Assessing new SSR markers for utility and informativeness in genetic studies of brown rust fungi on wheat,triticale, and rye

The aim of the present study was to validate new simple-sequence repeat (SSR) markers and use them to assess genetic variability among 24 isolates of Puccinia triticina collected from wheat (Pt-wheat) and triticale (Pt-triticale), and 15 isolates of P. recondita f. sp. secalis (Prs) collected from rye. The Pt and Prs isolates were tested for virulence on a set of 35 Thatcher wheat near-isogenic lines, eight rye lines with known resistance genes, and 53 triticale cultivars with uncharacterized leaf rust resistance. Molecular genotypes were determined using a newly developed set of 34 SSR microsatellite primer pairs. All SSR markers tested on Pt isolates successfully amplified fragments of appropriate size. When tested on the Prs isolates, 21 out of the 34 Pt SSRs amplified expected fragments. Sixteen of these 21 SSRs were polymorphic, providing for the first time microsatellite markers to study genetic variation in Prs. Based on virulence data, variation among Prs isolates was low, probably due to the small number of rye differential lines available. Much higher variation for virulence was observed within the collection of Pt isolates from wheat and triticale, and two separate groups were established with mixed host origin. Substantial genetic variation was detected among the isolates studied with the SSR markers, assuming two different models of SSR evolution (infinite alleles model and stepwise mutation model). The newly developed set of SSR markers proved their effectiveness in detecting genetic variation and should be useful in further population genetics investigations of the two pathogens.     

Russian populations of Puccinia triticina in distant regions are not differentiated for virulence and molecular genotype

The objective of this study was to determine whether genetically distinct groups of Puccinia triticina are present in four regions of the Russian Federation. Collections of P. triticina were obtained from the Central, North Caucasus, Volga and West Siberia regions from 2006 to 2010. Ninety‐nine single uredinial isolates were tested for virulence phenotype with 20 Thatcher near‐isogenic lines of wheat. Forty‐one virulence phenotypes were found in the four regions, with eight in common between the widely separated Central and West Siberia regions. A total of 72 isolates were tested for molecular genotype with 23 simple sequence repeat (SSR) primer pairs, and 66 isolates were used for further analysis after clone correction for virulence and molecular genotype. Analysis of variation showed no overall differentiation of SSR genotypes or virulence phenotypes based on region of origin. Linkage disequilibria for SSR genotypes were high across the entire population. The regional populations had higher than expected levels of allelic heterozygosity that indicated clonal reproduction. Based on cluster analysis of SSR genotypes there were two groups of P. triticina isolates that were widely distributed across Russia. The two SSR groups also differed significantly for virulence. Puccinia triticina may be dispersed from a common source of inoculum in the European or Caucasus regions of Russia. The Russian P. triticina populations were highly differentiated for SSR genotype from populations in Tajikistan, Kyrgyzstan, Uzbekistan, Armenia, Georgia and Azerbaijan and more similar to populations from southern Kazakhstan and northern Kazakhstan.     

Genetic differentiation of the wheat leaf rust fungus Puccinia triticina in Europe

The objective of this study was to determine whether genetically differentiated groups of Puccinia triticina are present in Europe. In total, 133 isolates of P. triticina collected from western Europe, central Europe and Turkey were tested for virulence on 20 lines of wheat with single leaf rust resistance genes, and for molecular genotypes with 23 simple sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotype within countries, 121 isolates were retained for further analysis. Isolates were grouped based on SSR genotypes using a Bayesian approach and a genetic distance method. Both methods optimally placed the isolates into eight European (EU) groups of P. triticina SSR genotypes. Seven of the groups had virulence characteristics of isolates collected from common hexaploid wheat, and one of the groups had virulence characteristics of isolates from tetraploid durum wheat. There was a significant correlation between the SSR genotypes and virulence phenotypes of the isolates. All EU groups had observed values of heterozygosity greater than expected and significant fixation values, which indicated the clonal reproduction of urediniospores in the overall population. Linkage disequilibria for SSR genotypes were high across the entire population and within countries. The overall values of R_ST and F_ST were lower when isolates were grouped by country, which indicated the migration of isolates within Europe. The European population of P. triticina had higher levels of genetic differentiation compared to other continental populations.     

