Simple Sequence Repeat Genotyping Research Articles

Simple sequence repeat genotyping of Colletotrichum species associated with soybean anthracnose in Uttarakhand and other states of India

AbstractAnthracnose is one of the most devastating diseases affecting soybean (Glycine max L. Merril) in India and is incited by the Colletotrichum truncatum species complex. In this study, 33 isolates of Colletotrichum spp. isolates were collected from major soybean‐growing states of India. The cultural, morphological and molecular characteristics (Internal Transcribed Spacer rDNA region) of all isolates were demonstrated. Twelve simple sequence repeat (SSR) markers were selected to amplify the genomic DNA of all isolates, to assess the genetic diversity. On the basis of morpho‐cultural and molecular characterization of three different Colletotrichum species, C. truncatum, C. cliviicola and C. cholorophyti were identified. Representative Colletotrichum isolates (Ct‐Pant, Ct‐Gur and Ct‐Gag) from each species group were found pathogenic to susceptible soybean variety (PS 1092) and fulfilled Koch's postulates. The SSR genotyping of 33 isolates revealed that all examined SSR markers exhibited a high degree of polymorphism, as indicated by the mean values of 0.51 for polymorphic information content, 1.97 for marker index and 1.99 for resolving power. For each primer, the average number of alleles per locus was 3.58. The results of Jaccard similarity coefficient‐based UPGMA (Un‐weighted Pair‐Group Method with Arithmetic Mean) cluster analysis and principal component analysis showed that isolates of the same species were clustered together, but no clear‐cut grouping was obtained on the basis of geographical distribution. Thus, it can be inferred from the study that C. truncatum accessions obtained from various geographical locations of India exhibit substantial genetic variation and a significant level of relatedness.

Machine Learning-Based Identification of Mating Type and Metalaxyl Response in Phytophthora infestans Using SSR Markers.

Phytophthora infestans is the causal agent of late blight in potato. The occurrence of P. infestans with both A1 and A2 mating types in the field may result in sexual reproduction and the generation of recombinant strains. Such strains with new combinations of traits can be highly aggressive, resistant to fungicides, and can make the disease difficult to control in the field. Metalaxyl-resistant isolates are now more prevalent in potato fields. Understanding the genetic structure and rapid identification of mating types and metalaxyl response of P. infestans in the field is a prerequisite for effective late blight disease monitoring and management. Molecular and phenotypic assays involving molecular and phenotypic markers such as mating types and metalaxyl response are typically conducted separately in the studies of the genotypic and phenotypic diversity of P. infestans. As a result, there is a pressing need to reduce the experimental workload and more efficiently assess the aggressiveness of different strains. We think that employing genetic markers to not only estimate genotypic diversity but also to identify the mating type and fungicide response using machine learning techniques can guide and speed up the decision-making process in late blight disease management, especially when the mating type and metalaxyl resistance data are not available. This technique can also be applied to determine these phenotypic traits for dead isolates. In this study, over 600 P. infestans isolates from different populations-Estonia, Pskov region, and Poland-were classified for mating types and metalaxyl response using machine learning techniques based on simple sequence repeat (SSR) markers. For both traits, random forest and the support vector machine demonstrated good accuracy of over 70%, compared to the decision tree and artificial neural network models whose accuracy was lower. There were also associations (p < 0.05) between the traits and some of the alleles detected, but machine learning prediction techniques based on multilocus SSR genotypes offered better prediction accuracy.

Phylogenetic relationships of Quercus species (Fagaceae) in Vietnam based on multiplexed inter simple sequence repeat genotyping by sequencing

Quercus is the largest genus of the family Fagaceae in the world and the second-largest in Vietnam, with approximately 50 species. However, the phylogeny study of the Quercus genus in Vietnam has yet to be thoroughly explored. In this study, we utilize the genome-wide single-nucleotide polymorphisms (SNPs) data obtained through Multiplexed Inter Simple Sequence Repeat Genotyping by sequencing (MIG-seq) to explore the phylogenetic relationships among Quercus species in Vietnam. The results of this study reveal that all Quercus species in Vietnam belong to subgenus Cerris and the phylogenetic analysis strongly supports the recognition of two infrageneric sections: Quercus and Ilex section for the Vietnamese Quercus. These results also confirm that the monophyly of Quercus in Vietnam is strongly supported by both morphological and molecular data. The results of this study also align entirely with previous research, indicating that Quercus species in Asia fall under the subgenus Cerris, comprising two sections: section Ilex and section Cyclobalanopsis.

