The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus

The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath.     

Inheritance and chromosome locations of scald-resistance genes derived from Iranian and Turkish wild barleys

 A set of advanced backcross barley lines derived from crosses between cv Clipper and different Iranian and Turkish wild barleys, which are homozygous for particular isozyme-marked donor intervals, was screened for resistance to barley scald. Eight lines that consistently exhibited scald resistance were identified, and genetic analysis indicated that single dominant genes encoded resistance in five of the lines, single recessive genes were present in two lines, and a pair of unlinked, dominant genes encoded the resistance in the last line. Linkage between the scald-resistance gene and the isozyme marking the introgressed donor chromosome interval was detected in four lines, allowing the chromosome locations of these resistance genes to be determined. One such resistance gene resides on barley chromosome 5, to which no other scald-resistance genes have been mapped; this gene has been designated Rrs14. A survey of the effectiveness of the eight resistance genes against a set of virulent pathotypes of the scald pathogen revealed that four of the lines were completely resistant to all of them. In two instances, the recovery of more than one scald-resistance gene from a single original donor parent could be demonstrated. These scald-resistance genes should provide additional opportunities for breeding programs that aim to develop scald-resistant barley cultivars. Received: 8 August 1996/Accepted: 27 September 1996     

青稞HvtAGO1基因的克隆及其在条纹病胁迫下的表达

AGO蛋白是RNA诱导沉默复合体的核心分子,在植物的生长、发育及胁迫响应中起重要作用.为探索青稞AGO基因在青稞抗条纹病病原菌过程中的作用机制,该研究以抗条纹病青稞品种‘昆仑14号’和感病品种‘Z1141’为材料,利用条纹病原菌侵染两品种,从感病前后的转录组测序结果中获得一个差异表达基因,克隆验证了该基因为HvtAGO...     

Molecular mapping of the Rph7.g leaf rust resistance gene in barley (Hordeum vulgare L.)

In many temperate areas of the world, leaf rust is becoming an important disease of barley. In the last decade, new races of Puccinia hordei G. Otth have emerged which are virulent against the so-far most-effective race-specific resistance genes, such as Rph7. Marker-assisted selection greatly facilitates the pyramidization of two or more resistance genes in a single variety in order to achieve a more comprehensive resistance. Such a strategy requires the development of efficient and reliable markers. Here, we have developed a linkage map and found RFLP markers closely linked to the Rph7.g resistance gene on chromosome 3HS of barley. The receptor-like kinase gene Hv3Lrk that maps at 3.2 cM from Rph7.g was used to develop a PCR-based marker by exploiting a single nucleotide polymorphism. This marker was detected in 11 out of 12 (92%) barley lines having Rph7 and represents a valuable tool for marker-assisted selection. In addition, the identification of markers flanking Rph7.g provides the basis for positional cloning of this gene. Received: 1 December 1999 / Accepted: 28 February 2000     

The effect of different virus isolates on the expression of tolerance to barley yellow dwarf virus in barley

A barley variety of Ethiopian origin, with a single Mendelian gene con-fering tolerance to barley yellow dwarf virus (BYDV), was equally tolerant to a number of isolates of the virus, whereas a susceptible European barley was more susceptible to isolates transmitted by Rhopalosiphum padi L. than to those transmitted by Macrosiphum (Sitobion) avenae (Fab). However, hybrids between these two varieties homozygous for the Ethiopian tolerance gene were more tolerant to ‘mild’ than to ‘severe’ isolates, irrespective of the vector specificity. The European variety was damaged more severely by all isolates when infected early than when infected late in its development, but the hybrids were damaged more severely by M. awraae-transmitted isolates when infected late. It is suggested that in susceptible plants the concentration, rather than the virulence, of the virus determines disease severity, whereas the reverse is true in plants possessing a gene which reduces virus multiplication. Virus concentration appears to determine the severity of R. padi-transmitted isolates, while virulence determines the severity of M. avenae-transmitted isolates. The latter would also seem to be adapted towards late infection.     

Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley

Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.     

