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.     

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.     

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.     

Identification of AFLP and RAPD markers linked to anthracnose resistance in grapes and their conversion to SCAR markers

Resistance to anthracnose or black spot ( Elsinoe ampelina ), a serious fungal pathogen in viticulture and table grape production, was investigated on 25 grape cultivars. Bioassays performed with culture filtrates produced by the pathogen revealed 14 resistant genotypes. In most plants resistance originated from Vitis labrucsa but also genotypes with V. rupestris and V. riparia  ×  V. rupestris background showed resistance. Genetic analysis was conducted in F₁, S₁ and BC₁ plants developed from various cultivars. In total, 326 F₁ plants were evaluated, 172 genotypes proofed to be resistant, whereas 154 were susceptible to anthracnose. A Mendelian segregation ratio of 1 : 1 (χ² = 0.30–0.65) indicating that anthracnose resistance is controlled by a single dominant gene. To facilitate the use of marker-assisted selection in grape-breeding PCR-based markers were developed by random amplified polymorphic DNA and amplified fragment length polymorphism in bulk segregant analysis. Finally, OPB 15₁₂₄₇ was developed as a sequence characterized amplified region marker being diagnostic for the locus of resistance to anthracnose in all resistant genotypes tested. Within the 25 grape cultivars OPB 15₁₂₄₇ is diagnostic in the genetic background of both V. labrucsa and V. rupestris and V. riparia  ×  V. rupestris .     

Inheritance of resistance to the Moroccan watermelon mosaic virus in the cucumber line TMG-1 and cosegregation with zucchini yellow mosaic virus resistance

Inbred lines derived from the Chinese cucumber (Cucumis sativus L.) cultivar, ‘Taichung Mou Gua’ (TMG), have been shown to be resistant to several potyviruses including: zucchini yellow mosaic virus (ZYMV), zucchini yellow fleck virus (ZYFV), watermelon mosaic virus (WMV) and the watermelon strain of papaya ringspot virus (PRSV-W). Recently, an additional virus that infects cucurbits, the Moroccan watermelon mosaic virus (MWMV), has been determined to be a distinct member of the potyvirus group. This study demonstrates that TMG-1 possesses resistance to MWMV. Rub or aphid inoculated TMG-1 seedlings remain free of symptoms. Progeny analyses of the F₁, F₂ and backcross generations show that resistance to MWMV is conferred by a single recessive gene (proposed designation, mwm). Sequential inoculation of progeny possessing resistance to ZYMV followed by MWMV (or MWMV followed by ZYMV) and analysis of F₃ families derived from F₂ individuals selected for resistance to ZYMV indicate that both resistances are conferred by the same gene, or two tightly linked genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Development of STS markers and QTL validation for common bacterial blight resistance in common bean

Common bacterial blight (CBB) of common bean ( Phaseolus vulgaris L.), is one of the major diseases that decrease yield and quality. A major quantitative trait locus (QTL) for CBB resistance from line XAN 159 was transferred into two bean lines, HR45 and HR67. Previous studies identified that two markers are linked to this QTL but the chromosome location was not consistent. To identify more tightly linked markers and to verify the chromosome location, 65 additional markers were mapped using 81 recombinant inbred lines (RILs) derived from a cross HR67 × OAC95-4. The QTL was mapped to a 13 cM region on chromosome 1 and defined by eight molecular markers that explained 25–52% of the phenotypic variation. Six tightly linked amplified fragment length polymorphism markers (0.6–9.7 cM from the QTL peak) were converted into seven sequence tagged site markers, three of which were mapped to this QTL. Five tightly linked markers were used to screen 907 F₂ plants derived from a cross HR45 × 'OAC Rex' and four of them were linked to each other within 4.2 cM. These markers may be useful in marker-assisted selection and map-based cloning of this major QTL.     

