Development of microsatellite markers and construction of genetic map in rice blast pathogen Magnaporthe grisea

Magnaporthe grisea is the most destructive fungal pathogen of rice and a model organism for studying plant-pathogen interaction. Molecular markers and genetic maps are useful tools for genetic studies. In this study, based on the released genome sequence data of M. grisea, we investigated 446 simple sequence repeat (SSR) loci and developed 313 SSR markers, which showed polymorphisms among nine isolates from rice (including a laboratory strain 2539). The number of alleles of each marker ranged 2-9 with an average of 3.3. The polymorphic information content (PIC) of each marker ranged 0.20-0.89 with an average of 0.53. Using a population derived from a cross between isolates Guy11 and 2539, we constructed a genetic map of M. grisea consisting of 176 SSR markers. The map covers a total length of 1247 cM, equivalent to a physical length of about 35.0 Mb or 93% of the genome, with an average distance of 7.1cM between adjacent markers. A web-based database of the SSR markers and the genetic map was established (http://ibi.zju.edu.cn/pgl/MGM/index.html).     

Development of near-isogenic Japonica rice lines with enhanced resistance to Magnaporthe grisea

Thirteen near-isogenic lines (NILs) of japonica rice were developed via a backcross method using the recurrent parent Chucheong, which is of good eating quality but is susceptible to Magnaporthe grisea, and three blast resistant japonica donors, Seolak, Daeseong and Bongkwang. The agro-morphological traits of these NILs, such as heading date, culm length, and panicle length, were similar to those of Chucheong. In a genome-wide scan using 158 SSR markers, chromosome segments of Chucheong were identified in most polymorphic regions of the 13 NIL plants, and only a few chromosome segments were found to have been substituted by donor alleles. The genetic similarities of the 13 NILs to the recurrent parent Chucheong averaged 0.961, with a range of 0.932-0.984. Analysis of 13 major blast resistance (R) genes in these lines using specific DNA markers showed that each NIL appeared to contain some combination of the four R genes, Pib, Pii, Pik-m and Pita-2, with the first three genes being present in each line. Screening of nine M. grisea isolates revealed that one NIL M7 was resistant to all nine isolates; the remaining NILs were each resistant to between three and seven isolates, except for NIL M106, which was resistant to only two isolates. In a blast nursery experiment, all the NILs proved to be more resistant than Chucheong. These newly developed NILs have potential as commercial rice varieties because of their increased resistance to M. grisea combined with the desirable agronomic traits of Chucheong. They also provide material for studying the genetic basis of blast resistance.     

The development of simple sequence repeat markers for Magnaporthe grisea and their integration into an established genetic linkage map

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.     

Population structure analysis and association mapping of bacterial blight resistance in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a serious disease in rice production worldwide. To understand the genetic diversity of bacterial blight resistance a population consisting of 175 indica accessions from nine countries was collected and detected their association between SSR (Simple Sequence Repeat) markers and resistance to six bacterial races. The resistance phenotypes of various rice accessions were evaluated through artificial inoculation under controlled conditions in 2013 and 2014. Association analysis showed that 17 SSR markers were significantly associated with resistance to four bacterial races and the phenotypic variations explained (PVE) ranged from 7.43 to 15.05%. Among the 17 associated SSR markers, two SSR markers located in previously reported genes regions, and 15 SSR markers were newly identified in this study. These results validated a new approach to map resistance genes of rice to bacterial blight. These markers could be used for marker-assisted selection (MAS) in rice bacterial blight resistance breeding programs.     

Simple sequence repeat markers for species in the Fusarium oxysporum complex

We describe nine simple sequence repeat (SSR) markers developed for studying Fusarium oxysporum. Allelic diversity at the nine loci ranged from 0.003 to 0.895, with a total of 71 alleles among 64 isolates. These markers will facilitate studies on relationships amongst isolates of F. oxysporum.     

Genetic analysis and molecular mapping of the avirulence gene PRE1, a gene for host-species specificity in the blast fungus Magnaporthe grisea.

