Comparative proteomic profiles of Pinus monticola needles during early compatible and incompatible interactions with Cronartium ribicola

The proteomic profiles of primary needles from Cr2-resistant and cr2-susceptible Pinus monticola seedlings were analysed post Cronartium ribicola inoculation by 2-DE. One hundred-and-five protein spots exhibiting significant differential expression were identified using LC–MS/MS. Functional classification showed that the most numerous proteins are involved in defence signalling, oxidative burst, metabolic pathways, and other physiological processes. Our results revealed that differential expression of proteins in response to C. ribicola inoculation was genotype- and infection-stage dependent. Responsive proteins in resistant seedlings with incompatible white pine blister rust (WPBR) interaction included such well-characterized proteins as heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, and intermediate factors functioning in the signal transduction pathways triggered by well-known plant R genes, as well as new candidates in plant defence like sugar epimerase, GTP-binding proteins, and chloroplastic ribonucleoproteins. Fewer proteins were regulated in susceptible seedlings; most of them were in common with resistant seedlings and related to photosynthesis among others. Quantitative RT-PCR analysis confirmed HSP- and ROS-related genes played an important role in host defence in response to C. ribicola infection. To the best of our knowledge, this is the first comparative proteomics study on WPBR interactions at the early stages of host defence, which provides a reference proteomic profile for other five-needle pines as well as resistance candidates for further understanding of host resistance in the WPBR pathosystem.     

Pinus monticola pathogenesis-related gene PmPR10-2 alleles as defense candidates for stem quantitative disease resistance against white pine blister rust (Cronartium ribicola)

White pine blister rust, caused by the invasive fungus Cronartium ribicola, has been responsible for extremely high mortality of native western white pine (Pinus monticola) and other five-needle pines in natural stands throughout western North America. The presence of this non-native fungus has also led to greatly restricted use of western white pine for reforestation. A few families of defense proteins have been found as functional candidates involved in tree resistance to rust infection. Here we report genetic variation of a gene encoding a family 10 pathogenesis-related (PR) protein (PmPR10-2) in open-pollinated seed families with different levels of stem quantitative disease resistance (QDR). Six novel alleles and five common genotypes were identified inside the PmPR10-2 locus: these genetic variations included 33 single nucleotide polymorphisms (SNPs) throughout the gene regions and copy variation of a rare octanucleotide simple sequence repeat (SSR), 5′-AATTATTT-3′, in the gene intron. PmPR10-2 exhibited a moderate level (average r ²?=?0.42) of linkage disequilibrium. Two-thirds of the identified SNPs and the SSR marker were significantly associated with stem QDR levels. The PmPR10-2 genotype (H3:H3) exhibited the highest level of stem QDR (P?<?0.01). Cost-effective and co-dominant SSR markers were developed and used for genotyping the PmPR10-2 locus using simple PCR, providing a potential molecular tool for accelerating screening efforts of white pine resistance against C. ribicola.     

Finding loci associated to partial resistance to white pine blister rust in sugar pine (<Emphasis Type="Italic">Pinus lambertiana</Emphasis> Dougl.).

White pine blister rust (WPBR) is an exotic disease threatening five-needle pines in North America. In spite of its relatively recent introduction, some five-needle pines such as sugar pine (Pinus lambertiana) have developed both complete (major) gene resistance and partial (quantitative) resistance to WPBR. While significant effort has been dedicated to clone and locate the position of the major gene of WPBR resistance in sugar pine, the genetic basis of quantitative resistance remains largely unknown in all Strobus pines. In this work, we took a preliminary approach to identify potential genotype × phenotype associations using the results of long-term survival and symptoms of infection in both experimental and applied breeding populations. Our study found significant associations between several genes and WPBR disease symptoms such as normal active cankers and blights, important symptoms in the development of partial resistance. No significant associations were found with percentage of survival, probably due to the complex inheritance of the disease and long time to infection. With this study, we hope to lay the ground for further genome-wide association studies using large phenotypic data sets in sugar pine and other Strobus pines.     

Genetic diversity and structure of western white pine (Pinus monticola) in North America: a baseline study for conservation, restoration, and addressing impacts of climate change

Western white pine (Pinus monticola) is an economically and ecologically important species in western North America that has declined in prominence over the past several decades, mainly due to the introduction of Cronartium ribicola (cause of white pine blister rust) and reduced opportunities for regeneration. Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity and structure among populations at 15 sites (e.g., provenances) across the native range of western white pine. The level of genetic diversity was different among 15 populations tested using 66 polymorphic AFLP loci. Nei’s gene diversity (H _E) at the population level ranged from 0.187 to 0.316. Genetic differentiation (G _ST) indicated that 20.1% of detected genetic variation was explained by differences among populations. In general, populations below 45^oN latitude exhibited a higher level of genetic diversity than higher latitude populations. Genetic distance analysis revealed two major clades between northern and southern populations, but other well-supported relationships are also apparent within each of the two clades. The complex relationships among populations are likely derived from multiple factors including migration, adaptation, and multiple glacial refugia, especially in higher latitudes. Genetic diversity and structure revealed by this study will aid recognition and selection of western white pine populations for species management and conservation programs, especially in consideration of current and future climate changes.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Comparative mapping in Pinus: sugar pine (Pinus lambertiana Dougl.) and loblolly pine (Pinus taeda L.)

