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.     

大豆品种郑97196抗疫霉病基因RpsZheng精细定位

大豆疫霉病是由大豆疫霉引起的一种重要大豆病害,可造成严重的经济损失。种植含有抗疫霉病基因的大豆品种是控制该病害最有效的途径。前人在大豆品种郑97196的3号染色体上鉴定了一个抗疫霉病基因RpsZheng。本研究的目的是验证幵精细定位抗疫霉病基因RpsZheng。以Williams和郑97196杂交衍生的188个F_(2:3)家系为作图群体,用大豆3号染色体上的SSR标记构建RpsZheng遗传连锁图,获得与RpsZheng紧密连锁的侧翼SSR标记SattWM82_39 (2.5 cM)和BARCSOYSSR_03_0269 (1.0 cM)。基于亲本间全基因组重测序数据鉴定和开发多态性InDel标记,进一步将RpsZheng候选区域缩小至105.2 kb,通过检测RpsZheng候选区域内的共分离标记特异性,获得了能够有效检测RpsZheng的分子标记WZInDel11。本研究明确了RpsZheng的候选基因组区间,鉴定出了能够有效用于基因功能研究和辅助选择育种的共分离分子标记。     

Resistance and partial resistance to Phytophthora sojae in early maturity group soybean plant introductions

Phytophthora sojae, an important yield limiting pathogen of soybean, causes seed, seedling, root, and stem rots. Losses caused by P. sojae can be controlled by both major gene and partial resistance. Early maturity group (MG) soybeans are an increasingly important crop in northwestern Minnesota and eastern North Dakota. Early MG plant introductions (PIs) from the USDA Soybean Germplasm Collection and early MG public and private cultivars were evaluated for resistance and partial resistance to P. sojae. Of the 113 PIs, PI438445, and PI438454 exhibited resistance to P. sojae races 4, 7, 17, and 28 indicating they may possess either Rps1c, Rps1k, previously unidentified or multiple resistance gene to Phytophthora sojae (Rps) genes. Because they exhibited partial resistance equal to or greater than the standard check cultivar Conrad, three early MG soybean cultivars (MN0902, MN0302, and 91B53) were selected as standard checks to evaluate early MG PIs for partial resistance. Sixty-nine PIs were evaluated for partial resistance to P. sojae races 7 and 25 using the inoculum layer method. Of this group of PIs, 22 had the same level of partial resistance as Conrad to P. sojae race 7 while 19 had the same degree of partial resistance to race 25. Twelve PIs had same level of partial resistance as Conrad to both P. sojae races 7 and 25. The PIs and cultivars identified in this study will be of great value in developing early MG soybean cultivars suitable for planting in Canada and the northern United States.     

Inheritance of coffee leaf rust resistance and identification of AFLP markers linked to the resistance gene

The most important disease of Coffea arabica is coffee leaf rust caused by the fungus Hemileia vastatrix. The purpose of this study was to characterize the inheritance of coffee resistance gene(s) to race II of this pathogen and to identify and map molecular markers linked to this trait. Different populations were used: F₂ (160 plants), BCr (20), and BCs (135), derived from a cross between the resistant genotype Híbrido de Timor UFV 427-15 and the susceptible cultivar Catuaí Amarelo UFV 2143-236 (IAC 30). The segregation analysis showed that the resistance of Híbrido de Timor to race II of the H. vastatrix is conferred by a single dominant gene. The amplification of 176 AFLP (Amplified fragment length polymorphism) primer combinations using bulked segregant analysis (BSA) allowed the identification of three molecular markers linked to the resistance gene. Genetic mapping of these three markers in the F₂ population indicated that they are distributed on both sides, flanking the resistance gene. The markers E.CTC/M.TTT405 and E.CGT/M.TGT300 were found linked to the resistance gene at 8.69 cM (LOD 18.91) and 25.10 cM (LOD 5.37), respectively, while E.CCT/M.TTC230 was localized on the other side of the gene, at 20.50 cM (LOD 6.15). These markers are the first rust resistance markers identified in Híbrido de Timor and can be useful for marker assisted selection in coffee breeding programs.     