转trxS基因大麦发芽种子水解酶活性的变化

利用转基因技术是改良大麦品种品质的有效途径.研究了转trxS基因对大麦种子发芽过程中水解酶活性的影响,结果表明转基因种子中α-淀粉酶、自由态β-淀粉酶和极限糊精酶的活性比未转基因种子高;转基因种子醇溶蛋白和谷蛋白中巯基的含量提高,说明该基因能够表达,为大麦育种和品质改良提供新的途径.     

Novel resistance to the Bymovirus BaMMV established by targeted mutagenesis of the PDIL5-1 susceptibility gene in barley

The Potyviridae are the largest family of plant-pathogenic viruses. Members of this family are the soil-borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), which, upon infection of young winter barley seedlings in autumn, can cause yield losses as high as 50%. Resistance breeding plays a major role in coping with these pathogens. However, some viral strains have overcome the most widely used resistance. Thus, there is a need for novel sources of resistance. In ancient landraces and wild relatives of cultivated barley, alleles of the susceptibility factor PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (PDIL5-1) were identified to confer resistance to all known strains of BaYMV and BaMMV. Although the gene is highly conserved throughout all eukaryotes, barley is thus far the only species for which PDIL5-1-based virus resistance has been reported. Whereas introgression by crossing to the European winter barley breeding pool is tedious, time-consuming and additionally associated with unwanted linkage drag, the present study exemplifies an approach to targeted mutagenesis of two barley cultivars employing CRISPR-associated endonuclease technology to induce site-directed mutations similar to those described for PDIL5-1 alleles that render certain landraces resistant. Homozygous primary mutants were produced in winter barley, and transgene-free homozygous M₂ mutants were produced in spring barley. A variety of mutants carrying novel PDIL5-1 alleles were mechanically inoculated with BaMMV, by which all frameshift mutations and certain in-frame mutations were demonstrated to confer resistance to this virus. Under greenhouse conditions, virus-resistant mutants showed no adverse effects in terms of growth and yield.     

PCR markers for selection of adult plant leaf rust resistance in barley (Hordeum vulgare L.)

Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust than seedling Rph (Resistance to Puccinia hordei) genes. A major gene for adult plant resistance to barley leaf rust has been mapped to the telomere region of chromosome 5HS. PCR-based molecular markers were developed for saturation of this region based on previously mapped simple sequence repeat, restriction fragment length polymorphism and Diversity Arrays Technology markers. In addition, defence gene homologue (DGH) and wheat expressed sequence tags mapped in specific bins were used to develop new PCR markers. Seventeen PCR-based markers were mapped to the short arm of chromosome 5H in 292 doubled haploid lines from a cross of Pompadour × Stirling, in which seven markers were mapped within 5 cM of the APR gene. The closest linked marker was about 0.7 cM from the APR gene. The wheat deletion bin map together with defence gene homologues was demonstrated to be an efficient tool for development of new molecular markers associated with the disease resistance gene. Four DGH markers were associated with the APR gene. The new molecular markers are a useful tool for marker-assisted selection of the APR gene and provided a better understanding of the molecular mechanism for leaf rust resistance.     

Mapping of powdery mildew resistance genes in a newly determined accession of Hordeum vulgare ssp. spontaneum

The accession PI466197 of wild barley (Hordeum vulgare ssp. spontaneum) with a newly identified resistance to powdery mildew caused by Blumeria graminis f.sp. hordei was studied with the aim to localise the genes determining resistance on a barley genetic map using DNA markers. Molecular analysis was performed in the F₂ population of the cross between the winter variety ‘Tiffany’ and the resistant accession PI466197, consisting of 113 plants. DNA markers, 17 simple sequence repeats (SSRs), four sequence-tagged sites (STSs) and one cleaved amplified polymorphic sequence (CAPS) marker developed from the Mla locus sequence were used for genetic mapping and a two-locus model of resistance was shown. One of the resistance genes originating from H. vulgare ssp. spontaneum PI466197 was localised between the markers RGH1aE1 and Bmac0213 on the short arm of chromosome 1H, which is the position consistent with the Mla locus. The other gene was proven to be highly significantly linked with GBMS247, Bmac0134 and MWG878 on the short arm of chromosome 2H. The flanking markers were Bmac0134 and MWG878, assigned 4 and 8 cM from the resistance gene, respectively. Until now, no gene conferring powdery mildew resistance originating from H. vulgare has been located on the short arm of barley chromosome 2H.     