SSR-based genetic linkage analysis of resistance to crown rust (Puccinia coronata f. sp. lolii) in perennial ryegrass (Lolium perenne)

Crown rust (caused by Puccinia coronata f. sp. lolii) is a serious foliar disease of the pasture and turfgrass perennial ryegrass (Lolium perenne). Previous genetic studies have detected both qualitative and quantitative resistance mechanisms, and interpretation of the genetic system is complicated by variation within the sexually reproducing pathogen. Resistant and susceptible parental genotypes of ryegrass were identified using a composite urediniospore population collected from three geographically distinct locations. A two-way pseudo-testcross mapping population was obtained as the F₁ progeny of the pair-cross between ryegrass parental genotypes Vedette₆ and Victorian₉. Both parents showed intermediate resistance against a pathogen population collected in a single geographical zone (Hamilton, Victoria), but in the F₁ population, significant variation for a range of resistance-associated characters was detected. Statistical analysis of phenotypic data suggested a major gene effect, hence bulked segregant analysis with map-assigned simple sequence repeat (SSR) markers was used to scan the genome. A marker showing strong association with resistance was assigned to linkage group (LG) 2 of perennial ryegrass. Analysis of 11 LG2 SSR markers defined an interval between loci xlpssrh03f03 and xlpssrk02e02 as containing the gene or genes (LpPc1) conferring crown rust resistance. Resistance gene determinants were inherited from both parents, with up to 80% of the total phenotypic variation explained by markers segregating from Vedette₆ and up to 26% of the variation explained by markers segregating from Victorian₉. The two contributions together resulted in an additive increase in effect, with fully resistant individuals requiring determinants from both parents. A conserved syntenic relationship was observed with linkage group B of Avena strigosa, which is the location of a cluster of resistance genes to the oat form of crown rust. The implications of this study for marker-assisted selection of disease resistance in perennial ryegrass are discussed.     

小麦叶锈菌越夏与气象因子相关性初步分析

小麦叶锈病是一种严重威胁小麦安全生产、依靠气流传播的真菌病害,近年来发生呈逐年加重趋势。为明确气象因子对小麦叶锈菌越夏的影响,本研究通过对全国698个气象站点7月-8月最热10 d的日均温和平均日最高气温进行回归分析,对7月-8月0 cm平均地温、平均风速、平均降水量、平均日照时数和平均相对湿度进行空间插值,提取了93个小麦叶锈菌越夏调查点的气象数据,再与调查点小麦叶锈菌能否越夏进行相关性分析,结果显示小麦叶锈菌越夏与7月-8月最热10 d日均温和最热10 d平均日最高气温之间存在极显著相关性(P<0.01),与其他气象因子相关性不显著(P> 0.05),结果为小麦叶锈病的越夏区划奠定了基础。     

Limited genetic diversity across pathogen populations responsible for the global emergence of boxwood blight identified using SSRs

Boxwood blight is an emerging disease of ornamental as well as native boxwood. The disease became widely established in Europe at the beginning of the 21st century, prior to its more recent discovery in North America and Asia. Two sister-species of fungi cause the disease, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Prior efforts to quantify intraspecific genetic polymorphisms of Cps and Che have yielded little information, limiting the ability to understand the evolution and migration of these pathogens. This study describes the development and implementation of simple sequence repeat (SSR) markers to analyse genetic diversity from a global collection of Cps and Che isolates, representing major blight outbreaks since the disease was first identified in the UK in the late 1990s. Analysis of the Cps CB002 genome sequence identified 180 single copy SSR loci using stringent search criteria, 11 of which were polymorphic and used to screen a global sample of 306 isolates. Fourteen multilocus genotypes of Cps and two multilocus genotypes of Che were identified. Twelve of the 14 Cps genotypes differed from each other by a single allele. The most common Cps genotype was found on all continents where boxwood blight is confirmed. Based on measurement of linkage disequilibrium, Cps showed no evidence of sexual recombination. Further in silico analysis identified 1594 SSRs using relaxed SSR definition criteria. Comparison of these SSR-containing loci with Cps and Che genome sequences representing three different genotypes demonstrated that single nucleotide polymorphisms might serve as informative genetic markers for future studies.     