Incorporation of Photoperiod Insensitivity and High-Yield Genes into an Indigenous Rice Variety from Myanmar, Paw San Hmwe

Paw San Hmwe (PSH) is an indigenous rice variety from Myanmar with a good taste, a pleasant fragrance, and excellent elongation ability during cooking. However, its low yield potential and strong photoperiod sensitivity reduce its productivity, and it is vulnerable to climate changes during growth. To improve the photoperiod insensitivity, yield, and plant stature of PSH, the high-yield genes Grain number 1a (Gn1a) and Wealthy Farmer’s Panicle (WFP), together with the photoperiod insensitivity trait, were introgressed into PSH via marker-assisted backcross breeding and phenotype selection. For the photoperiod insensitivity trait, phenotypic selection was performed under long-day conditions during the dry season. After foreground selection of Gn1a and WFP via simple sequence repeat genotyping, genotyping-by-sequencing was conducted to validate the introgression of target genes and determine the recurrent parent genome recovery of the selected lines. The improved lines were insensitive to photoperiod, and the Gn1a and WFP introgression lines showed significantly higher numbers of primary panicle branches and spikelets per panicle than the recurrent parent, with comparative similarity in cooking and eating qualities. This study successfully improved PSH by decreasing its photoperiod sensitivity and introducing high-yield genes via marker-assisted selection. The developed lines can be used for crop rotation and double-season cropping of better-quality rice.

Genetic Diversity and Population Structure of Maize (Zea mays L.) Inbred Lines in Association with Phenotypic and Grain Qualitative Traits Using SSR Genotyping

Maize (Zea mays L.) is an important cereal and is affected by climate change. Therefore, the production of climate-smart maize is urgently needed by preserving diverse genetic backgrounds through the exploration of their genetic diversity. To achieve this, 96 maize inbred lines were used to screen for phenotypic yield-associated traits and grain quality parameters. These traits were studied across two different environments (Anand and Godhra) and polymorphic simple sequence repeat (SSR) markers were employed to investigate the genetic diversity, population structure, and trait-linked association. Genotype–environment interaction (GEI) reveals that most of the phenotypic traits were governed by the genotype itself across the environments, except for plant and ear height, which largely interact with the environment. The genotypic correlation was found to be positive and significant among protein, lysine and tryptophan content. Similarly, yield-attributing traits like ear girth, kernel rows ear−1, kernels row−1 and number of kernels ear−1 were strongly correlated to each other. Pair-wise genetic distance ranged from 0.0983 (1820194/T1 and 1820192/4-20) to 0.7377 (IGI-1101 and 1820168/T1). The SSRs can discriminate the maize population into three distinct groups and shortlisted two genotypes (IGI-1101 and 1820168/T1) as highly diverse lines. Out of the studied 136 SSRs, 61 were polymorphic to amplify a total of 131 alleles (2–3 per loci) with 0.46 average gene diversity. The Polymorphism Information Content (PIC) ranged from 0.24 (umc1578) to 0.58 (umc2252). Similarly, population structure analysis revealed three distinct groups with 19.79% admixture among the genotypes. Genome-wide scanning through a mixed linear model identifies the stable association of the markers umc2038, umc2050 and umc2296 with protein, umc2296 and umc2252 with tryptophan, and umc1535 and umc1303 with total soluble sugar. The obtained maize lines and SSRs can be utilized in future maize breeding programs in relation to other trait characterizations, developments, and subsequent molecular breeding performances for trait introgression into elite genotypes.

First report of Phytophthora infestans lineage EU23 causing potato and tomato late blight in South Africa.

