapping population comprising 117 individuals was developed from a cross between the rust resistant parent VG 9514 and rust susceptible parent TAG 24. Rust resistance was governed by single dominant gene in this cross. We identified 11 (out of 160) RAPD primers that exhibited polymorphism between these two parents. Using a modified bulk segregant analysis, primer J7 (5′CCTCTCGACA3′) produced a single coupling phase marker (J7₁₃₅₀) and a repulsion phase marker (J7₁₃₀₀) linked to rust resistance. Screening of the entire F₂ population using primer J7 revealed that the coupling phase marker J7₁₃₅₀ was linked with the rust resistance gene at a distance of 18.5 cM. On the other hand, the repulsion phase marker J7₁₃₀₀ was completely linked with rust resistance. Additionally, both J7₁₃₀₀ (P = 0.00075) and J7₁₃₅₀ (P < 0.00001) were significantly associated with the rust resistance. Marker J7₁₃₀₀ identified all homozygous rust resistant genotypes in the F₂ population and was present in all the eight susceptible genotypes tested for validation. Thus, J7₁₃₀₀ should be applicable for marker-assisted selection (MAS) in the groundnut rust resistance breeding programme in India. To the best of our knowledge this is the first report on the identification of RAPD markers linked to rust resistance in groundnut.     

Sequence tagged site markers linked to the sbm1 gene for resistance to pea seedborne mosaic virus in pea

Resistance to pea seed‐borne mosaic virus (PSbMV) pathotype P‐1 in peas is conferred by sbm1 with recessive inheritance. PSbMV is an economically important pathogen with world‐wide distribution that causes significant losses in pea yield and reduces seed and produce quality. The sbm1 gene was previously mapped to linkage group VI on molecular linkage maps of the pea genome. To improve plant breeders’ ability to develop varieties resistant to PSbMV, two random amplified polymorphic DNA markers (G05_2537 and L01_910) and one restriction fragment length polymorphism (P446) linked to sbm1 have been identified. The genomic sequences for these markers have been characterized and the information used to develop three simple polymerase chain reaction‐based STS (sequence tagged site) assays. Linkage analysis in two F₂ populations showed that the most tightly linked of these three STS loci (sG05_2537) is approximately 4 cM from sbm1. Characterization of a collection of resistant and susceptible germplasm demonstrated a strong correlation between STS alleles and sbm1 alleles, indicating the utility of these markers for marker‐assisted selection in breeding programmes using a range of germplasm sources.     

Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn (Zea mays var. ceratina)

Exserohilum turcicum causes northern corn leaf blight (NCLB), an important disease occurring in maize producing areas throughout the world. Currently, the development of cultivars resistant to E. turcicum seems to be the most efficient method to control NCLB damage. Marker-assisted selection (MAS) enables breeders to improve selection efficiency. The objective of this work was to identify random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers associated with NCLB resistance. Bulked segregant analysis (BSA) was used to search for RAPD markers linked to NCLB resistance genes, using F₂ segregating population obtained by crossing a susceptible inbred ‘209W’ line with a resistant inbred ‘241W’ line. Two hundred and twenty-two decamer primers were screened to identify four RAPD markers: OPA07₅₂₁, OPA16₄₅₇, OPB09₅₂₀, and OPE20₅₃₆ linked to NCLB resistance phenotype. These markers were converted into dominant SCAR markers: SCA07₄₉₆, SCA16₄₂₀, SCB09₄₆₄, and SCE20₄₂₉, respectively. The RAPD and SCAR markers were developed successfully to identify NCLB resistant genotypes in segregating progenies carrying NCLB resistant traits. Thus, the markers identified in this study should be applicable for MAS for the NCLB resistance in waxy corn breeding programs.     

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 of SSR markers linked to the Phytophthora resistance gene Rps1-d in soybean

To identify markers for the Phytophthora resistance gene, Rps1‐d, 123 F_{2 : 3} families were produced from a cross between Glycine max (L.) Merr. ‘Tanbakuro’ (a Japanese traditional black soybean) and PI103091 (Rps1‐d) as an experimental population. The results of virulence tests produced 33 homozygous resistant, 61 segregating and 29 homozygous susceptible F_{2 : 3} families. The chi‐squared test gave a goodness‐of‐fit for the expected ratio of 1 : 2 : 1 for resistant, segregating and susceptible traits, suggesting that the inheritance of Rps1‐d is controlled by a monogenic dominant gene. Simple sequence repeat (SSR) analyses of this trait were carried out using the cultivars ‘Tanbakuro’ and PI103091. Sixteen SSR primers, which produced 19 polymorphic fragments between the two parents, were identified from 41 SSR primers in MLG N. Eight SSR markers were related to Rps1‐d, based on 32 of the 123 F_{2 : 3} families, consisting of 16 homozygous resistant and 16 homozygous susceptible lines. The remaining 91 families were analysed for these eight markers, and a linkage map was constructed using all 123 F_{2 : 3} families. The length of this linkage group is 44.0 cM. The closest markers, Sat_186 and Satt152, are mapped at 5.7 cM and 11.5 cM, respectively, on either side of the Rps1‐d gene. Three‐way contingency table analysis indicates that dual‐marker‐assisted selection using these two flanking markers would be efficient.