Genetic evidence for sexual reproduction and multiple infections of Norway spruce cones by the rust fungus Thekopsora areolata

Rust fungi are obligate parasites, of plants, with complex and in many cases poorly known life cycles which may include host alteration and up to five spore types with haploid, diploid, and dikaryotic nuclear stages. This study supports that Thekopasora areolata, the causal agent of cherry‐spruce rust in Norway spruce, is a macrocyclic heteroecious fungus with all five spore stages which uses two host plants Prunus padus and Picea abies to complete its life cycle. High genotypic diversity without population structure was found, which suggests predominantly sexual reproduction, random mating and a high gene flow within and between the populations in Fennoscandia. There was no evidence for an autoecious life cycle resulting from aeciospore infection of pistillate cones that would explain the previously reported rust epidemics without the alternate host. However, within cones and scales identical multilocus genotypes were repeatedly sampled which can be explained by vegetative growth of the fertilized mycelia or repeated mating of mycelium by spermatia of the same genotype. The high genotypic diversity within cones and haplotype inference show that each pistillate cone is infected by several basidiospores. This study provides genetic evidence for high gene flow, sexual reproduction, and multiple infections of Norway spruce cone by the rust fungus T. areolata which expands the general understanding of the biology of rust fungi.     

Quest to elucidate the life cycle of Puccinia psidii sensu lato

There is controversy surrounding the described life cycle of the rust fungus Puccinia psidii sensu lato, which causes disease on several plant species in the family Myrtaceae. The objective of this study was to determine whether P. psidii s.l. is autoecious by performing basidiospore inoculations, and microscopically examining the fate of basidiospores on the leaf surface and nuclear condition at different stages of rust development. No spermogonia developed on leaves of Agonis flexuosa inoculated either with a teliospore suspension or basidiospores naturally discharged from telia. Uredinial sori that developed in all three inoculations with teliospore suspensions and in one of the five inoculations with naturally-discharged basidiospores from telia were most likely the result of urediniospore infections. Microsatellite analysis revealed that isolates made from these uredinial sori had the same multilocus genotype as that of the original isolate. No signs of penetration of plant cells by basidiospores were observed on A. flexuosa and Syzygium jambos. The nuclear condition of mycelia of uredinial sori, urediniospores, teliospores, and four-celled metabasidia was typical of that in many rust fungi. Our study could not provide unequivocal proof that P. psidii s.l. is autoecious. While it is possible that it could be heteroecious, with an unknown alternate aecial host, it is also possible that basidiospores have lost the ability to infect Myrtaceae or are infrequently operational.     

Development of sequence characterized DNA markers linked to leaf rust (<Emphasis Type="Italic">Hemileia vastatrix</Emphasis>) resistance in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Coffee leaf rust due to Hemileia vastatrix is one of the most serious diseases in Arabica coffee (Coffea arabica). A resistance gene (S_H3) has been transferred from C. liberica into C. arabica. The present work aimed at developing sequence-characterized genetic markers for leaf rust resistance. Linkage between markers and leaf rust resistance was tested by analysing two segregating populations, one F₂ population of 101 individuals and one backcross (BC₂) population of 43 individuals, derived from a cross between a susceptible and a SH3-introgressed resistant genotype. A total of ten sequence-characterized genetic markers closely associated with the S_H3 leaf rust resistance gene were generated. These included simple sequence repeats (SSR) markers, sequence-characterised amplified regions (SCAR) markers resulting from the conversion of amplified fragment length polymorphism (AFLP) markers previously identified and SCAR markers derived from end-sequences of bacterial artificial chromosome (BAC) clones. Those BAC clones were identified by screening of C. arabica genomic BAC library using a cloned AFLP-marker as probe. The markers we developed are easy and inexpensive to run, requiring one PCR step followed by gel separation. While three markers were linked in repulsion with the S_H3 gene, seven markers were clustered in coupling around the S_H3 gene. Notably, two markers appeared to co-segregate perfectly with the S_H3 gene in the two plant populations analyzed. These markers are suitable for marker-assisted selection for leaf rust resistance and to facilitate pyramiding of the S_H3 gene with other leaf rust resistance genes.     