In South Africa, potato (Solanum tuberosum) late blight epidemics from 1996 to 2007 were caused by Phytophthora infestans clonal lineage US-1 (McLeod et al. 2001; Pule et al. 2013). Similarly, surveys on tomatoes in the mid-1990s only identified the US-1 clonal lineage in South Africa (McLeod et al., 2001). On potatoes, populations from the Southern Cape and Western Cape regions consisted of persistent mefenoxam-resistant populations (McLeod et al. 2001; Pule et al. 2013). Limited mefenoxam (R-enantiomer of metalaxyl) screening in 2021 in the Western Cape showed that potato isolates were sensitive, which prompted our study. Potato late blight samples were collected in 13 potato fields in the 2021 to 2023 seasons in the Western Cape (n = 4), Free State (n = 7), Limpopo (n = 1) and Kwazulu-Natal (n = 1) Provinces, and one tomato sample in 2022 in the Limpopo Province. Fourteen samples, one per field, were simple sequence repeat (SSR) genotyped for 12 loci (Li et al. 2013) using as DNA template, FTA cards, or genomic DNA extracted from cultures. P. infestans isolations from lesions and DNA culture extractions were conducted as previously described (Pule et al. 2013). SSR genotyping revealed that all 14 P. infestans samples belonged to clonal lineage EU_23_A1 (EU23), which has a phenotype (A1 and metalaxyl sensitive) and SSR genotype matching the US-23 lineage (Saville et al., 2021). As expected, minor polymorphisms were detected among the samples at loci Pi02, G11, D13 and SSR4. Mefenoxam sensitivity testing of seven potato isolates from the Free State (n = 3) and Western Cape (n = 4), and one tomato isolate was conducted as previously described (Mcleod et al. 2001). All isolates were sensitive to mefenoxam since no infection and sporulation occurred at 3 µg/ml. This was expected since EU23 has been reported as mefenoxam sensitive in other countries (Kawchuk et al., 2011; McGrath et al., 2015). Replacement of the US-1 clonal lineage by EU23 suggests that the latter lineage is more aggressive or fit than US-1, but this must be verified especially on potatoes. On tomatoes, on the other hand, EU23 is known as a highly aggressive lineage (Kawchuk et al., 2011; McGrath et al., 2015; Saville et al., 2021). Therefore, population displacements may have first occurred on tomatoes from where the lineage spread to potatoes. In the Cape coastal potato production regions, population displacement may have been supported by the withdrawal of mefenoxam/metalaxyl from the region since 1996 because the EU23 lineage is mefenoxam sensitive, as opposed to the previously prevailing US-1 mefenoxam-resistant lineage. More severe potato late blight epidemics has not been observed in recent years in South Africa. However, tomato late blight has increased and is more prevalent in the Limpopo province. The source of the introduction of EU23 into South Africa is unknown. Only test-tube plants and/or greenhouse tubers may be imported into South Africa since 1997. Therefore, the illegal importation of planting material may have introduced the new genotype. Whether this could have occurred from neighbouring African countries is unknown since P. infestans genotyping has not been conducted in these countries. In Africa, EU23 has been reported in northern African countries (Tunisia, Algeria and Egypt) (Saville et al., 2021; El-Ganainy et al., 2023). Mefenoxam and metalaxyl applications will likely be effective again in the Western Cape, but more samples will have to be tested to confirm this. This will provide growers with a more cost-effective fungicide (metalaxyl) since alternative actives with comparable systemic and curative activity are more expensive.

Simple Sequence Repeat Fingerprint Identification of Essential-Oil-Bearing Rosa rugosa via High-Resolution Melting (HRM) Analysis.

Oil-bearing Rosa rugosa are popular in the essential oil and perfume markets. The similar botanical characteristics between high-oil-yield or low-oil-yield cultivars are confusing and it is hard for farmers or breeders to identify the high-oil-yield cultivar by phenotype difference. High-resolution melting (HRM) analysis of simple sequence repeats (SSRs) can construct accurate DNA fingerprints quickly, which was shown to be effective for identification of closely related cultivars of R. rugosa. Optimization of HRM-SSR indicated that the 10 µL HRM reaction mixture containing 20 ng of genomic DNA of R. rugosa and 0.75 µL of 10 µmol/L of each primer with an annealing temperature of 64 °C was a robust SSR genotyping protocol. Using this protocol, 9 polymorphic SSR markers with 3-9 genotypes among the 19 R. rugosa cultivars were identified. The top three polymorphic makers SSR9, SSR12 and SSR19 constructed a fingerprint of all cultivars, and the rare insertion in the flanking sequences of the repeat motif of SSR19 generated three characteristic genotypes of three high-oil-yield cultivars. These results may be economical and practical for the identification of high-oil-yield R. rugosa and be helpful for the selection and breeding of oil-bearing roses.