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.     

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.     

Inheritance of angular leaf spot resistance in common bean line BAT 332 and identification of RAPD markers linked to the resistance gene

The existence of genetic variability for angular leaf spot (ALS) resistance in the common bean germplasm allows the development of breeding lines resistant to this disease. The BAT 332 line is an important resistance source to common bean ALS. In this work we determined the inheritance pattern and identified RAPD markers linked to a resistance gene present in BAT 332. Populations F₁, F₂,BCs and BCr derived from crosses between BAT 332 and cultivar Rudá were used. Rudá is a commercial cultivar with carioca type grains and susceptible to ALS. The resistance of BAT 332 to race 61.41 of the pathogen was confirmed. Segregation analysis of the plants indicated that a single dominant gene confers resistance. For identification of RAPD markers linked to the resistance gene, bulk segregant analysis (BSA) was used. Two RAPD markers,OPAA07₉₅₀ and OPAO12₉₅₀, linked in coupling phase at 5.10 and 5.83 cM of this gene, respectively, were identified. These molecular markers are important for common bean breeders and geneticists as source of genetic information and for marker assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of resistance to Fusarium head blight caused by Fusarium graminearum in Chinese wheat cultivar Sumai 3 and the Japanese cultivar Saikai 165

The genetic constitution of resistance to Fusarium head blight (FHB, scab) caused by Fusarium graminearum in the Chinese wheat cultivar Sumai 3 and the Japanese cultivar Saikai 165 was investigated using doubled haploid lines (DHLs) and recombinant inbred lines (RILs). Frequency distributions of DHLs derived from two F₁ crosses, Sumai 3 (very resistant to resistant; VR-R) / Gamenya (very susceptible; VS) and Sumai 3 / Emblem (VS), fitted well to 1: 2: 1 (resistant: moderately resistant: susceptible) ratios for reaction to FHB in the field. It is suggested that the resistance of Sumai 3 is controlled by two major genes with additive effects. One of the resistance genes may be linked in repulsion to the dominant suppressor B1 for awnedness with recombination values 15.1 ± 3.3% in Sumai 3 /Gamenya and 21.4 ± 4.3% in Sumai 3 / Emblem. Saikai 165 is a Japanese resistant line derived from an F₁ Sumai 3 / Asakaze-komugi (moderately resistant; MR). The data for RILs derived from the cross Emblem / Saikai 165, indicates that three resistance genes control the resistance of Saikai 165. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Development of novel microsatellite markers for effective applications in Anthurium cultivar identification

Anthurium andraeanum is one of the most economically important floral crops and potted flowers marketed worldwide. Microsatellite markers are currently the preferred molecular marker owing to the many desirable attributes, including hypervariability, codominance, and amenability to high-throughput genotyping; however, there are few polymorphic molecular markers available for Anthurium. The object of this study was to develop and characterize novel microsatellite markers using the Araceae sequences in GenBank of the National Center for Biotechnology Information (NCBI) to contribute to molecular identification for cultivar protection. Using 1,579 Araceae expressed sequence tags (ESTs) and the related nucleotide sequences, 100 candidates contained simple sequence repeat (SSR) motifs that were suitable for primer design. Furthermore, 100 pairs of SSR primers were screened against a set of 28 diverse genotypes representing 24 cultivars that included four registration cultivars which were bred from the Taiwan Agricultural Research Institute (TARI) and 20 commercial cultivars, appended with three hybrid progeny and a mutant line. From the selected six polymorphic SSR loci, 52 alleles were amplified and 27 distinct genotypes were found, except for ‘Tropical’ and its mutant, with a mean number of eight alleles per locus. The polymorphism information content (PIC) ranged from 0.86 to 0.93. Based on these results, we proposed a key identification set using four microsatellite markers that is sufficient to discriminate among 24 cultivars. Because the Anthurium microsatellite markers developed in this study are primarily from expressed sequence tags or related genomic sequences, they can be used for cultivar identification and, accordingly, contribute to genetic evaluations in breeding programs.     