Mapping of a gene for leaf scald resistance in barley line 'B87/14' and validation of microsatellite and RFLP markers for marker-assisted selection

Seedlings of the barley line ‘B87/14’ were resistant to 22 out of 23 Australian isolates of Rhynchosporium secalis, the causal agent of leaf scald.‘B87/14’‐based populations were developed to determine the location of the resistance locus. Scald resistance segregated as a single dominant trait in BC₁F₂ and BC₁F₃ populations. Bulked segregant analysis identified amplified fragment length polymorphisms (AFLPs) with close linkage to the resistance locus. Fully mapped populations not segregating for scald resistance located these AFLP markers on chromosome 3H, possibly within the complex Rrs1 scald locus. Microsatellite and restriction fragment length polymorphism markers adjacent to the AFLP markers were identified and validated for their linkage to scald resistance in a second segregating population, with the closest marker 2.2 cM from the resistance locus. These markers can be used for selection of the Rrs.B87 scald‐resistance locus, and other genes at the chromosome 3H Rrs1 locus.     

Molecular breeding for the BaMMV/BaYMV resistance gene ym4 in winter barley

The aim of this investigation was to develop a procedure for the largescale molecular breeding for ym4, allowing resistance to BaMMV/BaYMV to be fixed in early breeding generations of winter barley. A codominant STS marker derived from the restriction fragment length polymorphism marker MWG838 for the ym4 resistance gene was combined with a new and easy procedure for preparing leaf samples for polymerase chain reaction (PCR), theoretically allowing one person to extract DNA from 5000 samples in a single day. In the procedure for molecular breeding for ym4, all steps, including leaf sampling, DNA extraction, PCR amplification and digestion with restriction enzyme were assembled in microtitre plates allowing multipipetting throughout the procedure, including the loading of gels. The method is amenable to further automation with the aid of a robot arm. Double haploid (DH) lines, as well as F₂ and F₄ breeding lines were analysed and, based on markers, homozygous and heterozygous BaMMV/BaYMV resistant plants were identified for further breeding. The winter barley breeding programmes were modified to include marker-based selection for BaMMV/BaYMV resistance on DH or on F₂ individuals, which had been preselected for mildew and leaf rust resistance.     

An SSR marker in the nitrate reductase gene of common bean is tightly linked to a major gene conferring resistance to common bacterial blight

A simple sequence repeat (SSR) marker composed of a tetra nucleotide repeat is tightly linked to a major gene of common bean (Phaseolus vulgaris L.) conferring resistance to common bacterial blight (CBB) incited by Xanthomonas axonopodis pv. phasoli (Xap). This SSR is located in the third intron region of the common bean nitrate reductase (NR) gene, which is mapped to linkage group (LG) H7, corresponding to LG B7 of the bean Core map. Co-segregation analysis between the SSR marker and CBB resistance in a recombinant inbred line (RIL) population demonstrated a tight linkage between the NR gene-specific marker and the major gene for CBB resistance. In total, the marker explained approximately 70% of the phenotypic variation in the population. Because it is co-dominant, this SSR marker should be more efficient for marker-assisted selection (MAS) than dominant/recessive random amplified polymorphic DNA (RAPD) or sequence characterized amplified region (SCAR) markers that have been developed, especially for early generation selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Novel molecular marker associated with Tm2a gene conferring resistance to tomato mosaic virus in tomato

Tomato mosaic virus (ToMV) is an important Tobamovirus that causes significant crop losses. Resistance to the ToMV is conferred by the genes Tm1, Tm2 and Tm2^a. Among these three genes, Tm2^a confers resistance to most strains of the ToMV. Screening of genetic lines under field conditions based on phenotype is time‐consuming and challenging due to concerns associated with stability of the virus and its potential transmission to other plants. Tightly linked molecular markers associated with resistance genes can improve selection efficiency and avoid these problems. This study developed a PCR‐based marker based on restriction site differences from Tm2^a locus‐specific sequences, which was found to be useful in identifying the resistant and susceptible genotypes and was consistent with phenotypic data. The marker is a codominant cleaved amplified polymorphic sequence (CAPS) marker producing 270‐ and 600‐bp DNA fragments from resistant genotypes and an 870‐bp fragment from susceptible genotypes when digested with HaeIII restriction enzyme. This novel marker can be useful for tomato breeders to screen progeny from segregating populations for ToMV resistance.     

