Identification of a candidate gene for resistance to root‐knot nematode in a wild peach and screening of its polymorphisms

Root‐knot nematode disease, caused by Meloidogyne species, is an important soil‐borne disease of peach (Prunus persica L.) worldwide. To identify a major locus of genetic resistance to M. incognita, PkMi, in a wild peach species, we reconstructed a linkage group in a BC₁ population of 187 lines using resistance gene analogue markers surrounding the PkMi locus. A resistance gene analogue marker, ppa021062m, co‐segregated with the PkMi locus and was therefore considered a strong candidate for PkMi. Phylogenetic analysis of the deduced protein sequences of ppa021062m, together with the other seven genes for nematode resistance, allowed ppa021062m to be assigned to the Toll/Interleukin1 Receptor‐Nucleotide Binding Site‐Leucine Rich Repeat class, similar to Ma in myrobalan plum (P. cerasifera). Comparative analysis of the candidate gene sequence in four genotypes that had different levels of resistance to root‐knot nematode disease showed that most non‐synonymous SNPs in the genic region were distributed in the TIR and NBS motifs. This study enhances our understanding of the genetic and molecular control of resistance to root‐knot nematode disease in peach.     

Mapping of the apple powdery mildew resistance gene Pl1 and its genetic association with an NBS-LRR candidate resistance gene

Molecular markers for the major apple powdery mildew resistance gene Pl1 were identified and are presently used in marker-assisted selection in apple breeding. However, the precise map position of the Pl1 gene in the apple genome was not known. The objectives of this investigation were the identification of the Malus linkage group (LG) carrying the Pl1 locus, mapping of the resistance gene by simple sequence repeat (SSR) markers, and the analysis of genetic associations between the Pl1 gene and the numerous NBS-LRR resistance gene candidates already mapped in the apple genome. A two-step linkage mapping was used, based on two different apple families. The identification of LG 12 carrying Pl1 was performed indirectly by mapping the SCAR marker AT20 in an apple progeny for which there was a core genetic map but no mildew data available. Then, the position of Pl1 on LG 12 was determined by SSR markers in a second population which has been scored for mildew over 6 years in a greenhouse and in the field. The SSR Hi07f01, previously mapped on LG 12 [Tree Genet. Genomes, 2 (2006), 202] cosegregated with AT20 and was closely linked (∼1 cM) to the Pl1 gene. The TIR-NBS-LRR resistance gene analogue 15G11 mapped by the SSCP technique was also closely linked to the Pl1 resistance locus and might be a candidate for Pl1 itself, a second powdery mildew major resistance gene ( Pld , [Theor. Appl. Genet., 110 (2004), 175]), or two scab resistance genes ( Vg , [IOBC/WPRS Bull., 23 (2000), 245]; Vb , [Genome, 49 (2006), 1238]) which all seem to be located in a common R gene cluster at the distal end of apple LG 12.     

Mapping and candidate gene analysis for a new top spikelet abortion mutant in rice

Grain numbers is one of the determinations for rice yield and directly associated with spikelet numbers per panicle and its normal development. Lots of genes responsible for spikelet numbers and spikelet early development have been identified, but the molecular information about the spikelet development at later development is still limited. Here, we isolated a rice spikelet abnormal development mutant, which shows degenerated spikelet at the top panicle and named aborted top spikelet mutant 1(Ats1). The spikelets derived from the middle and bottom branches per panicle of Ats1 show normal development with those of wild type. However, a large number of branches and spikelets with arrested development were often observed only on apex panicle. The abnormality did not appear until the stage In8 when rachises elongate rapidly and reproductive organs get mature, based on observations through SEM analysis. The aborted spikelet could develop the complete floral organs with a pair of rudimentary glume, a pair of empty glume, two lodicule, six stamens and one carpel. But all these floral organs did not develop maturity. Genetic analysis on two F₂ populations indicated that the Ats1 was controlled by a single dominant gene. By using bulked segregant analysis of F₂ population developed from Ats1 crossing with Songjing6, ATS1 was mapped on chromosome 8 between RM3819 and RM5556. Then, the fine mapping was performed with 1078 F₂ population developed from Ats1 and IR36. The ATS1 locus was finely mapped in an 85.7 kb region between RM22448 and STS8‐2 with 8 genes according to the rice annotation project database. Sequence analysis of the candidate genes within the delimited region of the Ats1 and Akihikari showed two‐nucleotide changes, including single‐nucleotide substitutions corresponding to an amino acid substitution from asparagine to lysine acid in exons 3 and a 1‐bp deletion resulting in a premature stop codon in exon 22 at the candidate gene, LOC_Os08g06480. A cleaved amplified polymorphic sequence (CAPS) marker, CAPS‐ats1, was developed from the 1‐bp deletion site. The complete cosegregation of the CAPS genotypes with the matching phenotypes were observed in the F₂ populations. This suggested that Os08g06480 is most likely the ATS1 gene. These results will provide more information for better understanding of the molecular mechanism governing top spikelet abortion within a short developmental period.     