Genetic diversity and population genetic differentiation in the endangered annual weed,Bidens cernua (Compositae), and two common congeners in Japan

In this study, we examined the genetic diversity in three populations of the critically endangered annual, Bidens cernua, in Japan by using inter‐simple sequence repeat markers and compared our data with those from two common congeners: Bidens radiata var. pinnatifida and Bidens tripartita. In contrast to our expectations, the degree of genetic diversity at the species level was higher in B. cernua than in B. radiata var. pinnatifida or B. tripartita. At the population level, the degree of genetic diversity was highest in B. cernua. These results may be ascribed to the mating system and method of seedbank formation in B. cernua. An analysis of molecular variance revealed relatively high genetic differentiation among the populations of all three species. We concluded that the distribution width could not be an index of genetic variability in Bidens examined in this study.     

Low diversity and fast evolution in the population of Puccinia triticina causing durum wheat leaf rust in France from 1999 to 2009, as revealed by an adapted differential set

No internationally agreed differential set is available for characterization of virulences in populations of Puccinia triticina causing wheat leaf rust on durum wheat. In a first step, 73 potentially differential host genotypes were tested with 96 durum leaf rust isolates collected in France. A differential set, adapted to the local epidemiological context and useful for comparison with international studies was selected, including French commercial cultivars, Thatcher lines with Lr genes, and international cultivars. In the second step, a sample of 310 isolates collected in France from 1999 to 2009 was characterized on this set. Diversity was very low, as only five pathotypes were distinguished. Genotyping of a subset of 76 isolates according to 20 SSR markers confirmed this low diversity, with 73 isolates belonging to a single dominant genotype. Population was strongly shaped by cultivars, and the findings explain the successive breakdown of resistance sources deployed in French durum wheat cultivars. The gene Lr14a, suggested to be an efficient source of resistance in several European and American countries, was overcome by pathotypes frequent in France since 2000. Postulation of resistance genes in the commercial cultivars led to a proposed simplified version of the differential set. This study, providing new information about leaf rust resistance genes present in the French durum wheat germplasm, highlights the need to diversify sources of resistance to P. triticina in this germplasm. The results are also discussed in terms of relatedness and intercontinental migration of P. triticina on durum wheat.     

西南地区稻瘟病菌群体遗传多样性分析

为明确西南地区稻瘟病菌Magnaporthe grisea(Hebert)Barr群体遗传结构及其多样性水平,选用13对SSR引物对来自18个县(市)的221个稻瘟病菌单孢菌株进行PCR扩增,利用最长距离法和生物学软件进行聚类分析和群体遗传多样性分析。结果显示,13对SSR引物均能扩增出一条大小相同且清晰的条带,多态位点百分率高达100%。221个菌株在0.16相异水平上可划分为13个遗传宗谱,宗谱SCL01含205个菌株,占总菌株数的92.76%,为优势宗谱;宗谱SCL02~SCL013为劣势宗谱,差异极大。在群体水平上,菌源丰富的8个区域稻瘟病菌群体的Nei’s基因多样性指数为0.2133,Shannon信息指数为0.3588,具有丰富的遗传多样性,且群体间差异较大;这8个种群基于UPGMA法大都聚为一类,种群遗传谱系与地理区域分布呈一定相关性,群体遗传多样性均值为0.2518,存在一定的遗传分化,且群体内多样性大于群体间,总遗传变异的59.37%存在于群体内。总体上,西南地区稻瘟病菌群体结构既有明显的优势宗谱,又存在许多复杂多变的特异性小宗谱,具有丰富的遗传多样性,与地理分布关系较为密切。     