Distribution, Frequency and Variation of Stripe Rust Resistance Loci Yr10, Lr34/Yr18 and Yr36 in Chinese Wheat Cultivars

Wheat stripe rust is a devastating disease in many regions of the world. In wheat, 49 resistance genes for stripe rust have been officially documented, but only three genes are cloned, including the race-specific resistance Yr10 candidate gene (Yr10_CG) and slow-rusting genes Lr34/Yr18 (hereafter designated as Yr18) and Yr36. In this study, we developed gene-specific markers for these genes and used them to screen a collection of 659 wheat accessions, including 485 Chinese cultivars. Thirteen percent and eleven percent of the tested Chinese cultivars were positive for the markers for Yr10_CG and Yr18_RH (the resistant haplotype of Yr18), respectively, but none were positive for the Yr36 marker. Since there is a limited use of the Yr10 gene in Chinese wheat, the relatively high frequency of wheat varieties with the Yr10_CG marker suggests that the identity of the Yr10 gene is unknown. With regards to the Yr18 gene, 29% of the tested cultivars that are used in the Middle and Lower Yangtze Valleys' winter wheat zone were positive for Yr18_RH markers. A non-functional allele of Yr18_RH was identified in ‘Mingxian 169’, a commonly used susceptible check for studying stripe rust. The data presented here will provide useful information for marker-assisted selection for wheat stripe rust resistance.     

Identification and Validation of Molecular Markers Associated with Pachymetra Root Rot and Brown Rust Resistance in Sugarcane Using Map- and Association-based Approaches

Marker-assisted selection for traits that are difficult to screen for, such as resistance to many sugarcane diseases, has the potential to facilitate the development of improved cultivars in sugarcane. Pachymetra root rot (PRR) and brown rust resistance ratings were obtained over two years for 192 I1 progeny (progeny produced by two heterozygous, non-inbred parental lines) of a sugarcane (Saccharum spp. hybrid) cross between two elite sugarcane clones, Q117 and 74C42. Approximately 1000 single-dose markers, including microsatellite (SSR), amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers, were scored across the population and maps containing approximately 400 markers were constructed for each parent. At p ≤ 0.01, two genomic regions, one from the female Q117 map and a different region from the 74C42 male map, plus an unlinked bi-parental simplex marker (single-dose marker present in both parents) were identified as associated with PRR over both years of data collection. These regions explained between 6 and 16% of the phenotypic variation. An additional region was identified in the female map as associated with PRR at p ≤ 0.01 in one year and p ≤ 0.05 in the second year. This region explained between 4 and 8% of the phenotypic variation. For brown rust, two genomic regions, one from the female map and one from the male map, plus an unlinked marker from both maps, were identified as associated with brown rust resistance at p ≤ 0.01 over two years of phenotypic data. Each region explained between 7 and 18% of the phenotypic variation. Several additional regions were identified in both maps as associated with brown rust at p ≤ 0.01 in one year and p ≤ 0.05 in the second year. These regions also explained between 5 and 11% of the phenotypic variation. To validate these markers and determine whether they would be useful in alternative germplasm, markers from each genomic region associated with PRR or brown rust were screened across a set of 154 elite sugarcane clones; PRR and brown rust ratings were available for 131 and 72 of the clones, respectively. For PRR, three of the 6 markers tested remained significantly associated (p ≤ 0.01) with resistance ratings in the elite clone set. For brown rust, only one of the seven markers tested remained significantly associated (p ≤ 0.01) with resistance in the elite clone set, with one other marker associated at p ≤ 0.05. These results suggest that these markers could be broadly effective in selecting for PRR and/or brown rust resistance in sugarcane breeding programs.     

Species composition and distribution of Coleosporium species on the needles of Pinus densiflora at a semi-natural vegetation succession site in central Japan

Coleosporium species cause pine needle rust. Most species have heteromacrocyclic life cycles, and 12 species use Pinus densiflora as aecial hosts. To understand the biology of rust fungi and develop better methods for controlling rust diseases, it is necessary to clarify that which Coleosporium species affect pine trees. However, Coleosporium on pine trees have rarely been identified at the species level because of their morphological similarities. We used polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) to clarify the species composition, abundance, and distribution of Coleosporium in a P. densiflora forest. We surveyed a site where several Coleosporium species might complete their life cycles. PCR-RFLP revealed four species on the pines: C. asterum, C. clematidis-apiifoliae, C. lycopodis, and C. phellodendri. Coleosporium phellodendri was distributed throughout the forest and was the most abundant. Aecia of C. phellodendri formed mainly on 2-y-old needles. The abundance and distribution of C. phellodendri appeared to be affected by the longer effective dispersal range of basidiospores and the existence of abundant inoculum sources. The age of leaves where C. phellodendri form aecia mainly was thought to be influenced by the characteristic life cycle, with aecial formation requiring 2?y after basidiospore infection.     