We analyzed host-species specificity of Magnaporthe grisea on rice using 110 F1 progeny derived from a cross between the Oryza isolate CH87 (pathogenic to rice) and the Digitaria isolate 6023 (pathogenic to crabgrass). To elucidate the genetic mechanisms controlling species specificity in M. grisea, we performed a genetic analysis of species-specific avirulence on this rice population. Avirulent and virulent progeny segregated in a 1:1 ratio on the 2 rice cultivars 'Lijiangxintuanheigu' (LTH) and 'Shin2', suggesting that a single locus, designated PRE1, was involved in the specificity. In a combination between 'Kusabue' and 'Tsuyuake', the segregation of the 4 possible phenotypes of F1 progeny was significantly different from the expected 3:1:3:1 and instead fit a ratio of 2:0:1:1. This indicated that 2 loci, PRE1 and AVR2, were involved in specific parasitism on rice. These results suggest that the species specificity of M. grisea on rice is governed by species-dependent genetic mechanisms that are similar to the gene-for-gene interactions controlling cultivar specificity. Pathogenicity tests with various plant species revealed that the Digitaria isolate 6023 was exclusively parasitic on crabgrass. Genetic linkage analysis showed that PRE1 was mapped on chromosome 3 with respect to RAPD and SSR markers. RAPD marker S361 was linked to the avirulence gene at a distance of ~6.4 cM. Two SSR markers, m677-678 and m77-78, were linked to the PRE1 gene on M. grisea chromosome 3 at distances of 5.9 and 7.1 cM, respectively. Our results will facilitate positional cloning and functional studies of this gene.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

Genetic variation assessment of acid lime accessions collected from south of Iran using SSR and ISSR molecular markers

Iran has a long history of acid lime cultivation and propagation. In this study, genetic variation in 28 acid lime accessions from five regions of south of Iran, and their relatedness with other 19 citrus cultivars were analyzed using Simple Sequence Repeat (SSR) and Inter-Simple Sequence Repeat (ISSR) molecular markers. Nine primers for SSR and nine ISSR primers were used for allele scoring. In total, 49 SSR and 131 ISSR polymorphic alleles were detected. Cluster analysis of SSR and ISSR data showed that most of the acid lime accessions (19 genotypes) have hybrid origin and genetically distance with nucellar of Mexican lime (9 genotypes). As nucellar of Mexican lime are susceptible to phytoplasma, these acid lime genotypes can be used to evaluate their tolerance against biotic constricts like lime “witches’ broom disease”.     

A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea

Magnaporthe oryzae is described as a new species distinct from M. grisea. Gene trees were inferred for Magnaporthe species using portions of three genes: actin, beta-tubulin, and calmodulin. These gene trees were found to be concordant and distinguished two distinct clades within M. grisea. One clade is associated with the grass genus Digitaria and is therefore nomenclaturally tied to M. grisea. The other clade is associated with Oryza sativa and other cultivated grasses and is described as a new species, M. oryzae. While no morphological characters as yet distinguish them, M. oryzae is distinguished from M. grisea by several base substitutions in each of three loci as well as results from laboratory matings; M.oryzae and M. grisea are not interfertile. Given that M. oryzae is the scientifically correct name for isolates associated with rice blast and grey leaf spot, continued use of M. grisea for such isolates would require formal nomenclatural conservation.     

Predicting polymorphic EST‐SSRs in silico

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST‐SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co‐segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed.     

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.     

温室条件下条形柄锈菌体细胞重组的分子确证

为了获得温室条件下条形柄锈菌发生体细胞重组而导致毒性变异的直接证据,本研究选取7个美国条形柄锈菌小麦专化型菌系和2个美国条形柄锈菌大麦专化型菌系按照夏孢子颜色和专化型与毒性差异组成9对菌系组合,对于室内混合接种产生的子代菌系用具有不同抗性的小麦或大麦品种进行筛选,采用毒性分析及SSR分子标记技术对条形柄锈菌体细胞重组现象进行了研究。对获取的413个单孢子代菌系进行的毒性分析结果显示,有84个单孢子代菌系的毒性谱表现与亲本菌系不同,初步证明体细胞重组过程的存在。SSR标记分析结果显示,11对SSR引物中有6对引物在5对菌系组合的28个毒性谱不同的单孢子代菌系中,检测发现3个单孢菌系的扩增条带与其亲本菌系不同,且表现为亲本菌系扩增条带的重组,为体细胞重组菌系。这一结果从分子水平上证明了条形柄锈菌在室内接种条件下可以通过体细胞重组产生新小种而导致毒性变异。     

Genetic and Physical Mapping of Telomeres in the Rice Blast Fungus, Magnaporthe Grisea

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe, (TTAGGG)(3). Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set was used to minimize mapping errors. Twelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were ~1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was ~530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.     