Diversity of populations of Phytophthora infestans in relation to patterns of potato crop management in Latvia and Lithuania

Potato crop losses can be substantial when conditions for late blight (Phytophthora infestans) development and spread are favourable. In this study, drivers of differences between the P. infestans population structures in Latvia and Lithuania, two neighbouring countries with similar potato-growing traditions, were investigated. Genotypes of P. infestans and population genetic diversity were analysed using a 12-plex simple sequence repeat (SSR) marker assay. High genetic diversity was demonstrated in both populations, with population diversity being higher in Latvia. It would appear that local populations established from soilborne oospores early in the season are well adapted to the conditions in the region. However, somewhat greater spread and survival of local clones was detected in Lithuania, suggesting that potato cropping there is more vulnerable to clonal invasion than in Latvia. For effective disease management, current strategies should be adjusted according to the specific pathogen populations in the region, considering the reproduction and survival of the pathogen. Potato growers should implement late blight preventive measures such as longer field rotation to prevent oospore infections, especially in Latvia, and should use more disease resistant cultivars and high-quality seed potatoes.     

玉米大斑病菌ISSR反应体系的优化和遗传多样性分析

以玉米大斑病菌基因组DNA为模板,采用单因素水平优化的方法对DNA聚合酶的来源及浓度、引物浓度、dNTPs浓度、DNA模板浓度、Tm(退火温度)、PCR反应循环数等重要参数进行摸索和优化,建立了玉米大斑病菌ISSR-PCR优化反应体系,并从40条ISSR引物中筛选出9条多态性较好的ISSR引物。对来自河北、河南、辽宁等玉米主产区的44个菌株进行ISSR分析表明,ISSR标记在我国玉米大斑病菌中存在较高的多态性,多态性条带占40.3%。聚类分析显示,在阈值为0.8时菌株被分为7个类群。对ISSR揭示的玉米大斑病菌的遗传多样性与菌株交配型、地理来源之间的关系进行分析,结果显示菌株的遗传多样性与交配型间的关系密切,而与其地理来源无明显相关性。     

Population structure and mating system of Ascochyta rabiei in Tunisia: evidence for the recent introduction of mating type 2

The population structure of Ascochyta rabiei (teleomorph: Didymella rabiei ) in Tunisia was estimated among five populations sampled from the main chickpea growing regions using simple sequence repeat markers (SSR) and a mating type ( MAT ) marker. Mating type 2 isolates ( MAT1-2 ) had reduced genetic and genotypic diversity relative to mating type 1 isolates ( MAT1-1 ). This result, coupled with previous observations of lower overall frequency and restricted geographical distribution of MAT1-2 in Tunisia, and recent (2001) observation of the sexual stage, support the hypothesis of a recent introduction of MAT1-2 . Despite the presence of both mating types in Nabeul, Kef and Jendouba, the hypothesis of random mating was rejected in these locations with multilocus gametic disequilibrium tests. Highly significant genetic differentiation ( θ  = 0·32, G _ST = 0·28, P  < 0·001) was detected among populations and genetic distance and cluster analyses based on pooled allele frequencies revealed that populations from Nabeul and Kef were distinct from those in Beja, Bizerte and Jendouba. More than 70% of total gene diversity ( H _T = 0·55) detected was attributable to variation within populations compared to 28% among populations. This result, coupled with the occurrence of private alleles in each population, suggests that gene flow is currently limited among populations, even those separated by short geographic distances. The presence of two main genetic clusters was confirmed using Bayesian model-based population structure analyses of multilocus genotypes (MLGs) without regard to geographic origin of samples. The presence of MAT1-2 isolates in both clusters suggests at least two independent introductions of MAT1-2 into Tunisia that are likely to be the result of importation and planting of infected chickpea seeds.     