Phylogeny of an Albugo sp. infecting Barbarea vulgaris in Denmark and its frequency of symptom development in natural populations of two evolutionary divergent plant types

The oomycete Albugo candida has long been considered a broad spectrum generalist pathogen, but recent studies suggest that it is diverged into several more specialized species in addition to the generalist Albugo candida sensu stricto. Whereas these species cause the disease white blister rust in many crucifer plants, asymptomatic endophytic infections may be important in the epidemiology of others. One of the plant species attacked by Albugo sp. is the wild crucifer Barbarea vulgaris ssp. arcuata, which is diverged into two phytochemically and genetically different types with different geographical distributions in Europe. These were previously shown to differ strongly in propensity to develop white rust upon controlled infections in the greenhouse. Here, we analyse the phylogenetic relatedness of this local Albugo sp. field isolate to other species and lines of Albugo spp., including others collected on B. vulgaris. We further ask whether the difference in incidence of white rust between the two types of B. vulgaris are also expressed in natural populations.     

Seasonal aspects of life cycles in Calathus melanocephalus and C. micropterus (Coleoptera, Carabidae) in the northern taiga

The seasonal dynamics of the activity and the demographic structure of Calathus melanocephalus and C. micropterus populations were studied in the northern taiga of Arkhangelsk Province. The period of adult activity lasts from early June to mid-or late September with the maximum surface activity observed in the middle of summer. In C. melanocephalus, mature individuals are recorded from early June to early September, and in C. micropterus, during the entire season. The data on seasonal changes of the demographic structure of the population demonstrate that in the northern taiga, these species probably possess a biennial life cycle with summer reproduction. The geographical variability of the demographic structure of populations and of life cycles of two species of the genus Calathus was demonstrated. Northwards from the south, the period of activity decreases and the period of reproduction increases and shifts from autumn to the middle of summer. A hypothesis was formulated, according to which in the species studied the annual life cycle in the central part of the range is replaced by the biennial cycle in the north.     

Taxonomy of two host specialized Phakopsora populations on Meliosma in Japan

Phakopsora meliosmae, a macrocyclic autoecious rust fungus, is reported to occur on several Meliosma species widely distributed in Asia. Despite the apparent broad host range, a recent molecular phylogenetic study indicated that two rust populations on Meliosma myriantha and Meliosma tenuis respectively in Japan were biologically distinct. To clarify the biological and taxonomic relationships of these populations, cross inoculations and comparative morphological examinations were carried out. Cross inoculations using basidiospores and aeciospores confirmed the macrocyclic, autoecious nature of the life cycle in both rust populations and showed that the two populations were distinct in their host specificity. Furthermore, they were found to be distinct in the structure of the aecial peridium surface, the size and wall thickness of uredinial paraphyses, and the urediniospore size and shape. Consequently, the fungal population on M. tenuis is taxonomically separated from P. meliosmae originally proposed for the fungus on M. myriantha. A new name, Phakopsora orientalis, is proposed for the fungus on M. tenuis.     

Genetic implications of phylogeographical patterns in the conservation of the boreal wetland butterfly Colias palaeno (Pieridae)

The boreo‐montane wetland butterfly species Colias palaeno has a European distribution from the Alps to northern Fennoscandia. Within its European range, the species’ populations have shrunk dramatically in recent historical times. Therefore, detailed baseline knowledge of the genetic makeup of the species is pivotal in planning potential conservation strategies. We collected 523 individuals from 21 populations across the entire European range and analyzed nuclear (20 allozyme loci) and mitochondrial (600 bp of the cytochrome c oxidase subunit I gene) genetic markers. The markers revealed contrasting levels of genetic diversity and divergence: higher in allozymes and lower in mitochondrial sequences. Five main groups were identified by allozymes: Alps, two Czech groups, Baltic countries, Fennoscandia, and Poland. The haplotype mitochondrial network indicates a recent range expansion. The most parsimonious interpretation for our results is the existence of a continuous Würm glacial distribution in Central Europe, with secondary disjunction during the Last Glacial Maximum into a south‐western and a north‐eastern fragment and subsequent moderate differentiation. Both groups present signs of postglacial intermixing in the Czech Republic. However, even a complete extinction in this region would not considerably affect the species’ genetic basis, as long as the source populations in the Alps and in northern Europe, comprising the most relevant evolutionary units for conservation, are surviving.     