Development of an STS marker and SSRs suitable for marker-assisted selection for the BaMMV resistance gene rym9 in barley

Based on the RAPD marker OP‐C04H910 which is closely linked to the barley mild mosaic virus (BaMMV) resistance gene rym9 derived from the variety ‘Bulgarian 347’ the marker STS‐C04H910 cosegregating with OP‐C04H910 and generating a single additional band on plants carrying the recessive resistance encoding allele has been developed. Furthermore, the simple sequence repeats (SSRs) WMS6 and HVM67 have been integrated into the genetic map of the rym9 region on chromosome 4HL. Because of their close linkage to rym9 and distinct banding pattern STS‐C04H910 and HVM67 are well‐suited for marker‐ assisted selection, enhanced backcrossing procedures and pyramiding of resistance genes.     

A specific PCR assay for resistance to biotypes 1 and 2 of the rosy leaf curling aphid in apple based on an RFLP marker closely linked to the Sd1 gene

This report describes the conversion of a restriction fragment length polymorphism (RFLP) marker (the 2B12a locus). linked to the Sd₁ aphid resistance gene, to a polymerase chain reaction (PCR) based marker. A section of the 2BI2 probe was sequenced and two primers were designed lo amplify this sequence in the cultivars‘Prima’and‘Fiesta’: all the amplification products were the same size. After sequencing. two specific 24-mer oligonueleotides were synthesized (DdARM-5¹ and DdAR.M-3²) to exploit a single base-pair difference. These primers were used to screen 44 plants from the‘Prima’x‘Fiesta’family and generated a single amplification product (196bp). in approximately half of the seedlings, which was linked to the resistance gene Sd₁,. The DdARM primer combination was used to evaluate a range of apple cultivars and selections, including some varieties derived from‘Cox’and alternative sources of resistance reported in the literature. In parallel with this work, the phenotypic response of the same genotypes was either confirmed or determined in replicated glasshouse tests. The sequence characterized amplified regions (.SCAR) marker was amplified in all the resistant plants, with the exception of‘Northern Spy’and 3760 (the sources of Sd₂ and Sd₃ resistance, respectively), but never in the susceptible plants. The possible role of this marker in a marker-assisted breeding strategy, and its compatibility with a SCAR marker linked to the I, gene for resistance to apple scab. is discussed.     

Search for molecular markers linked to Liriomyza trifolii resistance in tomato

Resistance to many arthropods, including Liriomyza species, is known to be present in accessions of Lycopersicon hirsutum (f. typicum or f. glabratum). From the cross L. esculentum cv. Moneymaker and L. hirsutum f. glabratum G1561 100 F₂ plants were screened in a no-choice test for resistance to Liriomyza trifolii. The Bulked Segregant Analysis approach was used to find Random Amplified Polymorphic DNA markers linked to resistance. Two markers were located on chromosome 2. Restriction Fragment Length Polymorphisms constructed a more detailed genetic linkage map for part of chromosome 2. Kruskal-Wallis analysis showed that this chromosome harbored a Quantitative Trait Locus (QTL) for number of pupae, number of mines and damage. At least one major QTL is essential for resistance and this QTL is located on chromosome 2 nearby the location of the tomato probe TG451. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of molecular markers linked to the wheat powdery mildew resistance gene Pm4b and marker validation for molecular breeding

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici, is an important disease in wheat (Triticum aestivum L.). Bulk segregant analysis (BSA) was employed to identify SRAP (sequence‐related amplified polymorphism), sequence tagged site (STS) and simple sequence repeat (SSR) markers linked to the Pm4b gene, which confers good resistance to powdery mildew in wheat. Out of 240 SRAP primer combinations tested, primer combinations Me8/Em7 and Me12/Em7 yielded 220‐bp and 205‐bp band, respectively, each of them associated with Pm4b. STS‐₂₄₁ also linked to Pm4b with a genetic distance of 4.9 cM. Among the eight SSR markers located on wheat chromosome 2AL, Xgwm382 was found to be polymorphic and linked to Pm4b with a genetic distance of 11.8 cM. Further analysis was carried out using the four markers to investigate marker validation for marker‐assisted selection (MAS). The results showed that a combination of the linked markers STS_?241, Me8/Em7_?220 and Xgwm382 could be used for marker‐assisted selection of the resistance gene Pm4b in wheat breeding programmes.     