Genetic variation and cultivar identification of Jamaican yam germplasm by random amplified polymorphic DNA analysis

Summary We have used random amplified polymorphic DNA (RAPD) analysis to characterize eleven cultivars of the five economically most important yam species grown in Jamaica (Dioscorea alata, D. cayenensis, D. rotundata, D. trifida and D. esculenta). Amplification of genomic DNA samples with nine different arbitrary 10mer primers revealed a total of 338 different band positions, ranging in size from 0.3 to 2.5 kb. RAPD patterns proved to be highly reproducible and somatically stable. While no variation was observed among plants belonging to the same cultivar, a large number of intervarietal and interspecific polymorphisms enabled us to reliably discriminate between all Jamaican cultivars investigated.     

Identification of resistance gene analogue markers closely linked to wheat powdery mildew resistance gene Pm31

Specific oligonucleotide primers, designed for the sequences of known plant disease resistance genes, were used to amplify resistance gene analogues (RGAs) from wheat genomic DNA. This method was applied in a bulked segregant analysis to screen for the RGA markers linked to the powdery mildew resistance gene Pm31, introgressed into common wheat from wild emmer. Two RGA markers (RGA200 and RGA390) were found to be closely linked to Pm31 and completely co‐segregating with the marker allele of Xpsp3029 linked to Pm31, with a genetic distance of 0.6 cM. These two RGA markers were then integrated into the formerly established microsatellite map of Pm31 region. The result showed the effectiveness of the RGA approach for developing molecular markers linked to disease resistance genes and demonstrated the efficiency of denaturing polyacrylamide‐gel electrophoresis for detecting polymerase chain reaction polymorphism.     

Development of SCAR markers linked to a scald resistance gene derived from wild barley

The F₂ progeny of a third backcross(BC₃) line, BC line 240, derived from a Turkish accession of wild barley (Hordeum vulgare ssp. spontaneum),segregated for resistance to scald (Rhynchosporium secalis) in a manner indicating the presence of a single dominant resistance gene. Two SCAR marker slinked to this resistance were developed from AFLP markers. Screens of disomic and ditelosomic wheat-barley addition lines with the SCAR markers demonstrated that the scald resistance gene is located in the centromeric region of barley chromosome 3H,a region previously reported to contain a major scald resistance locus, Rrs1. Markers that flank the Rrs1 locus were used to screen the wild barley-derivedBC₃F₂ population. These markers also flank the wild barley-derived scald resistance, indicating that it maps to the same locus as Rrs1; it may beallelic, or a separate gene within a complex locus. However, BC line 240 does not respond to treatment with the Rhynchosporium secalis avirulence factorNIP1 in the same way as the Rrs1-carrying cultivar Atlas46. This suggests that the scald resistance gene derived from wild barley confers a different specificity of response to theRrs1 allele in Atlas46.In order to increase the durability of scald resistance in the field, we suggest that at least two scald resistances should be combined into barley cultivars before release. The scald resistance gene described here will be of value in the Australian environment, and the several markers linked to it will facilitate pyramiding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening and genetic analysis of resistance to peanut stunt virus in soybean: identification of the putative Rpsv1 resistance gene

The peanut stunt virus (PSV) causes yield losses in soybean and reduced seed quality due to seed mottling. The objectives of this study were to determine the phenotypic reactions of soybean germplasms to inoculation with two PSV isolates (PSV-K, PSV-T), the inheritance of PSV resistance in soybean cultivars, and the locus of the PSV resistance gene. We investigated the PSV resistance of 132 soybean cultivars to both PSV isolates; of these, 73 cultivars exhibited resistance to both PSV isolates. Three resistant cultivars (Harosoy, Tsurunotamago 1 and Hyuga) were crossed with the susceptible cultivar Enrei. The crosses were evaluated in the F₁, F₂ and F_2:3 generations for their reactions to inoculation with the two PSV isolates. In an allelism test, we crossed Harosoy and Tsurunotamago 1 with the resistant cultivar Hyuga. The results revealed that PSV resistance in these cultivars is controlled by a single dominant gene at the same locus. We have proposed Rpsv1, as the name of the resistance gene in Hyuga. We also constructed a linkage map using recombinant inbred lines between Hyuga × Enrei using 176 SSR markers. We mapped Rpsv1 near the Satt435 locus on soybean chromosome 7.     