Genetic mapping of rust resistance genes in confection sunflower line HA-R6 and oilseed line RHA 397

Few widely effective resistance sources to sunflower rust, incited by Puccinia helianthi Schwein., have been identified in confection sunflower (Helianthus annuus L.). The USDA inbred line HA-R6 is one of the few confection sunflower lines resistant to rust. A previous allelism test indicated that rust resistance genes in HA-R6 and RHA 397, an oilseed-type restorer line, are either allelic or closely linked; however, neither have been characterized nor molecularly mapped. The objectives of this study are (1) to locate the rust resistance genes in HA-R6 and RHA 397 on a molecular map, (2) to develop closely linked molecular markers for rust resistance diagnostics, and (3) to determine the resistance spectrum of two lines when compared with other rust-resistant lines. Two populations of 140 F_2:3 families each from the crosses of HA 89, as susceptible parent, with HA-R6 and RHA 397 were inoculated with race 336 of P. helianthi in the greenhouse. The resistance genes (R-genes) in HA-R6 and RHA 397 were molecularly mapped to the lower end of linkage group 13, which encompasses a large R-gene cluster, and were designated as R _13a and R _13b, respectively. In the initial maps, SSR (simple sequence repeat) and InDel (insertion and deletion) markers revealed 2.8 and 8.2 cM flanking regions for R _13a and R _13b, respectively, linked with a common marker set of four co-segregating markers, ORS191, ORS316, ORS581, and ZVG61, in the distal side and one marker ORS464 in the proximal side. To identify new markers closer to the genes, sunflower RGC (resistance gene candidate) markers linked to the downy mildew R-gene Pl ₈ and located at the same region as R _13a and R _13b were selected to screen the two F₂ populations. The RGC markers RGC15/16 and a newly developed marker SUN14 designed from a BAC contig anchored by RGC251 further narrowed down the region flanking R _13a and R _13b to 1.1 and 0.1 cM, respectively. Both R _13a and R _13b are highly effective against all rust races tested so far. Our newly developed molecular markers will facilitate breeding efforts to pyramid the R ₁₃ genes with other rust R-genes and accelerate the development of rust-resistant sunflower hybrids in both confection and oilseed sunflowers.     

Introgression of stem rust resistance genes SrTA10187 and SrTA10171 from Aegilops tauschii to wheat

Aegilops tauschii, the diploid progenitor of the wheat D genome, is a readily accessible germplasm pool for wheat breeding as genes can be transferred to elite wheat cultivars through direct hybridization followed by backcrossing. Gene transfer and genetic mapping can be integrated by developing mapping populations during backcrossing. Using direct crossing, two genes for resistance to the African stem rust fungus race TTKSK (Ug99), were transferred from the Ae. tauschii accessions TA10187 and TA10171 to an elite hard winter wheat line, KS05HW14. BC₂ mapping populations were created concurrently with developing advanced backcross lines carrying rust resistance. Bulked segregant analysis on the BC₂ populations identified marker loci on 6DS and 7DS linked to stem rust resistance genes transferred from TA10187 and TA10171, respectively. Linkage maps were developed for both genes and closely linked markers reported in this study will be useful for selection and pyramiding with other Ug99-effective stem rust resistance genes. The Ae. tauschii-derived resistance genes were temporarily designated SrTA10187 and SrTA10171 and will serve as valuable resources for stem rust resistance breeding.     

Differentiation of Fusarium subglutinans f. sp. pini by Histone Gene Sequence Data

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.     