鮸鱼EST 序列中微卫星标记的初步筛选及特征分析

为大规模发掘和利用Ⅰ型微卫星标记, 通过建立全长均一化cDNA 文库和随机序列测定, 对鱼鮸转录基因组进行较大规模的微卫星序列特征分析和初步筛选。研究从4609 条高质量ESTs 中筛选到382 条微卫星序列, 筛出率为8.29%, 平均每318 bp 的ESTs 中就有一段不小于14 bp 的微卫星序列。筛选到的微卫星全部在低拷贝区间, 且重复类型丰富, 其中, 二核苷酸和三核苷酸重复类型出现频率较高, 分别占微卫星总序列的37.43%和32.98%。这两种类型的微卫星序列占到所有微卫星序列数目的70.41%, 而每种重复类型中的优势拷贝类型又存在差异, 两核苷酸微卫星中AC 型含量最高, 达到75.4%; 三核苷酸重复的优势重复类型是A、G 组合的基元重复类型(AAG 和AGG), 占三核苷酸类型的44.75%。根据设计的45 对引物的多态检测, 在可以扩增的33 对引物中筛选到9 个多态位点, 多态位点的比例达到20%。这表明黑鮸EST-SSR 中的多态位点比较丰富, 适于进行大规模EST-SSR 多态标记的筛选。以上结果为近缘物种间的微卫星分布频率和丰度的比较、鮸鱼及近缘物种可用Ⅰ型微卫星标记开发等工作提供基础和分析平台。       

Development and genetic mapping of microsatellite markers from whole genome shotgun sequences in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The availability of whole genome shotgun sequences (WGSs) in Brassica oleracea provides an unprecedented opportunity for development of microsatellite or simple sequence repeat (SSR) markers for genome analysis and genetic improvement in Brassica species. In this study, a total of 56,465 non-redundant SSRs were identified from the WGSs in B. oleracea, with dinucleotide repeats being the most abundant, followed by tri-, tetra- and pentanucleotide repeats. From these, 1,398 new SSR markers (designated as BoGMS) with repeat length ≥25 bp were developed and used to survey polymorphisms with a panel of six rapeseed varieties, which is the largest number of SSR markers developed for the C genome in a single study. Of these SSR markers, 752 (69.5%) showed polymorphism among the six varieties. Of these, 266 markers that showed clear scorable polymorphisms between B. napus varieties No. 2127 and ZY821 were integrated into an existing B. napus genetic linkage map. These new markers are preferentially distributed on the linkage groups in the C genome, and significantly increased the number of SSR markers in the C genome. These SSR markers will be very useful for gene mapping and marker-assisted selection of important agronomic traits in Brassica species.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.     

Genome-Wide Analysis of Simple Sequence Repeats and Efficient Development of Polymorphic SSR Markers Based on Whole Genome Re-Sequencing of Multiple Isolates of the Wheat Stripe Rust Fungus

The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs) are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.     

Repeat-induced point mutation (RIP) in Magnaporthe grisea: implications for its sexual cycle in the natural field context

Repeat-induced point mutation (RIP) is a process that detects DNA duplications and peppers their sequences with C:G to T:A transitions in the sexual phase of the life cycle. So far, this unique mechanism has been identified as a currently active process in only two fungal species, Neurospora crassa and Podospora anserina. To determine whether a RIP-like process operates in the plant pathogenic fungus Magnaporthe grisea, the retrotransposon MAGGY and the hygromycin B phosphotransferase gene were introduced into the fungus as multiple transgenes and examined for sequence alterations after a cross. Frequent C:G to T:A transitions in the transgenes were found in the descendants, preferentially in (A/Tp)Cp(A/T)contexts, suggesting that a process similar to RIP functions in M.grisea. We also examined the sequence of another retrotransposon Pyret in six field isolates of M. grisea. Even though no perfect stage has been known in M. grisea under field conditions to date, RIP-like transitions were found in all the field isolates tested. Interestingly, the frequency of the transitions mostly correlated with the fertility of the isolates examined under laboratory conditions. These results imply that the sexual cycle of this fungus exists or existed in the natural field context.