Identification and Characterization of Random Amplified Polymorphic DNA Markers Linked to a Major Gene (Cr2) for Resistance to Cronartium ribicola in Pinus monticola

ABSTRACT DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.     

Barrier to Gene Flow Between Eastern and Western Populations of Cronartium ribicola in North America

ABSTRACT The population structure of Cronartium ribicola from eastern and western North America was studied to test the null hypothesis that populations are panmictic across the continent. Random amplified polymorphic DNA markers previously characterized in eastern populations were mostly fixed in western populations, yielding high levels of genetic differentiation between eastern and western populations (phi(st) = 0.55; theta = 0.36; P < 0.001). An unweighted pair-group method, arithmetic mean dendro-gram based on genetic distances separated the four eastern and four western populations into two distinct clusters along geographic lines. Similarly, a principal component analysis using marker frequency yielded one cluster of eastern populations and a second cluster of western populations. The population from New Mexico was clearly within the western cluster in both analyses, confirming the western origin of this recent introduction. This population was completely fixed (H(j) = 0.000; n = 45) at all loci suggesting a severe recent population bottleneck. Genetic distances were low among populations of western North America (0.00 to 0.02) and among eastern populations (0.00 to 0.02), indicating a very similar genetic composition. In contrast, genetic distances between eastern and western populations were large, and all were significantly different from 0 (0.07 to 0.19; P < 0.001). Indirect estimates of migration were high among western populations, including the number of migrants among pairs of populations (Nm > 1) between New Mexico and British Columbia populations, but were smaller than one migrant per generation between eastern and western populations. These results suggest the presence of a barrier to gene flow between C. ribicola populations from eastern and western North America.     

The CC-NBS-LRR Subfamily in Pinus monticola: Targeted Identification, Gene Expression, and Genetic Linkage with Resistance to Cronartium ribicola

ABSTRACT To investigate disease resistance gene analogs (RGAs) encoding coiled-coil-nucelotide-binding site-leucine-rich repeats (CC-NBS-LRR) proteins in western white pine, degenerate primers targeting the conserved motifs in the NBS domain were designed to amplify RGAs from genomic DNA and cDNA. Sixty-one distinct RGAs were identified with identities to well-known R proteins of the CC-NBS-LRR subfamily. These RGAs exhibited variation of putative amino acid sequences from 13% to 98%, representing a complex CC-NBS-LRR subfamily. A phylogenetic tree constructed from the amino acid sequence alignment revealed that these 61 RGAs were grouped with other CC-NBS-LRR members from angiosperms, and could be further divided into six classes with an identity threshold of 68%. To map RGAs, RGA polymorphisms and a modified amplified fragment length polymorphism (AFLP) method with incorporated sequences from the NBS domain were used to reveal NBS or NBS-AFLP markers. RGA polymorphism study revealed that three off the identified RGAs were not linked to the Cr2 gene imparting resistance to white pine blister rust. However, the AFLP strategy, using bulk segregant analysis (BSA) and haploid segregation analysis, identified 11 NBS-AFLP markers localized in the Cr2 linkage, the closest two to the gene being 0.41 cM and 1.22 cM away at either side. Eight of these markers showed significant amino acid sequence homologies with RGAs.     

Genetic Structure of Cronartium ribicola Populations in Eastern Canada

ABSTRACT The genetic structure of populations of Cronartium ribicola was studied by sampling nine populations from five provinces in eastern Canada and generating DNA profiles using nine random amplified polymorphic DNA markers. Most of the total gene diversity (H(t) = 0.386) was present within populations (H(w) = 0.370), resulting in a low level of genetic differentiation among populations in northeastern North America (F(st) = 0.062). A hierarchical analysis of genetic structure using an analysis of molecular variance (AMOVA) revealed no statistically significant genetic differentiation among provinces or among regions. Yet, genetic differentiation among populations within regions or provinces was small (AMOVA phi(st) = 0.078) but statistically significant (P < 0.001) and was several orders of magnitude larger than differentiation among provinces. This is consistent with a scenario of subpopulations within a metapopulation, in which random drift following migration and new colonization are major evolutionary forces. A phenetic analysis using genetic distances revealed no apparent correlation between genetic distance and the province of origin of the populations. The hypothesis of isolation-by-distance in the eastern populations of C. ribicola was rejected by computing Mantel correlation coefficients between genetic and geographic distance matrices (P > 0.05). These results show that eastern Canadian provinces are part of the same white pine blister rust epidemiological unit. Nursery distribution systems are controlled provincially, with virtually no seedling movement among provinces; therefore, infected nursery material may not play an important role in the dissemination of this disease. Long-distance spore dispersal across provincial boundaries appears to be an epidemiologically important factor for this pathogen.     