Detection of RAPD markers linked to the everbearing gene in Japanese cultivated strawberry

To identify markers for the everbearing gene in strawberries, 199 F₁ progeny plants were produced from a cross between ‘Ever Berry’ (a Japanese everbearing strawberry) and ‘Toyonoka’ (a Japanese Junebearing strawberry) as the experimental population. The results of flowering tests produced 97 everbears and 102 Junebears. The chi‐square test gave a goodness of fit for the expected ratio of 1 : 1 for everbears to Junebears, suggesting the inheritance of the everbearing trait is controlled by a monogenic dominant gene. RAPD analyses on this trait were carried out using ‘Ever Berry’ and ‘Toyonoka’. Seventy‐one primers, which produced 89 polymorphic fragments between the two parents, were identified from a total of 175 primers. Five markers relating to the everbearing trait were selected from 26 of the 199 progeny plants. The remaining 173 seedlings were analysed with these five markers and a linkage map was constructed using all of the 199 F₁ progeny plants. The length of this linkage group is 39.7 cM. The closest markers found, OPE07‐1 and OPB05‐1, are respectively mapped at 11.8 and 15.8 cM on each side of the everbearing gene.     

User‐friendly markers linked to Fusarium wilt race 1 resistance Fw gene for marker‐assisted selection in pea

Fusarium wilt is one of the most widespread diseases of pea. Resistance to Fusarium wilt race 1 was reported as a single gene, Fw, located on linkage group III. The previously reported AFLP and RAPD markers linked to Fw have limited usage in marker‐assisted selection due to their map distance and linkage phase. Using 80 F₈ recombinant inbred lines (RILs) derived from the cross of Green Arrow × PI 179449, we amplified 72 polymorphic markers between resistant and susceptible lines with the target region amplified polymorphism (TRAP) technique. Marker–trait association analysis revealed a significant association. Five candidate markers were identified and three were converted into user‐friendly dominant SCAR markers. Forty‐eight pea cultivars with known resistant or susceptible phenotypes to Fusarium wilt race 1 verified the marker–trait association. These three markers, Fw_Trap_480, Fw_Trap_340 and Fw_Trap_220, are tightly linked to and only 1.2 cM away from the Fw locus and are therefore ideal for marker‐assisted selection. These newly identified markers are useful to assist in the isolation of the Fusarium wilt race 1 resistance gene in pea.     

Genetic analysis and identification of two soybean mosaic virus resistance genes in soybean [Glycine max (L.) Merr]

Soybean mosaic virus (SMV) commonly affects soybean production worldwide, and the SC18 strain has been widespread in China. This study aimed to characterize and map the SC18 resistance genes present in soybean cultivars ‘Kefeng No. 1’ and ‘Qihuang 22’. Inheritance analysis revealed that two independent single dominant genes in Kefeng No. 1 and Qihuang 22 confer resistance to SC18. Using simple sequence repeat (SSR) markers and bulked segregant analysis, the Kefeng No. 1 and Qihuang 22 resistance genes were located on soybean chromosomes 2 and 13, respectively. We further screened two populations of recombinant inbred lines with 32 SSR markers in the target region, where the resistance gene in Kefeng No. 1 was fine mapped to an 80‐kb region containing six putative genes. Sequence and expression analyses of these genes revealed that SMV resistance in Kefeng No. 1 was probably attributable to three of the candidate genes (i.e. Glyma.02G127800, Glyma.02G128200 and Glyma.02G128300). Collectively, the results of this study will greatly facilitate the cloning of SC18 resistance genes and marker‐assisted breeding of SMV‐resistant soybean cultivars.     

Development of SCAR markers linked to the Pm21 gene conferring resistance to powdery mildew in common wheat

Powdery mildew is an important disease in most of the wheat production areas of the world. The resistance gene Pm21 (6AL/6VS trans-location) derived from Haynaldia villosa confers resistance to all available isolates of Erysiphe (Blumeria) graminis f. sp. tritici in China and Europe. The objective of this study was to develop fast and reliable sequence characterized amplified region (SCAR) markers linked to the Pm21 gene. A random amplified polymorphic DNA (RAPD) marker for Pm21, OPH17₁₄₀₀, was converted to SCAR markers after sequencing the two ends of the polymorphic DNA fragment. Two SCAR markers, SCAR₁₂₆₅ and SCAR₁₄₀₀, were developed to detect the Pm21 gene in different genetic backgrounds. The specific SCAR₁₂₆₅ marker enable large-scale accurate screening for the presence/absence of Pm21 allele.