The majority of genomic research in conifers has been conducted in the Pinus subgenus Pinus mostly due to the high economic importance of the species within this taxon. Genetic maps have been constructed for several of these pines and comparative mapping analyses have consistently revealed notable synteny. In contrast, little genomic research has been conducted on the Pinus subgenus Strobus, even though these pines have strong ecological relevance. We report a consensus genetic linkage map for sugar pine (Pinus lambertiana Dougl.) constructed with 399 single nucleotide polymorphisms markers derived from annotated genes. The map is 1,231 cM in length and organized into 19 linkage groups. Two of the mapping populations were derived from trees that were segregating for the major gene of resistance (Cr1) to Cronartium ribicola, the fungal pathogen responsible for white pine blister rust. The third mapping population was derived from a full-sib cross segregating for partial resistance to white pine blister rust. In addition, we report the first comparative mapping study between subgenera Strobus and Pinus. Sixty mapped markers were found in common between sugar pine and the loblolly pine reference map with 56 of them (93%) showing collinearity. All 19 linkage groups of the sugar pine consensus map coaligned to the 12 linkage groups of the loblolly pine reference map. The syntenic relationship observed between these two clades of pines provides a foundation for advancing genomic research and genetic resources in subgenus Strobus.     

Conversion of AFLP markers to high-throughput markers in a complex polyploid,sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.     

SCAR markers linked to the common bean rust resistance gene Ur-13

Rust in common bean (Phaseolus vulgaris L.) is caused by Uromyces appendiculatus Pers.:Pers. (Unger) which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a considerable number of genes. Pyramiding resistance genes is desirable and could be simplified by the use of molecular markers closely linked to the genes. The resistance gene Ur-13, present in the South African large seeded cultivar Kranskop, has been used extensively in the local breeding program. The purpose of this study was the development of a molecular marker linked to Ur-13. An F₂ population derived from a cross between Kranskop and a susceptible (South African) cultivar Bonus was used in combination with bulked segregant analysis utilizing the amplified fragment length polymorphism (AFLP) technique. Seven AFLP fragments linked significantly to the rust resistance and five were successfully converted to sequence characterized amplified region (SCAR) markers. The co-dominant SCAR markers derived from a 405 bp EAACMACC fragment, KB126, was located 1.6 cM from the gene. Two additional SCAR markers and one cleaved amplified polymorphic sequence marker were located further from the gene. The gene was mapped to linkage group B8 on the BAT 93/Jalo EEP 558 core map (chromosome 3).     

Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple.

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.     

<Emphasis Type="Italic">Rpp1</Emphasis>, a dominant gene providing race-specific resistance to rose powdery mildew (<Emphasis Type="Italic">Podosphaera pannosa</Emphasis>): molecular mapping,SCAR development and confirmation of disease resistance data

We have previously demonstrated that in the diploid rose population 97/9 resistance to the powdery mildew race 9 is controlled by a major dominant resistance gene, Rpp1. In the study reported here, we isolated several molecular markers closely linked to Rpp1 via bulked segregant analysis, with the gene being tagged in an interval of 5 cM between the two most adjacent markers. It was possible to convert the most closely linked amplified fragment length polymorphic (AFLP) marker into a sequence-characterised amplified region (SCAR) segregating in the same manner. Indirect mapping of Rpp1 in relation to the black spot resistance gene Rdr1 revealed no linkage between the two R genes. Furthermore, the genetic model based on a single dominant resistance gene was supported by the marker data.     