Genetic diversity of herbicide-resistant and -susceptible Avena fatua populations in North Dakota and Minnesota

Genetic diversity within and among 20 herbicide-resistant (HR) and 16 herbicide-susceptible (HS) Avena fatua multi-field populations was determined using 82 polymorphic loci resulting from two intersimple sequence repeat (ISSR) primers and one long-primer random amplified polymorphic DNA (LP-RAPD) primer. Collections from the Red River Valley of North Dakota and Minnesota, sampled in 1964 and 2000, represented A. fatua populations before and after intensive exposure to herbicides. A 1995 collection from south-west North Dakota represented A. fatua exposed to low herbicide selection. Despite differences in years of herbicide exposure among collections, both HR and HS populations from every collection maintained nearly similar levels of ISSR and RAPD diversity. Genetic differentiation among populations (G_ST) varied from 11% to 13% among HR populations and from 9% to 16% among HS populations, indicating that 84–91% of total variation remained within HS or within HR populations. Minimal difference in gene diversity between HR and HS is consistent with multiple origins of resistance, where HR A. fatua most likely evolved from diverse founding individuals.     

Genetic diversity within and between sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides in Japan

To reveal the effects of herbicide selection on genetic diversity in the outcrossing weed species Schoenoplectus juncoides, six sulfonylurea‐resistant (SU‐R) and eight sulfonylurea‐susceptible (SU‐S) populations were analysed using 40 polymorphic inter‐simple sequence repeat loci. The plants were collected from three widely separated regions: the Tohoku, Kanto and Kyushu districts of Japan. Genetic diversity values (Nei's gene diversity, h) within each SU‐S population ranged from h = 0.125 to h = 0.235. The average genetic diversity within the SU‐S populations was H_S = 0.161, and the total genetic diversity was H_T = 0.271. Although the H_S of the SU‐R populations (0.051) was lower than that of the SU‐S populations, the H_T of the SU‐R populations (0.202) was comparable with that of the SU‐S populations. Most of the genetic variation was found within the region for both the SU‐S and SU‐R populations (88% of the genetic variation respectively). Two of the SU‐R populations showed relatively high genetic diversity (h = 0.117 and 0.161), which were comparable with those of the SU‐S populations. In contrast, the genetic diversity within four SU‐R populations was much lower (from h = 0 to 0.018) than in the SU‐S populations. The results suggest that selection by sulfonylurea herbicides has decreased genetic diversity within some SU‐R populations of S. juncoides. The different level of genetic diversity in the SU‐R populations is most likely due to different levels of inbreeding in the populations.     

西双版纳自然保护区球孢白僵菌的遗传多样性

为了解西双版纳自然保护区球孢白僵菌Beauveria bassiana的遗传多样性,采用SSR分子标记技术对西双版纳自然保护区的240株球孢白僵菌的遗传结构、基因流、菌株分化以及遗传变异相关性进行了测定和分析。结果表明,9条引物扩增240株球孢白僵菌菌株多态位点数为77个,多态位点比率为97.40%。采集的240株球孢白僵菌的总体等位基因数、有效等位基因数、Nei’s基因多样性指数和Shannon信息多样性指数分别为1.97、1.31、0.18和0.29。从寄主居群来看,鞘翅目昆虫球孢白僵菌群体遗传多样性最高,螳螂目最低,其Nei’s基因多样性指数和Shannon信息多样性指数分别为0.20和0.30、0和0;总遗传变异的29.27%存在于寄主居群间。从生境水平来看,五十五生境中球孢白僵菌群体遗传多样性最高,丫口寨生境中最低,其Nei’s基因多样性指数和Shannon信息多样性指数最高分别为0.19和0.31、0.06和0.09;总遗传变异的14.92%存在于生境居群间。从季节水平来看,球孢白僵菌秋季居群的遗传多样性最高,夏季最低,其Nei’s基因多样性指数和Shannon信息多样性指数分别为0.20和0.32、0.17和0.26;总遗传变异的3.45%存在于季节居群间。表明西双版纳自然保护区球孢白僵菌在寄主昆虫、生境分布和季节间存在明显的遗传多样性,与寄主昆虫类群的关系密切。     

Discriminating microsatellites from Macrophomina phaseolina and their potential association to biological functions