Quercus ilex L. dieback is genetically determined: Evidence provided by dendrochronology, δ13C and SSR genotyping

Quercus ilex L. dieback has been reported in several Mediterranean forests, revealing different degree of crown damages even in close sites, as observed in two Q. ilex forest stands in southern Tuscany (IT). In this work, we applied a novel approach combining dendrochronological, tree-ring δ13C and genetic analysis to test the hypothesis that different damage levels observed in a declining (D) and non-declining (ND) Q. ilex stands are connected to population features linked to distinct response to drought. Furthermore, we investigated the impact of two major drought events (2012 and 2017), that occurred in the last fifteen years in central Italy, on Q. ilex growth and intrinsic water use efficiency (WUEi). Overall, Q. ilex showed slightly different ring-width patterns between the two stands, suggesting a lower responsiveness to seasonal climatic variations for trees at D stand, while Q. ilex at ND stand showed changes in the relationship between climatic parameters and growth across time. The strong divergence in δ13C signals between the two stands suggested a more conservative use of water for Q. ilex at ND compared to D stand that may be genetically driven. Q. ilex at ND resulted more resilient to drought compared to trees at D, probably thanks to its safer water strategy. Genotyping analysis based on simple-sequence repeat (SSR) markers revealed the presence of different Q. ilex populations at D and ND stands. Our study shows intraspecific variations in drought response among trees grown in close. In addition, it highlights the potential of combining tree-ring δ13C data with SSR genotyping for the selection of seed-bearing genotypes aimed to preserve Mediterranean holm oak ecosystem and improve its forest management.

Evaluation of the Genetic Diversity of Greek Garlic (Allium sativum L.) Accessions Using DNA Markers and Association with Phenotypic and Chemical Variation

The genetic diversity of 27 garlic accessions (local varieties/landraces) was investigated using five simple sequence repeat (SSR) and six inter-simple sequence repeat (ISSR) markers. The SSR genotyping revealed 26 different alleles, while 84 polymorphic bands were produced using the ISSR markers. The average polymorphism information content (PIC) was 0.471 and 0.465 for the SSR and the ISSR markers, respectively. Compared to ISSRs, SSR markers revealed a higher level of redundancy, indicating potential duplicates among the accessions. The analysis of molecular variance (AMOVA) based on both marker systems showed that most of the molecular diversity was attributable to differences within accessions, rather than among them. UPGMA, STRUCTURE, and Principal Coordinate Analysis (PCoA) based on the SSRs produced similar clustering results, although not in agreement with those produced by the ISSR markers. Fisher’s exact tests and multinomial logistic regression analyses identified significant associations between the chemical compounds, the morphological traits of the bulb, and specific SSR and ISSR loci. Our results provide a molecular basis for understanding the genetic diversity of Greek garlic landraces, which could be useful for the conservation and sustainable management of this crop. Potential markers fostering the selection of genotypes in garlic breeding have also been revealed.

Genetic Variation of Puccinia triticina Populations in Iran from 2010 to 2017 as Revealed by SSR and ISSR Markers.

Puccinia triticina is a major wheat pathogen worldwide. Although Iran is within the Fertile Crescent, which is supposed to be the center of origin of both wheat and P. triticina, the knowledge of the genetic variability of local populations of this basidiomycete is limited. We analyzed 12 inter simple sequence repeats (ISSRs) and 18 simple sequence repeats (SSRs) of 175 P. triticina isolates sampled between 2010 and 2017 from wheat and other Poaceae in 14 provinces of Iran. SSRs revealed more polymorphisms than ISSRs, indicating they were more effective in differentiating P. triticina populations. Based on a dissimilarity matrix with a variable mutation rate for SSRs and a Dice coefficient for ISSRs, the isolates were separated into three large groups, each including isolates from diverse geographic origins and hosts. The grouping of SSR genotypes in UPGMA dendrograms was consistent with the grouping inferred from the Bayesian approach. However, isolates with a common origin clustered into separate subgroups within each group. The high proportion of heterozygous alleles suggests that in Iran clonal reproduction prevails over sexual reproduction of the pathogen. A significant correlation was found between SSR and ISSR genotypes and the virulence phenotypes of the isolates, as determined in a previous study.