Molecular polymorphism and linkage analysis in sweet passion fruit,an outcrossing species

One of the current challenges of tropical fruit crop improvement is to incorporate molecular marker‐based approaches into conventional breeding programmes. This study was designed to build an integrated genetic map of the sweet passion fruit (Passiflora alata), a diploid (2n = 18) outcrossing species which is greatly appreciated for in natura consumption, and reported to inspire cosmetic and pharmaceutical companies to create plant‐derived compounds. With this in mind, a full‐sib family of 180 individuals was genotyped using different molecular marker types, such as amplified fragment length polymorphisms (AFLP), microsatellite‐AFLP (M‐AFLP), simple sequence repeats (SSR), resistance gene analogues (RGA) and target region amplification polymorphism (TRAP). On average, the rate of polymorphism between the parental genotypes was 20.3%. We also searched for single nucleotide polymorphisms (SNPs) in some AFLP bands and in seven gene fragments, and found one SNP every 87 bp. All SNPs were biallelic and occurred most frequently in putative gene fragments (81.5%) rather than in AFLP bands (60.0%) analyzed. Excellent gel profiles were obtained allowing the recognition of all types of segregation expected for a progeny of an outcrossing species. Multipoint linkage analysis was performed using OneMap software, with logarithm of the odds (LOD) score ≥ 5.6 and recombination fraction <0.5. The resulting integrated map consists of 549 markers, 2.0% of which fit a segregation ratio of 1:1:1:1, 1.3% a ratio of 1:2:1, 27.3% a ratio of 3:1 and 69.4% a ratio of 1:1. The map spanned a total of 2073.0 cM, with an average distance between adjacent markers of 3.8 cM. This is the first linkage study on sweet passion fruit and should prove useful for quantitative trait loci mapping.     

A genetic map of an interspecific cross in Allium based on amplified fragment length polymorphism (AFLPTM) markers

Segregation of 692 polymorphic AFLP^TM (amplified fragment length polymorphism) fragments was determined in an F₂ of the interspecific cross A. roylei x A. cepa. Two different enzyme combinations were used, PstI/MseIand EcoRI/MseI; in the latter one extra selective nucleotide was added to the MseI primer. The map based on A. cepa markers consisted of eight linkage groups with 262 markers covering 694 cM of the expected 800 cM. The map based on A. roylei markers comprised 15 linkage groups with 243 markers and had a length of 626 cM. The two maps were not integrated, and 25% of the markers remained unlinked. One of the alliinase genes and a SCAR marker linked to the disease resistance gene to downy mildew are present on this map. Of the AFLP markers, 50—80% were polymorphic between A. cepa and A. roylei; the level of polymorphic markers between different A. cepa accessions was4-8%. Received: 28 August 1998 / Accepted: 31 March 1999     

Efficient fine mapping of the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F₂ population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.Communicated by G. Wenzel     

An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence

In an attempt to identify a specific marker for biotype 2 of the Asian rice gall midge (Orseolia oryzae, Wood-Mason), we used AFLP (amplified fragment length polymorphism) fingerprinting. We identified an AFLP marker that is specifically amplified in biotypes 1, 2 and 5 of the rice gall midge, but not in biotype 4. Biotypes 1, 2 and 5 are avirulent to hosts bearing the Gm2 resistance gene (found in rice variety Phalguna), whereas biotype 4 is virulent to Gm2. Based on the sequence of this AFLP marker, SCAR (sequence characterized amplified region) primers were designed and used in combination with previously developed SCAR primers to distinguish effectively all five biotypes in a multiplex PCR-based assay. The inheritance pattern of this marker in the progenies of inter-biotype crosses between biotypes 1, 2 and 4 shows that the marker can be amplified by PCR from all F₁ females, irrespective of the biotype status of their parents. However, the marker is present only in those male progenies whose mother was of a Gm2 avirulent biotype. The specific amplification of this marker in the avirulent biotypes and its pattern of inheritance show that avirulence with respect to carriers of the Gm2 gene in rice gall midge is sex-linked. Received: 16 August 1999 / Accepted: 27 December 1999     

Isolation of a full-length CC–NBS–LRR resistance gene analog candidate from sugar pine showing low nucleotide diversity

The nucleotide-binding-site and leucine-rich-repeat (NBS–LRR) class of R proteins is abundant and widely distributed in plants. By using degenerate primers designed on the NBS domain in lettuce, we amplified sequences in sugar pine that shared sequence identity with many of the NBS–LRR class resistance genes catalogued in GenBank. The polymerase chain reaction products were used to probe a cDNA library constructed from needle tissue of sugar pine seedlings. A full-length cDNA was obtained that demonstrated high predicted amino acid sequence similarity to the coiled coil (CC)–NBS–LRR subclass of NBS–LRR resistance proteins in GenBank. Sequence analyses of this gene in megagametophytes from two sugar pine trees segregating for the hypersensitive response to white pine blister rust revealed zero nucleotide variation. Moreover, there was no variation found in 24 unrelated sugar pine trees except for three single-nucleotide polymorphisms located in the 3′ untranslated region. Compared to other genes sequenced in Pinaceae, such a low level of sequence variation in unrelated individuals is unusual. Although, numerous studies have reported that plant R genes are under diversifying selection for specificity to evolving pathogens, the resistance gene analog discussed here appears to be under intense purifying selection.An erratum to this article can be found at