One hundred and eighty‐two microsatellites or simple‐sequence‐repeat (SSR) markers for Macrophomina phaseolina were developed. These were tested on 24 isolates of M. phaseolina obtained from seven plant species, and the genetic variation of isolates was studied in relation to potential biological processes that could be affected in this fungus. A total of 120 SSR markers were polymorphic, amplifying >90% of the 24 isolates tested. Thirty percent of the markers showed multiple alleles on individual samples. A large number of markers showed unique alleles in isolates collected from pumpkin and snap bean. DNA sequences corresponding to 43 markers had significant hits on blast x and/or blast2go , and the polymorphism of 36 of those markers showed specific allele patterns for one or more plant host origin of the isolates. Additional tests on growth rate and copper resistance of the isolates identified markers that could be related to those traits. In addition, 27 markers were monomorphic and amplified all 24 isolates. Whereas polymorphic markers can be used for population genetics studies of M. phaseolina, the group of 27 monomorphic markers could help in the fast identification of this species in clinical specimens. The SSR markers developed here will enrich the limited molecular marker resource in M. phaseolina and could be used as the basis for more in‐depth studies of the host‐pathogen interactions of M. phaseolina.     

Clonality and long-distance migration of Puccinia striiformis f.sp. tritici in north-west Europe

The biotrophic fungus Puccinia striiformis f.sp. tritici , a basidiomycete that causes yellow rust on wheat, is spread by wind-dispersed spores. Analysis of amplified fragment length polymorphism (AFLP) variation showed that the fungus frequently migrates between the UK, Germany, France and Denmark. There is no biological evidence for sexual or parasexual reproduction under natural conditions, and this was supported by the lack of recombination, as revealed by AFLP, over the time and area represented by the samples in this study. A phylogeographic analysis revealed that there was effectively a single, clonal population in the four countries, up to 1700 km apart, consistent with a 'continent-island' model in which Denmark is the recipient of migrants from other countries. In five cases, specific pathogen clones were dispersed between the UK and Denmark, and on at least two recent occasions clones were also spread from the UK to Germany and France, causing outbreaks of yellow rust on wheat cultivars that were previously resistant to the disease in these countries. The agronomic consequences of migration were enhanced because of the limited genetic diversity for yellow rust resistance in wheat cultivars in the area. These results demonstrate that long-distance migration of pathogen clones, coupled with low diversity in the host species, may cause previously useful resistance genes to become ineffective for disease control on a continental scale.     

Genetic diversity of the wheat yellow rust population in Pakistan and its relationship with host resistance

Yellow rust, caused by Puccinia striiformis f.sp. tritici (PST), is an important disease that threatens wheat production in Pakistan. This study was designed to explore the virulence and simple sequence repeat (SSR) diversity of the Pakistani PST population and the ongoing selective pressures of widely grown wheat cultivars. Analyses of 49 isolates sampled from the North‐West Frontier Province of Pakistan led to the identification of 12 distinct pathotypes. The virulence frequencies of v2 (virulent against Yr2), v6, v7, v9, vSU and v27 ranged from 63% to 100%. Virulences v3, v4, v17 and vSD were uncommon, whilst v5, v10, v15, v24, v32 and vSp were not detected. The pathotypes thus described were then classified into 27 distinct genotypes. Bayesian structure analysis clustered these genotypes into five groups (in addition to one hybrid isolate) which represent three distinct lineages of the SSR‐based phylogenetic tree. Of the studied isolates, 80%, represented by three predominant pathotypes (P1–P3), belonged to the same characteristic Pakistani lineage, whilst the other isolates were close to either a Mediterranean lineage or a Northern European lineage. Genetic recombination was detected within P2 isolates. Resistance genes postulated in 40 Pakistani wheat cultivars indicated the high frequency of Yr2, Yr6, Yr7, Yr9, Yr27 and YrSU. Only 11 cultivars were found to be resistant to P1–P3. Migration and varietal diversity factors might contribute to maintaining the currently high genetic diversity in Pakistani PST, and have serious regional implications for wheat improvement programmes.