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.     

Mapping a dominant negative mutation for triforine sensitivity in lettuce and its use as a selectable marker for detecting hybrids

Some lettuce cultivars are highly sensitive to triforine, an inhibitor of sterol biosynthesis found in some commercial systemic fungicides. First symptoms of a sensitive reaction are usually observed within 24–48 h after treatment and include severe wilting, necrosis and rapid plant death. We mapped a single dominant gene (Tr) that confers sensitivity of lettuce to triforine to linkage group 1 of the integrated genetic map of lettuce. The occurrence of sensitivity is not uniform across horticultural types of lettuce. While over 80% of green-romaine lettuce cultivars tested were sensitive, most cultivars of all other lettuce types were insensitive to triforine. All accessions of wild Lactuca spp. were insensitive to triforine. Allelism tests using F₁ and F₂ progeny revealed that sensitive cultivars of all horticultural types likely carry the same Tr gene. The dominant allele for sensitivity found in cultivated lettuce probably had a monophyletic origin. The reaction to triforine can be used as a marker for detecting hybrids originating from a cross between phenotypically similar parents with different responses to triforine treatment. It also provides an indication of genotypes for which applications of triforine-containing fungicides are inappropriate.     

甘蔗抗黄锈病G1标记的分子检测及候选抗病基因WAK的分析

甘蔗黄锈病是屈恩柄锈菌(Puccinia kuehnii Butler)引起的一种世界性真菌病害,导致产量减少和糖分降低,给甘蔗产业造成严重损失。本研究采用抗黄锈病分子标记G1,检测我国和世界上重要的甘蔗栽培品种、野生种和近缘属的抗黄锈病基因,并对扩增的代表性特异条带进行克隆测序、功能注释和聚类分析,推测其抗性基因的起源和进化。G1检测结果表明,国内124份甘蔗栽培品种检测到G1标记的有83份,占66.9%;国外46份甘蔗栽培品种检测到G1标记的有31份,占67.4%。34份甘蔗野生种和近缘属中检测到G1标记的有17份,占50%,其中割手密种含有该基因比例最高,为100%。功能注释揭示,G1标记的候选基因编码一种细胞壁连接的类受体激酶,并在甘蔗栽培品种的单倍体蛋白组数据库中鉴定到3个相似度较高的蛋白,这些蛋白都有细胞壁受体激酶结构的胞外域、跨膜域和激酶活性的胞内域。聚类结果则清晰展示了抗病候选基因的起源及进化关系,具体可分为3组,第1组来源于割手密种和大茎野生种;第2组来源于大茎野生种、热带种和河八王属;第3组来源于割手密种、大茎野生种、中国种和栽培品种。研究结果为抗黄锈病甘蔗品种的选育提供重要的抗源支撑,并为抗性分子机制的解析奠定基础。     

Mapping and validation of PCR-based markers associated with a major QTL for seed dormancy in wheat

Seed dormancy is one of the important factors controlling pre-harvest sprouting (PHS) resistance in wheat. We identified a major quantitative trait locus (QTL) for seed dormancy on the long arm of wheat chromosome 4A (4AL) via simple sequence repeat (SSR)-based genetic mapping using doubled haploid lines from a cross between Japanese PHS resistant variety ‘Kitamoe’ and the Alpine non-resistant variety “Münstertaler” (K/M). The QTL explained 43.3% of total phenotypic variation for seed dormancy under greenhouse conditions. SSR markers flanking the QTL were assigned to the chromosome long arm fraction length 0.59–0.66 on the basis of chromosome deletion analysis, suggesting that the gene(s) controlling seed dormancy are probably located within this region. Under greenhouse conditions, the QTL explained 28.5 and 39.0% of total phenotypic variation for seed dormancy in Haruyutaka/Leader (HT/L) and OS21-5/Haruyokoi (O/HK) populations, respectively. However, in field conditions, the effect was relatively low or not significant in both the K/M and HT/L populations. These markers were considered to be widely useful in common with various genetic backgrounds for improvement of seed dormancy through the use of marker-assisted selection. Further detailed research using near isogenic lines will be needed to define how this major QTL interacts with environmental conditions in our area.     

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.     

Linkage between a major gene for powdery mildew resistance and an RFLP marker on chromosome 1R of rye

DNA samples from an F₂ progeny which segregated for resistance to powdery mildew were bulked for resistant and susceptible individuals. In a segregant analysis, genomic rye probes which had been localized previously in a linkage map of rye were systematically screened for polymorphisms between these bulks. An RFLP marker located on linkage group 1RS was found to be tightly linked to a dominant mildew resistance gene. This is the first publication mapping a major gene for mildew resistance in rye.     

Genetic mapping,marker assisted selection and allelic relationships for the Pu6 gene conferring rust resistance in sunflower

Rust resistance in the sunflower line P386 is controlled by Pu₆, a gene which was reported to segregate independently from other rust resistant genes, such as R₄. The objectives of this work were to map Pu₆, to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu₆ and R₄. Genetic mapping of Pu₆ with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu₆ was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu₆ and R₄ indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R_adv/R₄/R₁₁/ R_13a/R_13b/Pu₆ cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.     