A Class IV Chitinase Is Up-Regulated by Fungal Infection and Abiotic Stresses and Associated with Slow-Canker-Growth Resistance to Cronartium ribicola in Western White Pine (Pinus monticola)

ABSTRACT In the present study, in a candidate gene approach, a class IV chitinase gene (PmCh4A) of pathogenesis-related family three was cloned and characterized in western white pine (Pinus monticola). PmCh4A chitinase expression in the different organs of healthy seedlings was below levels detectable by western immunoblot analysis using an antibody raised against PmCh4A protein. However, a 27-kDa isozyme of PmCh4A accu mulated in both susceptible and slow-canker-growth (SCG) resistant seedlings after infection by Cronartium ribicola. As with fungal infection, the application of a signal chemical (methyl jasmonate) and a protein phosphatase 1 and 2A inhibitor (okadaic acid) increased the PmCh4A protein accumulation. Furthermore, another 26-kDa isozyme was expressed specifically in SCG resistant seedlings, providing a potential tool for marker-assisted selection in forest breeding. Wounding treatment also induced expression of the protein. These data suggest that the class IV chitinase PmCh4A is involved in the defense response of western white pine to infection and abiotic stresses.     

Association of a novel Pinus monticola chitinase gene (PmCh4B) with quantitative resistance to Cronartium ribicola

Multiple families of pathogenesis-related (PR) proteins are believed to contribute to plant quantitative resistance to various pathogens. Along with other host PR proteins, PR3 chitinase is one protein component participating in genetic resistance of western white pine (Pinus monticola) to the white pine blister rust (WPBR) pathogen (Cronartium ribicola). In the present study, we characterized a novel P. monticola class IV chitinase gene (PmCh4B) and further analyzed its nucleotide variations in the open-pollinated seed families of diverse geographical distribution and variable levels of quantitative resistance to C. ribicola infection. PmCh4B showed high haplotype diversity (Hd=0.94) and nucleotide diversity (π=0.00965), similar to those of other conifer genes related to environmental stresses. A low level of intragenic linkage disequilibrium (LD) (but most of the levels with statistical significance) was found within a distance of ≈800 bp. Based on PmCh4B haplotype frequency, moderate to high levels of population structure were observed among P. monticola seed families currently used in breeding programs for WPBR resistance (average FST=0.163, P<0.001). Association analysis revealed that allelic variants and multiple single-nucleotide polymorphisms of PmCh4B were significantly associated with quantitative levels of P. monticola resistance against C. ribicola. This work represents the first association study for quantitative resistance in western white pine pathosystem and provides a potential for marker-assisted selection in white pine breeding.     

Major Gene and Polygenic Resistance to Leptosphaeria maculans in Oilseed Rape (Brassica napus)

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is through the breeding of resistant cultivars. Race specific major genes that mediate resistance from the seedling stage have been identified in B. napus or have been introgressed from related species. Many race specific major genes have been described and some of them are probably identical in B. napus (allotetraploid AACC) and the parental species B. rapa (diploid AA). More work is needed using a set of well-characterised isolates to determine the number of different major resistance genes available. In some B. napus cultivars, there is resistance which is polygenic (mediated by Quantitative Trait Loci) and postulated to be race non-specific. Many of these major genes and Quantitative Trait Loci for resistance to L. maculans have been located on B. napus genetic maps. Genes involved in race specific and polygenic resistance are generally distinct.     

Inheritance of Race-Specific Resistance to Mycosphaerella graminicola in Wheat

ABSTRACT Mycosphaerella graminicola causes Septoria tritici blotch of hexaploid and tetraploid wheat. The inheritance of high-level resistance to Septoria tritici blotch was studied in controlled environment experiments. Intraspecific reciprocal crosses were made between hexaploid wheat lines Salamouni, ST6, Katepwa, and Erik, and the tetraploid wheat lines Coulter and 4B1149. Parental, F(1), F(2), F(3), BC(1)F(1), and BC(1)F(2) populations were evaluated for reaction to isolates MG2 and MG96-36 of M. graminicola. Resistance was controlled by incompletely dominant nuclear genes in all cases. Salamouni had three independent resistance genes to isolate MG2, two of which also controlled resistance to isolate MG96-36. ST6 had a single resistance gene to isolate MG2 and none to isolate MG96-36. The resistance genes in Salamouni and ST6 were not allelic. Two independent genes control resistance to isolate MG2 in Coulter, one of which also controlled resistance to isolate MG96-36. These data are consistent with a gene-for-gene interaction in the wheat-M. graminicola pathosystem.     