An UDP-Glucosyltransferase Gene from Barley Confers Disease Resistance to <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight

UDP-glucosyltransferases (UGTs) contribute to Fusarium head blight (FHB) resistance of wheat and barley by glycosylating the deoxynivalenol (DON), which is produced by Fusarium fungus. In this study, seven alleles of barley HvUGT14077 (GenBank No.GU170356.1) were cloned using RT-PCR. Among them, HvUGT-10W1, which was isolated from a FHB resistant barley variety 10W1, was significantly up-regulated in young spikes after F. graminearum (F.g) inoculation. HvUGT-10W1::GFP was subcellularly located in the plasma membrane and cytoplasm of the wheat protoplasts. In vitro antifungal activity assay showed that the HvUGT-10W1 protein exerted obvious inhibition against the growth of F.g. The silencing of the HvUGT-10W1 by virus-induced gene silencing (VIGS) resulted in compromised FHB resistance of 10W1, which was shown by the increased infected colonies on the leaves. These indicated that the barley HvUGT-10W1 may also contribute to F.g resistance in barley and provided a potential candidate gene to develop transgenic barley with enhanced FHB resistance.     

Population growth rate and relative virulence of the two South African biotypes of Russian wheat aphid,Diuraphis noxia,and bird cherry‐oat aphid,Rhopalosiphum padi,on resistant and non‐resistant barley

In South Africa a new biotype of the Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), RWASA2, has appeared which exhibits an improved performance compared to the original biotype (RWASA1) on wheat containing the Dn1 resistance gene. We examined population growth rates as well as damage caused by RWASA1 and RWASA2, in addition to a different aphid species, the bird cherry‐oat aphid (BCA), Rhopalosiphum padi L. (Hemiptera: Aphididae), on three RWA‐resistant barley [Hordeum vulgare L. (Poaceae)] lines (STARS‐9577B, STARS‐0502B, and STARS‐9301B) and one susceptible control (PUMA). RWASA2 had a higher reproductive rate than RWASA1 on all barley lines tested, which is consistent with previous results on wheat. Two of the RWA‐resistant lines (STARS‐0502B and STARS‐9301B) also exhibited a similar resistance phenotype against BCA. In our experiments, severe chlorosis and leaf roll appeared earlier on the control PUMA barley variety as a result of RWASA2 feeding than was the case with RWASA1, probably due to the differences in reproductive rate. Although chlorosis appeared earlier on resistant plants after RWASA2 feeding, this symptom developed much faster during RWASA1 feeding on all three resistant lines tested. As chlorosis did not correlate well with aphid population numbers, we surmise that the differential chlorosis effects may be related to differences in the amount of saliva introduced by the two aphid clones during feeding. Our results indicate that the difference between RWASA2 and RWASA1 are broader than a ‘gene for gene’ interaction with the Dn1 resistance (R) gene in wheat, and that these biotypes also differ in important aspects of their biology.     

Variation to cause host injury between Russian wheat aphid (Homoptera: Aphididae) clones virulent to Dn4 wheat

Biotypes are infraspecific classifications based on biological rather than morphological characteristics. Cereal aphids are managed primarily by host plant resistance, and they often develop biotypes that injure or kill previously resistant plants. Although molecular genetic variation within aphid biotypes has been well documented, little is known about phenotypic variation, especially virulence or the biotype's ability to cause injury to cultivars with specific resistance genes. Five clones (single maternal lineages) of Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), determined to be injurious to wheat, Triticum aestivum L., with the Dn4 gene, were evaluated on resistant and susceptible wheat and barley, Hordeum vulgare L., for their ability to cause chlorosis, reduction in plant height, and reduction in shoot dry weight. Variation to cause injury on resistant 'Halt' wheat, susceptible 'Jagger' wheat, and resistant 'STARS-9301B' barley was found among the Dn4 virulent clones. One clone caused up to 30.0 and 59.5% more reduction in plant height and shoot dry weight, respectively, on resistant Halt than other clones. It also caused up to 29.9 and 55.5% more reduction in plant height and shoot dry weight, respectively, on susceptible Jagger wheat. Although STARS-9301B barley exhibited an equal resistant response to feeding by all five clones based on chlorosis, two clones caused approximately 20% more reduction in plant height and shoot dry weight than three other clones. The most injurious clones on wheat were not the most injurious clones on barley. This is the first report of variation to cause varying degrees of plant damage within an aphid biotype virulent to a single host resistance gene. A single aphid clone may not accurately represent the true virulent nature of a biotype population in the field.     

