Multilocus Simple Sequence Repeat Research Articles

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.

Population Structure of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in the San Joaquin Valley of California.

Xylella fastidiosa causes disease in many commercial crops, including almond leaf scorch (ALS) disease in susceptible almond (Prunus dulcis). In this study, genetic diversity and population structure of X. fastidiosa associated with ALS disease were evaluated. Isolates obtained from two almond orchards in Fresno and Kern County in the San Joaquin Valley of California were analyzed for two successive years. Multilocus simple-sequence repeat (SSR) analysis revealed two major genetic clusters that were associated with two host cultivars, 'Sonora' and 'Nonpareil', respectively, regardless of the year of study or location of the orchard. These relationships suggest that host cultivar selection and adaptation are major driving forces shaping ALS X. fastidiosa population structure in the San Joaquin Valley. This finding will provide insight into understanding pathogen adaptation and host selection in the context of ALS disease dynamics.

Molecular diversity of the entomopathogenic fungal Metarhizium community within an agroecosystem

The entomopathogenic fungal Metarhizium anisopliae lineage harbors cryptic diversity and was recently split into several species. Metarhizium spp. are frequently isolated from soil environments, but the abundance and distribution of the separate species in local communities is still largely unknown. Entomopathogenic isolates of Metarhizium spp. were obtained from 32 bulked soil samples of a single agroecosystem in Denmark using Tenebrio molitor as bait insect. To assess the Metarhizium community in soil from the agricultural field and surrounding hedgerow, 123 isolates were identified by sequence analysis of 5′ end of elongation factor 1-α and their genotypic diversity characterized by multilocus simple sequence repeat (SSR) typing. Metarhizium brunneum was most frequent (78.8%) followed by M. robertsii (14.6%), while M. majus and M. flavoviride were infrequent (3.3% each) revealing co-occurrence of at least four Metarhizium species in the soil of the same agroecosystem. Based on SSR fragment length analysis five genotypes of M. brunneum and six genotypes of M. robertsii were identified among the isolates. A single genotype within M. brunneum predominated (72.3% of all genotypes) while the remaining genotypes of M. brunneum and M. robertsii were found at low frequencies throughout the investigated area indicating a diverse Metarhizium community. The results may indicate potentially favorable adaptations of the predominant M. brunneum genotype to the agricultural soil environment.

Analysis of population structure and genetic diversity in balloon flower (Platycodon grandiflorum (Jacq.) A. DC.) germplasm using Simple Sequence Repeat (SSR) markers

Balloon flower (Platycodon grandiflorum A. DC) is widely distributed in South Korea and there are some local landraces that are cultivated as a vegetable crop or medicinal plant. Making use of the gene resources of wild-type and landraces is a way to increase the genetic diversity of the cultivars. However, few tools or information are available on an efficient identification system for maintaining and management of these landraces. To improve the genetic resources for balloon flower, 22 simple sequence repeat (SSR) markers, also known as microsatellite markers, were evaluated in a collection of 42 balloon flower landraces, 34 of which were from Korea and eight from China. All microsatellite markers produced the 107 alleles ranging from 2 to 10 with a mean of 4.864 alleles per each locus (NA). The values of observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.00 to 0.667 (mean of 0.285) and from 0.024 to 0.741 (mean of 0.416), respectively. An average value of polymorphic information contents (PIC) were 0.382 with a range of 0.023 to 0.703. Results of population structure and phylogenetic and principal coordinate analysis (PCoA) indicated that P. grandiflorum germplasm formed two largely distinct clusters according to their origins and the genetic differentiation. There was a high level of genotypic diversity at broad geographic regions between Korea and China, but the low genetic differentiation was found within the collections from Korea. The results of the genetic diversity will be useful for the selection of the parents for developing balloon flower breeding and the multi-locus SSR markers developed herein will be a valuable resource for germplasm assessments, evaluation of genetic diversity, and population genetic studies of balloon flower.

Cultivar Identification, Pedigree Verification, and Diversity Analysis among Peach Cultivars Based on Simple Sequence Repeat Markers

Information on genetic relationships and pedigree structure in germplasm collections is vital to breeders in crop improvement programs. In this study, we assessed genetic identity, kinship distance, and parentage–sibship relationships among 37 peach (Prunus persica) accessions and breeding lines using simple sequence repeat (SSR) markers. Pairwise comparisons based on multilocus SSR profiles led to the identification of two synonymous groups including five accessions. Two pairs of parent–offspring and one full sibling relationships were identified using the likelihood method, and Bayesian cluster analysis partitioned the accessions into groups that were partially compatible with the known pedigree, origin, and flesh color. The 37 accessions were grouped into four clusters, which were largely supported by the known pedigree and origin of these accessions. Although the observed mean heterozygosity was 0.219, mean inbreeding coefficient was 0.635, indicating a high degree of inbreeding among the accessions. Eleven of the 15 SSR markers (73.3%) tested were transferable to nine related Prunus species. Results of the study demonstrate that these SSRs could facilitate the assessment of genetic identity and pedigree structure.

Genetic diversity of ‘Cadidatus Liberibacter solanacearum’ strains in the United States and Mexico revealed by simple sequence repeat markers

‘Candidatus Liberibacter solanacearum’ is associated with the Zebra Chip (ZC) disorder of potatoes. A panel of eight simple sequence repeat (SSR) markers was developed and used to genetically characterize ‘Ca. L. solanacearum’ strains obtained from ZC-affected potato plants in the United States and Mexico. The multilocus SSR markers in this study effectively differentiated genotypes and estimated genetic diversity of ‘Ca. L. solanacearum’ strains. Genotype assignment analyses identified two major lineages of ‘Ca. L. solanacearum’ in the North American populations while only one lineage type was identified in Mexican population. No clear genetic structure was found among haplotypes based on geographical proximity or host. The high resolution power of the SSR marker system developed in this study provides a useful tool for genotyping closely related strains and tracking sources of the pathogen. Genotype information combined with epidemiological data will advance knowledge of ZC disease and will facilitate development of effective disease management.

Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping projects.

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.

Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa

A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.

Targeted development of informative microsatellite (SSR) markers.

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.