Inheritance of Resistance to Leptosphaeria maculans in Brassica juncea

ABSTRACT The inheritance of resistance to Leptosphaeria maculans, the causal agent of black leg of crucifers, was studied in Brassica juncea. Three resistant accessions (UM3021, UM3043, and UM3323) and one susceptible accession (UM3132) of B. juncea were crossed in a complete diallel. Parents, F(1), and F(2) progenies were evaluated for all crosses using both cotyledon and stem inoculation. Cotyledon reaction was evaluated with two isolates of L. maculans, but stem reaction was evaluated with one isolate. Disease reactions observed for individual plants were the same for both inoculation methods and for both isolates of the pathogen for cotyledon reaction. No segregation was observed for the crosses between resistant accessions (UM3043 x UM3323 and UM3021 x UM3323), but a few susceptible plants were observed in the F(2) progeny of crosses between resistant parents (UM3021 x UM3043). This was probably due to heterozygosity in some parental plants of UM3021. For crosses be tween the susceptible parent and resistant parents, F(1) plants for two crosses were all resistant. For cross UM3132 x UM3021, some susceptible plants occurred, which was also suggestive of heterozygosity in UM3021. Although resistance in F(1) was dominant, for F(2) populations, segregation fit either 13:3, 3:1, or 1:3 ratios, indicating that resistance can be either adominant or recessive trait. F(3) families derived from some susceptible F(2) plants from crosses UM3021 x UM3132 and UM3043 x UM3132 were evaluated using the cotyledon inoculation method only. Segregation of F(2) plants and F(3) families in crosses involving resistant and susceptible parents indicated that the resistance to L. maculans in B. juncea is controlled by two nuclear genes with dominant recessive epistatic gene action.     

Inheritance of Resistance to Colletotrichum acutatum in Fragaria x ananassa

ABSTRACT Anthracnose, caused by Colletotrichum acutatum, is a major disease of the octoploid cultivated strawberry, Fragaria x ananassa The inheritance of high and intermediate level plant resistances to C. acutatum, pathogenicity group 2, was investigated in an 8 x 8 factorial design. A single dominant gene (Rca2) controlled the high-level resistance, although minor genes may also contribute to resistance in cultivars such as Belrubi. The intermediate level of resistance was quantitative and controlled by minor genes. Analysis of 26 genotypes and cultivars from Fragaria spp. showed that the dominant gene was not rare in the germ plasm of F. x ananassa and that anthracnose resistance was also present in other species of Fragaria. These findings have important implications for anthracnose resistance breeding.     

水稻抗病性的遗传研究——Ⅱ.水稻对稻瘟病抗性的遗传

 本文续接"Ⅰ.水稻对白叶枯病抗性的遗传"一文(见《植物病理学报》,1986,(3):161-168)。     

Biometric Analyses of the Inheritance of Resistance to Didymella rabiei in Chickpea

ABSTRACT Historically, the response of chickpea (Cicer arietinum L.) to Didymella rabiei (causal agent of Ascochyta blight) has been mainly related to as complete resistance and it was commonly assayed with qualitative (nonparametric) scales. Two reciprocal populations, derived from intra-specific crosses between a moderately resistant late flowering Israeli cultivar and a highly susceptible early flowering Indian accession, were tested at F(3) and F(4) generations in 1998 and 1999, respectively. A quantitative (parametric) assessment (percent disease severity) was used to evaluate the chickpea field response to Ascochyta blight. The transformed relative area under the disease progress curve (tRAUDPC) was calculated for each experimental unit for further analyses. Heritability estimates of the tRAUDPC were relatively high (0.67 to 0.85) in both generations for both reciprocal populations. The frequency distributions of tRAUDPC of the populations were continuous and significantly departed from normality (Shapiro-Wilk W test; P of W < 0.0001), being all platykurtic and skewed toward either the resistant or the susceptible parental lines. The presence of major genes was examined by testing the relationship between the F(3) and F(4) family means and the within-family variances (Fain's test). Analyses of these relationships suggested that segregation of a single (or few) quantitative trait locus with major effect and possibly other minor loci was the predominant mode of inheritance. The correlation estimates between the resistance and days to flower (r = -0.19 to -0.44) were negative and significantly (P = 0.054 to 0.001) different from zero, which represents a breeding constraint in the development of early flowering cultivars with Ascochyta blight resistance.     