Genetic analysis and identification of SSR marker linked to Phomopsis blight resistance in eggplant (Solanum melongena L.)

The present investigation was carried out to decipher inheritance of resistance and to identify linked SSR markers for Phomopsis blight resistance in eggplant. An F₂ population comprising 161 plants was developed from the cross of Pusa Kranti and BR-40-7. Genetic analysis was carried out using Chi square test. Artificial inoculation of fruits was carried out using pin prick method, and scoring was done as per the standard scoring scale. The F₂ plants segregated into 92 susceptible (77—highly susceptible, 15—susceptible): 69 resistant (17—highly resistant, 27—resistant, 25—moderately resistant) suggesting complimentary epistasis with ratio of 9:7. To identify the putatively linked markers to resistance gene, parental polymorphic markers were subjected to bulk segregant analysis (BSA), and two markers (emk03O04 and emf11A03) could differentiate resistant and susceptible bulk and co-segregated with resistance gene. The genetic distance between the identified markers was found to be 18.12 cM using QTL IciMapping V3.2 software depicting two new QTLs on chromosome number 6. The identified QTLs have great significant importance in marker assisted breeding programme.     

Identification and mapping of random amplified polymorphic DNA markers linked to a rhizomania resistance gene in sugar beet (Beta vulgaris L.) by bulked segregant analysis

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to a gene that confers rhizomania resistance to a sugar beet line created from a Holly Sugar Company breeding population (USA). Polymorphism revealed with 160 arbitrary 10-mer oligonucleotide primers was screened in two bulks produced by separately pooling the individual DNAs from the six most resistant and the six most susceptible plants of an F₂ population segregating for rhizomania resistance. A study of the F₂ individuals showed that 19 primers generated 44 polymorphic markers which were then grouped into nine linkage groups. By analysis of variance, 12 were shown to have a significant effect upon the level of resistance and were mapped on a segment 22.3 cM long. A quantitative trait locus (QTL) of resistance was identified and located in a 4.6cM interval between two markers. It accounted for 67.4% of the observed variation and almost all the genetic variation. These results suggest that the identified QTL corresponds to a unique major gene conditioning the Holly resistance studied, which we have named Rz-l.     

Development and validation of DNA markers linked to Sdvy-1, a common bean gene conferring resistance to the yellowing strain of Soybean dwarf virus

The yellowing strain of Soybean dwarf virus (SbDV-YS) causes yellowing and yield loss in common bean (Phaseolus vulgaris). The most effective control is achieved through breeding for resistance. An indeterminate climbing cultivar with a white seed coat, ‘Oofuku’, is resistant to SbDV-YS in inoculation tests. We crossed ‘Oofuku’ with an elite cultivar, ‘Taisho-Kintoki’, which is SbDV-YS-susceptible, determinate dwarf with a red-purple seed coat, and performed amplified-fragment-length polymorphism analysis of F₃ lines. From nucleotide sequences of the resistant-specific fragments and their flanking regions, we developed five DNA markers, of which DV86, DV386, and DV398 were closely linked to Sdvy-1, a resistance gene. Using the markers, we developed ‘Toiku-B79’ and ‘Toiku-B80’, the near-isogenic lines (NILs) incorporating Sdvy-1 in the background of ‘Taisho-Kintoki’. The NILs had similar growth habit, maturity date and seed shape to those of ‘Taisho-Kintoki’. The quality of boiled beans was also similar, except that the NILs had more seed coat cracking than ‘Taisho-Kintoki’. The NILs showed no SbDV-YS infection in inoculation tests. We suggest that Sdvy-1 is a useful source of SbDV-YS resistance in common bean.