Genome-wide identification and characterization of parthenocarpic fruit set-related gene homologs in cucumber (Cucumis sativus L.)

Cucumber (Cucumis sativus L.), a major horticultural crop, in the family Cucurbitaceae is grown and consumed globally. Parthenocarpy is an ideal trait for many fruit and vegetables which produces seedless fruit desired by consumers. The seedlessness occurs when fruit develops without fertilization which can be either natural or induced. So far, a limited number of genes regulating parthenocarpic fruit set have been reported in several fruit or vegetable crops, most of which are involved in hormone biosynthesis or signalling. Although parthenocarpic cucumber has been widely used in commercial production for a long time; its genetic basis is not well understood. In this study, we retrieved thirty five parthenocarpy fruit-set related genes (PRGs) from bibliomic data in various plants. Thirty-five PRG homologs were identified in the cucumber genome via homology-based search. An in silico analysis was performed on phylogenetic tree, exon–intron structure, cis-regulatory elements in the promoter region, and conserved domains of their deduced proteins, which provided insights into the genetic make-up of parthenocarpy-related genes in cucumber. Simple sequence repeat (SSR) sequences were mined in these PRGs, and 31 SSR markers were designed. SSR genotyping identified three SSRs in two polymorphic genes. Quantitative real-time PCR of selected genes was conducted in five cucumber lines with varying degrees of parthenocarpic fruit set capacities, which revealed possible association of their expression with parthenocarpy. The results revealed that homologs CsWD40 and CsPIN-4 could be considered potential genes for determination of parthenocarpy as these genes showed parental polymorphism and differential gene expression in case of parthenocarpic and non-parthenocarpic parents.

SSR variability of stem rust pathogen on spring bread wheat in Russia.

Wheat stem rust caused by Puccinia graminis f. sp. tritici Erikss. & E. Henning (Pgt), which used to be a harmful disease of winter wheats in the southern part of Russia, has been largely affecting the yield of spring bread wheat in the territories of temperate climate zone since 2009. A total of 222 Pgt isolates were obtained from samples of susceptible cultivars of spring bread wheat in Central and Volga regions, and Omsk and Novosibirsk provinces in 2019. Genotyping of the isolates was carried out at 16 SSR loci. Number of alleles, proportion of heterozygotes and deviation from Hardy-Weinberg equilibrium were determined at each SSR locus. Based on genetic variability of SSR genotypes, it was shown that the Pgt population is subdivided into two large clusters in the territory of Russian temperate climate zone: "European" population (the Central region) and "Asian" one (the Volga region and two main wheat provinces of Western Siberia). Both the Pgt populations are characterized by a mixed mode of reproduction (sexual and clonal), but different sources of inoculum seem to shape a genotype structure within them. A group of Pgt genotypes with high variability, the inbreeding coefficient closed to zero and low observed heterozygosity was revealed among samples from Omsk. Moreover, two singular SSR genotypes identified among the Asian samples of Pgt isolates should attract a special attention in monitoring of stem rust in order to disclose unexpected rapid changes of the pathogen in the corresponding regions and to prevent the disease outbreak.

Genetic diversity analysis and variety identification using SSR and SNP markers in melon

Melon is an important horticultural crop with a pleasant aromatic flavor and abundance of health-promoting substances. Numerous melon varieties have been cultivated worldwide in recent years, but the high number of varieties and the high similarity between them poses a major challenge for variety evaluation, discrimination, as well as innovation in breeding. Recently, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), two robust molecular markers, have been utilized as a rapid and reliable method for variety identification. To elucidate the genetic structure and diversity of melon varieties, we screened out 136 perfect SSRs and 164 perfect SNPs from the resequencing data of 149 accessions, including the most representative lines worldwide. This study established the DNA fingerprint of 259 widely-cultivated melon varieties in China using Target-seq technology. All melon varieties were classified into five subgruops, including ssp. agrestis, ssp. melo, muskmelon and two subgroups of foreign individuals. Compared with ssp. melo, the ssp. agrestis varieties might be exposed to a high risk of genetic erosion due to their extremely narrow genetic background. Increasing the gene exchange between ssp. melo and ssp. agrestis is therefore necessary in the breeding procedure. In addition, analysis of the DNA fingerprints of the 259 melon varieties showed a good linear correlation (R2 = 0.9722) between the SSR genotyping and SNP genotyping methods in variety identification. The pedigree analysis based on the DNA fingerprint of ‘Jingyu’ and ‘Jingmi’ series melon varieties was consistent with their breeding history. Based on the SNP index analysis, ssp. agrestis had low gene exchange with ssp. melo in chromosome 4, 7, 10, 11and 12, two specific SNP loci were verified to distinguish ssp. agrestis and ssp. melon varieties. Finally, 23 SSRs and 40 SNPs were selected as the core sets of markers for application in variety identification, which could be efficiently applied to variety authentication, variety monitoring, as well as the protection of intellectual property rights in melon.