Genetics of Virulence in Cochliobolus sativus and Resistance in Barley

ABSTRACT Spot blotch, caused by Cochliobolus sativus, is one of the most common foliar diseases of barley in the upper midwest region of the United States. To examine the genetics of host-specific virulence in C. sativus, a cross was made between isolate ND90Pr (which exhibits high virulence on barley genotype Bowman and low virulence on genotype ND 5883) and ND93-1 (which exhibits low virulence on both genotypes). Ascospore progeny segregated 48:55 for low virulence/high virulence on Bowman, indicating the presence of a single virulence gene in isolate ND90Pr. To complement the study of host-specific virulence in the pathogen, an experiment also was conducted on the genetics of specific resistance in the host. Progeny from a Bowman/ND 5883 cross were evaluated for their infection responses (IRs) to isolate ND90Pr at the seedling stage. The F(2) population segregated 1:3 for low IRs (resistant)/high IRs (susceptible), indicating the presence of a single resistance gene in genotype ND 5883. This result was confirmed in the F(3) generation, as a 1:2:1 ratio was found for homozygous resistant, segregating, and homozygous susceptible families, respectively. The data from this study demonstrate that both virulence in the pathogen and resistance in the host are under monogenic control in this specific host genotype/fungal isolate combination.     

Inheritance of Race-Specific Resistance to Xanthomonas campestris pv. campestris in Brassica Genomes

ABSTRACT The inheritance of resistance to three Xanthomonas campestris pv. campestris races was studied in crosses between resistant and susceptible lines of Brassica oleracea (C genome), B. carinata (BC genome), and B. napus (AC genome). Resistance to race 3 in the B. oleracea doubled haploid line BOH 85c and in PI 436606 was controlled by a single dominant locus (Xca3). Resistance to races 1 and 3 in the B. oleracea line Badger Inbred-16 was quantitative and recessive. Strong resistance to races 1 and 4 was controlled by a single dominant locus (Xca1) in the B. carinata line PI 199947. This resistance probably originates from the B genome. Resistance to race 4 in three B. napus lines, cv. Cobra, the rapid cycling line CrGC5, and the doubled haploid line N-o-1, was controlled by a single dominant locus (Xca4). A set of doubled haploid lines, selected from a population used previously to develop a restriction fragment length polymorphism map, was used to map this locus. Xca4 was positioned on linkage group N5 of the B. napus A genome, indicating that this resistance originated from B. rapa. Xca4 is the first major locus to be mapped that controls race-specific resistance to X. campestris pv. campestris in Brassica spp.     

Resistance of Cereals to Heterodera avenae: Methods of Investigation, Sources and Inheritance of Resistance1

Techniques used in cereal breeding programmes for selecting for resistance to cereal cyst nematodes are described. Routine screening is done in field nurseries and in pots of infested soil in glasshouse and controlled environments. Other investigations of resistance use a laboratory technique which permits identification of the penetration sites of individual nematodes. Erosion of resistance, as indicated by 1) the development of only one or two females or 2) the occasional development of more females on plants with resistance genes, is discussed in relation to variation in nematode virulence and to environmental, especially temperature effects. Identification of tolerance is more difficult unless the differences are great, or the experimentation carefully controlled and replicated. Direct selection for tolerance in segregating generations is not presently practicable. Indirectly selecting for mechanisms of tolerance may eventually prove possible but has not yet been exploited. Available sources of resistance are described in relation to genetic studies and complementary studies of nematode pathotype and host range. There are three distinct resistances in barley which may have been responsible for selecting present pathotypes. Two, Rha1 and Rha2, are useful in breeding and linkage and allelism at these loci are described. In oats, as in wheat, only one locus has been used by breeders. There are probably additional loci in some of the other resistance sources but polyploidy of the host and different virulence gene frequencies in unselected nematode populations make it more difficult to identify genetic relationships in oats and wheat.     

A Cluster of Major Specific Resistance Genes to Leptosphaeria maculans in Brassica napus

ABSTRACT Two types of genetic resistance to Leptosphaeria maculans usually are distinguished in Brassica napus: qualitative, total resistance expressed at the seedling stage and quantitative, partial resistance expressed at the adult plant stage. The latter is under the control of many genetic factors that have been mapped through quantitative trait loci (QTL) studies using 'Darmor' resistance. The former usually is ascribed to race-specific resistance controlled by single resistance to L. maculans (Rlm) genes. Three B. napus-originating specific Rlm genes (Rlm1, Rlm2, and Rlm4) previously were characterized. Here, we report on the genetic identification of two novel resistance genes, Rlm3 and Rlm7, corresponding to the avirulence genes AvrLm3 and AvrLm7. The identification of a novel L. maculans- B. napus specific interaction allowed the detection of another putative new specific resistance gene, Rlm9. The resistance genes were mapped in two genomic regions on LG10 and LG16 linkage groups. A cluster of five resistance genes (Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9) was strongly suggested on LG10. The relation between all these specific resistance genes and their potential role in adult-plant field resistance is discussed. These two Rlm-carrying regions do not correspond to major QTL for Darmor quantitative resistance.     