The Yd2 resistance to barley yellow dwarf virus is effective in barley plants but not in their leaf protoplasts

The Yd2 gene for "resistance" to barley yellow dwarf virus (BYDV) has been widely used in barley ( Hordeum vulgare ). We have tested Australian isolates of BYDV of varying severity against barley genotypes with and without the Yd2 gene and report here a positive relationship between symptoms and virus levels determined by ELISA. Cultivar Shannon is the result of backcrossing the resistant line CI 3208 to cultivar Proctor, a susceptible line. It appears to be intermediate in reaction to BYDV between Proctor and CI 3208, although it carries the major gene, Yd2. Unlike the whole plant studies, no significant differences were observed with regard to the ability of protoplasts derived from these various genotypes to support BYDV replication. It is therefore demonstrated for the first time that the Yd2 gene is not among the small number of resistance genes which are effective against virus replication in isolated protoplasts.     

The effect of increasing dosage of barley yellow dwarf virus on some resistant and susceptible barleys

Five spring barleys, grown either in pots out of doors or in the field, were inoculated with barley yellow dwarf virus (BYDV) using 5, 10, 20 or 50 infective aphids (Rhopalosiphum padi) per plant. Control plants of each variety received no aphids. Infection with all aphid numbers had highly significant adverse effects on all varieties except Cb 1029, an early maturing BYDV-resistant barley of Ethiopian origin. 12583 Co, a locally bred, late maturing barley possessing the same resistance gene as Cb 1029 suffered more in a pot experiment, but less than three susceptible varieties all of which were severely damaged even when few infective aphids were used. Progressive effects with increasing aphid numbers, indicative of dosage response, occurred in some varieties. These effects included delay in heading and increased stunting, but not less yield. In Cb 1029, BYDV infection caused a reduction in the number of heads per plant, but this was partly compensated for by an increase in the number of grains per head. Conversely, BYDV infection in 12583 Co caused an increase in the number of heads, partly offset by a decrease in the number of Brains tier head.     

Acquired resistance functions in mlo barley,which is hypersusceptible to Magnaporthe grisea

Barley plants carrying a mutation in the Mlo (barley [Hordeum vulgare L.] cultivar Ingrid) locus conferring a durable resistance against powdery mildew are hypersusceptible to the rice blast fungus Magnaporthe grisea. It has been speculated that a functional Mlo gene is required for the expression of basic pathogen resistance and that the loss of Mlo function mediating powdery mildew resistance is an exception for this particular disease. Here, we report that the onset of acquired resistance (AR) after chemical as well as biological treatments is sufficient to overcome the hypersusceptible phenotype of backcross line BCIngridmlo5 (mlo) barley plants against M. grisea. Moreover, even barley plants bearing a functional Mlo gene and thus showing a moderate infection phenotype against rice blast exhibit a further enhanced resistance after induction of AR. Cytological investigations reveal that acquired resistance in mlo genotypes is manifested by the restoration of the ability to form an effective papilla at sites of attempted penetration, similarly to wild-type Mlo plants. In addition, the rate of effective papillae formation in Mlo plants was further enhanced after the onset of AR. These results demonstrate that treatments leading to the AR state in barley function independently of the Mlo/mlo phenotype and suggest that the Mlo protein is not a component of the AR signaling network. Moreover, it seems that only concomitant action of Mlo together with AR permits high level resistance in barley against blast. Higher steady state levels of PR1 and barley chemically induced mRNA correlate with higher disease severity rather than with the degree of resistance observed in this particular interaction.