SSR Genotypes of the Puccinia triticina in 15 Provinces of China Indicate Regional Migration in One Season from East to West and South to North

Leaf rust of wheat caused by Puccinia triticina (Pt) is one of the most common fungal diseases in the southwest and northwest of China, the middle and lower reaches of the Yangtze River, and the southern part of the Huang-Huai-Hai river basin. Using 13 simple sequence repeat (SSR) markers, we systematically revealed the genotypic diversities, population differentiation and reproduction of Pt isolates in 15 wheat-producing areas in China. A total of 622 isolates were divided into 3 predominant populations, including the eastern Pt populations, consisting of Pt samples from 8 eastern provinces of Beijing, Hebei, Shanxi, Shaanxi, Anhui, Shandong, Henan, and Heilongjiang; the 4 western Pt populations from Gansu, Qinghai, Sichuan, and Inner Mongolia provinces; and the bridge Pt populations including Jiangsu, Hubei, and Yunnan, which communicated the other 2 populations as a “bridge”. The pathogen transmission of eastern Pt populations was more frequent than western Pt populations. The linkage disequilibrium test indicated that the whole Pt population was in a state of linkage disequilibrium. However, populations of Beijing, Hebei, Shaanxi, Jiangsu, Henan, and Heilongjiang provinces showed obvious linkage equilibrium, while the five provinces of Qinghai, Hubei, Anhui, Shandong, and Inner Mongolia supported clonal modes of reproduction.

Genetic characterization of cultivated apple (Malus x domestica Borkh.) in Morocco using microsatellite (SSR) markers

Genetic diversity of 27 apple genotypes from four geographical regions of Morocco was analyzed using 26 simple sequence repeat (SSR) primers. In total, 195 polymorphic SSR alleles were detected, with sizes ranging from 78 to 209 bp. The results obtained for the average number of alleles per locus (7.5), effective number of alleles (4.62), Shannon information index (1.66), expected heterozygosity (0.76), observed heterozygosity (0.74) and polymorphism information content (0.76) showed a moderate level of polymorphism, which suggests that Moroccan apple cultivars exhibit significant genetic diversity. Over the 26 SSR loci used, the probability of identity P (ID) was 4.62 10 −29 indicating that most markers were highly discriminating and powerful for cultivars characterization. The principal coordinate analysis and hierarchical classification suggested the presence of four well defined groups, which was confirmed later by model based population structure. Furthermore, strong genetic differentiation between these four groups was also detected (FST = 0.28) . Most of the individuals segregated into the four groups showing a membership coefficient greater than 0.80, which indicates their better genetic integrity. A parentage analysis, however, shows that cultivars from the four regions are minimally hybridized. After comparing SSR profiles and genotypes names, we conclude that the problem of homonyms and/or labeling errors appear in the studied cultivars.Thus, based on genetic profiles, two individuals (Beldi 1/Beldi 2 and Laakri 5/Laakri 6) found to be duplicated. Additionally, some cultivars with the same name are grouped in different clusters suggesting the existence of possible homonymy. Lastly, the present work confirms the usefulness of SSR markers for the elimination of duplications and characterization of diversity and hybrid characters of apple genotypes in Morocco.

Genetic Diversity and Population Structure Reveal Cryptic Genetic Variation and Long Distance Migration of Puccinia graminis f. sp. tritici in the Indian Subcontinent.

Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of wheat worldwide since time immemorial. Several wheat stem rust outbreaks have been reported worldwide including India. Approximately 7 mha wheat area in central and peninsular India is highly vulnerable to stem rust epidemics. In this study, a repository of 29 single genotype uredospore pathotypes, representing five geographical regions, was characterized by investigating their virulence phenotype and simple sequence repeat (SSR) genotypes using 37 reproducible polymorphic SSR markers, 32 of which had ≥ 0.50 polymorphic information content (PIC) value. Virulence phenotypes were used to evaluate the virulence frequency (VF) and construct a hypothetical evolutionary hierarchy of these pathotypes. We projected seven lineages to explain the evolutionary pattern of the Pgt population. The VF of these pathotypes ranged between 0% and 100%. The virulence-based neighbor-joining (NJ) cluster analysis grouped Pgt pathotypes into five virulence groups. Likewise, five molecular groups were categorized using molecular genotypes. The molecular grouping was supported by principal coordinate analysis (PCoA), which revealed 25% of the cumulative variance contributed by the first two axes. Analysis of molecular variance (AMOVA) revealed 8 and 92% of the variation among and within the populations, respectively. The Mantel test confirmed a positive but weak correlation (R2 = 0.15) between virulence phenotypes and SSR genotypes. The highest and lowest values of different genetic diversity parameters (Na, Ne, I, He, uHe, and %P) revealed maximum and minimum variability in the Pgt population from Maharashtra and Uttar Pradesh, respectively. The population structure analysis clustered 29 Pgt pathotypes into two subpopulations and an admixture. Our results demonstrated that there was significant genetic diversity among Pgt pathotypes resulting from their long-distance dispersal ability complemented by gene flow. These findings provide insights into the virulence patterns, genetic variations, and possible evolution of Pgt pathotypes, which would support strategic stem rust resistance breeding.

An integrated strategy for target SSR genotyping with toleration of nucleotide variations in the SSRs and flanking regions

BackgroundWith the broad application of high-throughput sequencing and its reduced cost, simple sequence repeat (SSR) genotyping by sequencing (SSR-GBS) has been widely used for interpreting genetic data across different fields, including population genetic diversity and structure analysis, the construction of genetic maps, and the investigation of intraspecies relationships. The development of accurate and efficient typing strategies for SSR-GBS is urgently needed and several tools have been published. However, to date, no suitable accurate genotyping method can tolerate single nucleotide variations (SNVs) in SSRs and flanking regions. These SNVs may be caused by PCR and sequencing errors or SNPs among varieties, and they directly affect sequence alignment and genotyping accuracy.ResultsHere, we report a new integrated strategy named the accurate microsatellite genotyping tool based on targeted sequencing (AMGT-TS) and provide a user-friendly web-based platform and command-line version of AMGT-TS. To handle SNVs in the SSRs or flanking regions, we developed a broad matching algorithm (BMA) that can quickly and accurately achieve SSR typing for ultradeep coverage and high-throughput analysis of loci with SNVs compatibility and grouping of typed reads for further in-depth information mining. To evaluate this tool, we tested 21 randomly sampled loci in eight maize varieties, accompanied by experimental validation on actual and simulated sequencing data. Our evaluation showed that, compared to other tools, AMGT-TS presented extremely accurate typing results with single base resolution for both homozygous and heterozygous samples.ConclusionThis integrated strategy can achieve accurate SSR genotyping based on targeted sequencing, and it can tolerate single nucleotide variations in the SSRs and flanking regions. This method can be readily applied to divergent sequencing platforms and species and has excellent application prospects in genetic and population biology research. The web-based platform and command-line version of AMGT-TS are available at https://amgt-ts.plantdna.site:8445 and https://github.com/plantdna/amgt-ts, respectively.

Pathogenic and genetic diversity ofPuccinia triticinafrom triticale in Poland between 2012 and 2015

AbstractVariation amongPuccinia triticinaisolates collected from triticale in Poland between 2012 and 2015 was studied based on virulence and simple sequence repeat (SSR) markers. Two hundred and forty‐two single‐uredinial isolates from four geographically separated locations were tested for virulence against 33 near‐isogenic Thatcher lines containing knownLrresistance genes, and their molecular genotypes were characterized with 34 SSR markers. Structure and relationships of the regional and annual populations ofP.triticinawere analysed using an assignment‐based approach for both the virulence and SSR data. The molecular marker analysis was based on two different models of SSR evolution: the stepwise mutation model with a variable mutation rate (SMMv) and the infinite alleles model (IAM). A highly significant deviation from Hardy–Weinberg equilibrium among SSR genotypes, a high proportion of heterozygotes, and a moderate association of relationships between virulence phenotypes and SSR genotypes were consistent with the occurrence of clonal lineages of related races within the population. While the results suggest that genetic drift and mutation affect variation within the pathogen population, it seems that migration has the most significant role in shaping the population structure ofP.triticinaoccurring on triticale in Poland.