Genetic Variability and Structuring of Arctic Charr (Salvelinus alpinus) Populations in Northern Fennoscandia

Variation in presumably neutral genetic markers can inform us about evolvability, historical effective population sizes and phylogeographic history of contemporary populations. We studied genetic variability in 15 microsatellite loci in six native landlocked Arctic charr (Salvelinus alpinus) populations in northern Fennoscandia, where this species is considered near threatened. We discovered that all populations were genetically highly (mean F _ST ≈ 0.26) differentiated and isolated from each other. Evidence was found for historical, but not for recent population size bottlenecks. Estimates of contemporary effective population size (N _e) ranged from seven to 228 and were significantly correlated with those of historical N _e but not with lake size. A census size (N _C) was estimated to be approximately 300 individuals in a pond (0.14 ha), which exhibited the smallest N _e (i.e. N _e/N _C = 0.02). Genetic variability in this pond and a connected lake is severely reduced, and both genetic and empirical estimates of migration rates indicate a lack of gene flow between them. Hence, albeit currently thriving, some northern Fennoscandian populations appear to be vulnerable to further loss of genetic variability and are likely to have limited capacity to adapt if selection pressures change.     

Molecular markers linked to white rust resistance in mustard Brassica juncea

 White rust, caused by Albugo candida (Pers.) Kuntze, is an economically important disease of Brassica juncea (L.) Czern. and Coss mustard, particularly in India. The most efficient and cost-effective way of protecting mustard plants from white rust disease is through genetic resistance. The objective of this study was to identify RAPD markers for white rust resistance in an F₁-derived doubled-haploid (DH) population originating from a cross between white rust-susceptible and white rust-resistant breeding lines of B. juncea from the canola-quality B. juncea breeding project of the Agriculture and Agri-Food Canada-Saskatoon Research Centre. The DH population was used to screen for RAPD markers associated with white rust resistance/susceptibility using bulked segregant analysis. Two markers, WR2 and WR3, linked to white rust resistance, flanked the resistance locus Ac2 ₁ and were highly effective in identifying the presence or absence of the resistance gene in the DH population. These two markers were shown to be specific to the Russian source of white rust resistance utilized in this project. It is concluded that the availability of these RAPD markers will enhance the breeding for white rust resistance in B. juncea. Received: 17 December 1997 / Accepted: 7 April 1998     

Seasonal aspects of the life cycles of Carabus granulatus and C. glabratus (coleoptera, carabidae) in the northern taiga

The seasonal dynamics of the activity and the demographic structure of Carabus granulatus and C. glabratus populations was studied in the northern taiga of European Russia. The period of adult activity lasts for 90 days from early June to late August with the maximal activity observed in June. At the northern boundary of the range, the spring species Carabus granulatus retains its annual life cycle typical of this species in the other part of the range. At the same time, the reproductive period decreases, resulting in the synchronization of the development of specimens within the population. In the autumn species C. glabratus, a biennial life cycle with early summer reproduction is formed in the zone of broad-leaved forests of the northern taiga instead of the annual cycle. This species is characterized by the presence of two groups of individuals developing for two years each, but their development is shifted by a year. Such strategy results in the annual breeding of the species.     

Molecular and Quantitative Genetic Differentiation in Sitobion avenae Populations from Both Sides of the Qinling Mountains

Quantitative trait differences are often assumed to be correlated with molecular variation, but the relationship is not certain, and empirical evidence is still scarce. To address this issue, we sampled six populations of the cereal aphid Sitobion avenae from areas north and south of the Qinling Mountains, and characterized their molecular variation at seven microsatellite loci and quantitative variation at nine life-history traits. Our results demonstrated that southern populations had slightly longer developmental times of nymphs but much higher lifetime fecundity, compared to northern populations. Of the nine tested quantitative characters, eight differed significantly among populations within regions, as well as between northern and southern regions. Genetic differentiation in neutral markers was likely to have been caused by founder events and drift. Increased subdivision for quantitative characters was found in northern populations, but reduced in southern populations. This phenomenon was not found for molecular characters, suggesting the decoupling between molecular and quantitative variation. The pattern of relationships between F_ST and Q_ST indicated divergent selection and suggested that local adaptation play a role in the differentiation of life-history traits in tested S. avenae populations, particularly in those traits closely related to reproduction. The main role of natural selection over genetic drift was also supported by strong structural differences in G-matrices among S. avenae populations. However, cluster analyses did not result in two groups corresponding to northern and southern regions. Genetic differentiation between northern and southern populations in neutral markers was low, indicating considerable gene flow between them. The relationship between molecular and quantitative variation, as well as its implications for differentiation and evolution of S. avenae populations, was discussed.     