Fine mapping and candidate gene analysis of a new mutant gene for panicle apical abortion in rice

The rice panicle architecture depends on the arrangement of branches and spikelets which directly affect grain yield. We identified a mutant for panicle apical abortion (PAA-Hwa) from a japonica cultivar Hwacheongbyeo treated with N-methyl-N-nitrosourea. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as a slight reduction in plant height, narrow and dark green leaf blades, and small erect panicles with clear PAA compared to the wild-type plants. Genetic analysis revealed that the PAA was controlled by a single recessive gene, which is tentatively designated as paa-h. The paa-h gene was fine mapped at an interval of 71 kb flanked by sequence tagged site markers aptn3 and S6685-1 at the long arm of chromosome 4. Sequence analysis of the candidate genes within the delimited region showed a single base-pair change corresponding to an amino acid substitution from glycine to glutamic acid at the candidate gene, LOC_Os04g56160. We expect that the paa-h gene will be a clue to uncover the molecular mechanism of PAA and to maintain the panicle identity for grain yield in rice breeding programs.     

Tryptophan synthesis block-associated sugar response, a physiological marker for rapid selection of Arabidopsis thaliana transformants, marked with feedback-inhibition-insensitive anthranilate synthase gene

The feedback-insensitive anthranilate synthase (AS) gene was used as a selection marker for transformants of Arabidopsis thaliana . The mutant gene ( mAS1-2 ) was constructed by substituting nucleotide at the effector-binding site of the intrinsic AS gene via PCR-mediated site-directed mutagenesis and flanked with the myrosinase promoter pyk10 to drive its expression during initial root elongation. This inducible gene cassette was first introduced into Agrobacterium tumefaciens and then delivered into A. thaliana by floral-dip inoculation. 5-methyltryptophan (5-MT) inhibited AS and suppressed seedling growth of wild type plants as a result of tryptophan starvation. With the addition of sucrose (10 mg/ml), 5-MT inhibited cotyledon opening and caused anthocyanin to accumulate in juvenile seedlings. The present mutant reversed the tryptophan starvation caused by 5-MT and blocked subsequent sugar responses. The sugar responses were detected in non-transformed plants grown on a selection medium containing 10 mg/ml of sucrose and 10 μg/ml of 5-MT after 3 days of incubation. Thus, true transformants could be selected after a short incubation, compared to the conventional kanamycin-selection method that did not eliminate all non-transformed plants.     

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.     

Validation of a major QTL for scab resistance with SSR markers and use of marker-assisted selection in wheat

The objectives of this study were to validate the major quantitative trait locus (QTL) for scab resistance on the short arm of chromosome 3B in bread wheat and to isolate near‐isogenic lines for this QTL using marker‐assisted selection (MAS). Two resistant by susceptible populations, both using ‘Ning7840’ as the source of resistance, were developed to examine the effect of the 3BS QTL in different genetic backgrounds. Data for scab resistance and simple sequence repeat (SSR) markers linked to the resistance QTL were analyzed in the F_2:3 lines of one population and in the F_3:4 lines of the other. Markers linked to the major QTL on chromosome 3BS in the original mapping population (‘Ning7840’/‘Clark’) were closely associated with scab resistance in both validation populations. Marker‐assisted selection for the QTL with the SSR markers combined with phenotypic selection was more effective than selection based solely on phenotypic evaluation in early generations. Marker‐assisted selection of the major QTL during the seedling stage plus phenotypic selection after flowering effectively identified scab resistant lines in this experiment. Near‐isogenic lines for this 3BS QTL were isolated from the F₆ generation of the cross ‘Ning7840’/‘IL89‐7978’ based on two flanking SSR markers, Xgwm389 and Xbarc147. Based on these results, MAS for the major scab resistance QTL can improve selection efficiency and may facilitate stacking of scab resistance genes from different sources.     

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 mapping of the fertility restoration locus Rf1 in sunflower and development of diagnostic markers for the restorer gene

Fertility restoration by dominant nuclear genes is essential for hybrid breeding based on cytoplasmic male sterility (CMS) to obtain heterotic effects and high seed yields. In sunflower, only the PET1 sterility inducing cytoplasm has been used in commercial hybrid breeding until now. This particular male sterility was derived from an interspecific hybrid Helianthus petiolaris × H. annuus. For the recent work we used the segregating population RHA325(CMS) × HA342, based on the PET1 cytoplasm. Molecular markers were mapped within 1.1 cM around the restoration locus Rf1. At the distal side, the marker OP-K13_454 mapped at a distance of 0.9 cM and E32M36-155R at 0.7 cM from Rf1. At the proximal side the markers E44M70-275A, E42M76-125A and E33M61-136R were mapped at 0.1, 0.2, and 0.3 cM from the restorer locus, respectively. These markers provide an excellent basis for a map based cloning approach and for marker-assisted sunflower breeding.