Population Diversity, Dynamics, and Differentiation of Wheat Stripe Rust Pathogen Puccinia striiformis f. sp. tritici From 2010 to 2017 and Comparison With 1968 to 2009 in the United States.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease on wheat in the United States, especially after 2000. In the present study, 2,247 Pst isolates collected over all stripe rust epidemiological regions in the United States from 2010 to 2017 were genotyped at 14 simple sequence repeat (SSR) loci to investigate the population diversity, dynamics, and differentiation. A total of 1,454 multilocus genotypes (MLGs) were detected. In general, the populations in the west (regions 1–6) had more MLGs and higher diversities than the populations in the east (regions 7–12). The populations of 2010 and 2011 were more different from the other years. Genetic variation was higher among years than among regions, indicating the fast changes of the population. The divergence (Gst) was bigger between the west population and east population than among regions within either the west or east population. Gene flow was stronger among the regional populations in the east than in the west. Clustering analyses revealed 3 major molecular groups (MGs) and 10 sub-MGs by combining the genotypic data of 2010–2017 isolates with those of 1968–2009. MG1 contained both 1968–2009 isolates (23.1%) and 2010–2017 isolates (76.9%). MG2 had 99.4% of isolates from 1968–2009. MG3, which was the most recent and distinct group, had 99.1% of isolates from 2010–2017. Of the 10 sub-MGs, 5 (MG1-3, MG1-5, MG3-2, MG3-3, and MG3-4) were detected only from 2011 to 2017. The SSR genotypes had a moderate, but significant correlation (r = 0.325; p < 0.0001) with the virulence phenotype data. The standard index values of association (rbarD = 0.11) based on either regional or yearly populations suggest clonal reproduction. This study indicated high diversity, fast dynamics, and various levels of differentiation of the Pst population over the years and among epidemiological regions, and the results should be useful for managing wheat stripe rust.

Physiologic Specialization and Genetic Differentiation of Puccinia triticina Causing Leaf Rust of Wheat on the Indian Subcontinent During 2016 to 2019.

Wheat is the second most cultivated cereal crop in the world and is an important crop in India. Leaf (brown) rust, caused by Puccinia triticina, was the most prevalent among the three rusts found in all the wheat-growing areas of India, Bhutan, and Nepal during 2016 to 2019. Leaf rust samples from wheat crops in these countries were pathotyped using the wheat differential genotypes and binomial Indian system of nomenclature. To facilitate international communication, each pathotype identified was also tested using the North American differentials. A total of 33 pathotypes were identified from 1,086 samples, including three new pathotypes: 61R47 (162-5 = KHTPM) and 93R49 (49 = NHKTN) from India and 93R57 (20-1 = NHKTN) from Nepal. Two pathotypes, 121R60-1 (77-9/52 = MHTKL) and 121R63-1 (77-5 = THTTM), accounted for 79.46% of the population. Virulence on Lr19 was identified in 0.27% of the samples from Nepal only. The proportion of pathotype 121R60-1 (77-9 = MHTKL) increased to 57.55% during these years. Virulence was not observed on Lr9, Lr24, Lr25, Lr28, Lr32, Lr39, Lr45, and Lr47 in the population of the Indian subcontinent. Eighteen polymorphic simple sequence repeat (SSR) primer pairs tested on the isolates amplified 48 alleles with an average of 2.66 alleles per primer pair. Based on SSR genotyping, these pathotypes could be grouped into two clades with another two subclades each. Many of the Lr genes present in Indian wheat germplasm (Lr1, Lr3a, Lr10, Lr11, Lr14a, Lr15, Lr16, Lr17, Lr20, Lr23, and Lr26) were ineffective for a majority of pathotypes. Most of these varieties possessed a high degree of leaf rust resistance. The field resistance of wheat varieties could be attributed to the interaction of genes, unknown resistance, or adult plant resistance.