Studies on the rustMaravalia cryptostegiae,a potential biological control agent of rubber-vine weed (Cryptostegia grandiflora,Asclepiadaceae: Periplocoideae) in Australia,I: Life-cycle

Three spore types are described forMaravalia cryptostegiae: hemileioid urediniospores; thin-walled, hyaline, ellipsoidal, non-resting teliospores and ovoid to lacrimoid basidiospores. Field surveys in the Madagascan native range of rubber-vine failed to confirm the existence of spermogonia and morphologically distinct aecia within the life-cycle. Greenhouse inoculations with basidiospores were unsuccessful. Cytological studies revealed that rubber-vine rust has a similar nuclear cycle to that reported for coffee leaf rust,Hemileia vastatrix. The working hypothesis is proposed thatMaravalia cryptostegiae is a primitive, autoecious tropical forest rust with only a short or partially expanded life-cycle represented by two teliospore forms. The predominant, functional form is uredinioid with a novel nuclear cycle, in which there is a delayed meiotic division (the Kamat phenomenon). The non-dispersed form appears to be vestigial or non-functional since it germinates to produce a metabasidium with genetically variable and unstable basidiospores. The relationships and evolutionary significance of the generaMaravalia andHemileia are discussed.     

Molecular mapping of stripe rust resistance gene YrH921 in a wheat introgression line H921-11-1

Several novel stripe rust pathogen races emerging in the wheat-planting regions of China in recent years were virulent to a majority of the designated wheat seedling resistance genes. Therefore, it is of great significance to continuously select more new and valuable materials for enriching resistant sources diversity, pyramiding different excellent resistance genes and achieving durable resistance. In this study, a resistance gene, temporarily designated as YrH921, was identified from wheat–Psathyrostachys huashanica introgression line H921-11-1. Two hybrid populations, 160 F₂ plants and corresponding derived F_2:3 families, of the two parents about Mingxian169 as female and H921-11-1 as male, were used to evaluate stripe rust resistance in seedling stage and as a mapping population. At last, a genetic map which comprises of four simple sequence repeat (SSR) markers and six expressed sequence tag (EST) markers was constructed. YrH921 was located on the long arm of chromosome 5A. Two closely linked EST-STS markers (BF483937 and BF484913) were screened, and the genetic distance linked to YrH921 was 3.0 and 4.3 cM, respectively. There was great value in research and production if the two closest markers were effectively used for marker-assisted selection of YrH921 in breeding.     

Fine-mapping of the leaf rust Lr34 locus in Triticum aestivum (L.) and characterization of large germplasm collections support the ABC transporter as essential for gene function

Leaf rust resistance gene Lr34 is likely the most important leaf rust gene characterized to date. It has been characterized as an adult plant resistance gene and is known to enhance the resistance of other leaf rust resistance genes and to condition resistance to a number of other diseases. Located on chromosome 7D, this gene was identified to be one of six co-located genes of which, an ABC transporter was shown to be the only valid candidate. Ten new molecular markers were developed spanning the Lr34 locus, including six novel microsatellite markers (cam), one insertion site-based polymorphism marker (caISBP), two single nucleotide polymorphisms (caSNP), and one gene-specific marker (caIND). Using these new markers and others that were previously published, a comparative fine map of the locus was constructed from five segregating populations representing 1,742 lines. Identification of a susceptible line with a recombination in the 4.9 kb interval between caSNP4 located in the ABC transporter gene and cam8 located just upstream of this gene provided further evidence to support the identity of the ABC transporter as Lr34 by ruling out four of the adjacent genes. Originally, three mutations forming two haplotypes had been described for the ABC transporter gene. A third combination of the three mutations and an additional rare mutation in exon 22 were subsequently described. We identified an additional novel mutation in exon 10 that would cause a frameshift and is likely non-functional. This mutation was only found in Lr34? lines and constituted a novel molecular haplotype. Characterization of two germplasm collections of 700 Triticum aestivum lines permitted us to gain an understanding of the frequency of the ABC haplotypes characterized to date and their distribution in germplasm from and around the world. In addition to the four haplotypes previously described, a fifth haplotype was found in two of the 700 lines from the germplasm collections. These lines displayed the deletion in indel 11 characteristic of Lr34+ lines, but are likely susceptible to leaf rust. Mapping and haplotyping data suggest that of all the markers described herein, marker caIND11 is the best diagnostic marker for marker-assisted selection of Lr34 because it is co-dominant, robust and with the exception of 2/700 lines, it is highly diagnostic. Other markers are also described to provide alternatives for laboratories with different technologies.