Association of Mmd1, a Major Gene for Resistance to Melampsora medusae f. sp. deltoidae, with Quantitative Traits in Poplar Rust

ABSTRACT A single gene, Mmd1, which conditions resistance and a necrotic flecking response to a monouredinial isolate of Melampsora medusae f. sp. deltoidae and is presumed to possess the corresponding avirulence gene, was previously shown to segregate 3:1 (resistant to susceptible) in an interspecific hybrid poplar F(2) progeny. Some inoculated clones of the resistant phenotypic class of this progeny were completely resistant and bore no uredinia, but most bore some sporulating uredinia with accompanying necrotic flecking. The dominant allele at the Mmd1 locus in these incompletely resistant clones was significantly associated with reduced uredinial density and diameter and longer latent period in a growth-room assay and with reduced disease incidence and infection efficiency and longer latent period in a leaf-disk assay. However, high clone-mean heritabilities within the susceptible phenotypic class indicated that genes other than Mmd1 also contribute to control of quantitative traits. Leaf age had a significant effect on uredinial density and latent period but not on uredinial diameter in the growth-room assay. All quantitative traits were intercorrelated to varying extents. A principal components analysis (PCA) demonstrated that uncorrelated components associated with uredinial diameter and uredinial density explained two-thirds of the total variation. Since uredinial diameter (PC1) and necrotic flecking are the visual components of an infection-type rating scale, genetic analyses of poplar rust should be based on infection type. Mmd1 is a major gene for resistance associated with a significant effect on all quantitative traits measured. Gene complexes in domesticated, agricultural rust pathosystems known to provide durable resistance consist of similar "pivotal" major genes and inferred ancillary genes or quantitative trait loci.     

Epicuticular Wax and White Pine Blister Rust Resistance in Resistant and Susceptible Selections of Eastern White Pine (Pinus strobus)

ABSTRACT Epicuticular wax on needles was evaluated for its influence on Cronartium ribicola infection of resistant and susceptible selections of Pinus strobus. Environmental scanning electron microscopy comparisons revealed that needles from a resistant selection of eastern white pine, P327, had a significantly higher percentage of stomata that were occluded with wax, fewer basidiospores germinating at 48 h after inoculation, and fewer germ tubes penetrating stomata than needles from a susceptible selection H111. In addition, needles from seedlings that failed to develop symptoms 6 weeks after inoculation, from a cross between P327 and susceptible parent H109, had a significantly higher percentage of stomata occluded with wax compared with needles from seedlings that developed symptoms. In experiments where epicuticular waxes were removed from needles before seedlings were infected, resistant seedlings without wax developed approximately the same number of infection spots (as measured by spot index) as susceptible seedlings with wax intact. Gas chromatography/mass spectrometry comparisons of extracted epicuticular waxes revealed several peaks that were specific to P327 and not found in susceptible H111 suggesting biochemical differences in wax composition. These results implicate the role of epicuticular waxes as a resistance mechanism in P. strobus selection P327 and suggest a role for waxes in reducing spore germination and subsequent infection through stomatal openings.     

Inheritance of Carboxin Resistance in a European Field Isolate of Ustilago nuda

ABSTRACT Two carboxin-resistant field isolates of Ustilago nuda from Europe were crossed with a carboxin-sensitive field isolate from North America. Meiotic tetrads isolated from germinating F(1) teliospores of one of the hybrids were tested for carboxin resistance and mating type. Carboxin resistance was shown to be controlled by a single gene (CBX1R), because a 1:1 segregation of carboxin resistance was observed in all 27 tetrads. Tetrad analysis indicated that the loci for carboxin resistance (Cbx1) and mating type (MAT1) segregate independently but may be located on the same chromosome. Tetrad analysis was not possible with the F(1) hybrid of he other field isolate, and its resistance cannot yet be attributed to CBX1R. Carboxin resistance was qualitatively dominant to sensitivity in vitro, as demonstrated by triad analysis of germinating F(1) teliospores. Quantitative in planta infection percents supported the conclusion that CBX1R is dominant, although incompletely, in the F(1) hybrid of one of the field isolates. Also, fewer than expected carboxin-sensitive F(2) individuals were observed in planta. However, inoculations of host plants with U. nuda have resulted in similar, unexpected variation in the past.