Simple Sequence Repeat Genotyping Research Articles

A novel (ATC)n microsatellite locus is associated with litter size in an indigenous Chinese pig.

Simple sequence repeats (SSRs) are an important part of the genome and have become powerful auxiliary DNA markers in animal breeding using marker‐assisted selection (MAS). Based on previous sequencing data of Qinghai Bamei pigs, a total of three novel candidate SSR loci were analysed in this study. Time‐of‐flight mass spectrometry (TOF‐MS) was used for SSR genotyping, and association analyses between SSRs and the litter size of Qinghai Bamei sows was also performed. The results of genotyping showed that the (ATC)n‐P1, (AC)n‐P2 and (AC)n‐P3 loci had 2, 3 and 18 genotypes, respectively; 2, 3 and 8 alleles were also identified at these loci. Except for the (AC)n‐P2 locus, the polymorphism information content (PIC) values of other loci were greater than 0.25. Association analyses indicated that only the (ATC)n‐P1 locus was significantly associated with the litter size of Qinghai Bamei sows (p = .047). Compared to 189‐/189‐ genotype, individuals with the 189‐/195‐ genotype had the senior litter size, which was 9.04 ± 0.21. Our results enrich the data on SSRs in Qinghai Bamei pigs and indicate that (ATC)n‐P1 is a candidate locus for MAS in the pig industry.

Race Characterization and Molecular Genotyping of Puccinia triticina Populations from Durum Wheat in Russia.

Variability of the Russian population of Puccinia triticina from durum wheat was studied with virulence and simple sequence repeat (SSR) markers. The pathogen was sampled during 2017 to 2019 in all regions with sizable durum wheat (Triticum durum) growing areas from winter (North Caucasus) and spring (Middle Volga, Ural, and West Siberia) wheat. A total of 474 isolates were tested on a set of 20 Lr-gene lines. Molecular genotypes for 105 selected isolates were determined at 11 SSR loci. Variable virulence/avirulence reaction was observed only on three Lr-gene lines, whereas just five SSR loci were polymorphic with two alleles at each. Seven different virulence phenotypes and 11 SSR genotypes were found among 474 and 105 isolates, respectively, indicating a very low variability of the pathogen. One virulence phenotype and three SSR genotypes occurred in all Russian regions. However, two phenotypes were specific to the European regions of Russia (North Caucasus and Middle Volga), while another two were found only in the Asian part of Russia (Ural and West Siberia). Significant differentiation between six populations of P. triticina from durum wheat in the Asian and European (mainly North Caucasus) regions was also shown with numerous metrics and approaches for data with and without clone correction. Relationships among the regional populations of P. triticina from durum wheat established with virulence phenotypes significantly associated with those for SSR genotypes and was similar to the relationships among the regional populations of the pathogen from common wheat.

QTL mapping using GBS and SSR genotyping reveals genomic regions controlling wheat coleoptile length and seedling emergence

Seedling emergence (SE) is an essential trait in wheat (Triticum aestivum L.) that allows desirable stand establishment even when adequate seed-zone soil moisture is present at depths of 10 cm or greater. The SE trait bestows several other advantages, including lodging resistance and improved water use efficiency. The aim of this study was to understand the genetic basis of SE. It has previously been reported that the cultivar Spinkcota has a high rate of SE and Bounty 309 has a slow rate of SE. An F2 population of 190 plants from a cross between these two parents was developed and evaluated for coleoptile length (CL) and in a specialized field SE test. The population was genotyped by simple sequence repeat (SSR) and genotyping-by-sequencing (GBS) for genetic mapping and marker discovery of the two traits. The GBS analysis was performed using 128,848,348 sequence reads with an average of 671,085 per F2 plant. From a total of 2,639 raw SNPs discovered in the F2 population, 243 high-quality SNPs along with 68 SSR markers were utilized to construct a genetic linkage map of wheat spanning 5,532.9 cM on the 21 chromosomes. Using this map, eight QTLs linked to CL and SE were detected on chromosomes 1A, 1B, 2B, 3A, and 7D. Bioinformatic dissection of the intervals under these QTLs on the wheat whole-genome sequence led to the identification of ~ 60 genes. Validated markers may enhance the understanding of this critical trait and provide a basis for marker-assisted selection.

Genomic Resource Development for Hydrangea (Hydrangea macrophylla (Thunb.) Ser.)—A Transcriptome Assembly and a High-Density Genetic Linkage Map

Hydrangea (Hydrangea macrophylla) is an important ornamental crop that has been cultivated for more than 300 years. Despite the economic importance, genetic studies for hydrangea have been limited by the lack of genetic resources. Genetic linkage maps and subsequent trait mapping are essential tools to identify and make markers available for marker-assisted breeding. A transcriptomic study was performed on two important cultivars, Veitchii and Endless Summer, to discover simple sequence repeat (SSR) markers and an F1 population based on the cross ‘Veitchii’ × ‘Endless Summer’ was established for genetic linkage map construction. Genotyping by sequencing (GBS) was performed on the mapping population along with SSR genotyping. From an analysis of 42,682 putative transcripts, 8780 SSRs were identified and 1535 were validated in the mapping parents. A total of 267 polymorphic SSRs were selected for linkage map construction. The GBS yielded 3923 high quality single nucleotide polymorphisms (SNPs) in the mapping population, resulting in a total of 4190 markers that were used to generate maps for each parent and a consensus map. The consensus linkage map contained 1767 positioned markers (146 SSRs and 1621 SNPs), spanned 1383.4 centiMorgans (cM), and was comprised of 18 linkage groups, with an average mapping interval of 0.8 cM. The transcriptome information and large-scale marker development in this study greatly expanded the genetic resources that are available for hydrangea. The high-density genetic linkage maps presented here will serve as an important foundation for quantitative trait loci mapping, map-based gene cloning, and marker-assisted selection of H. macrophylla.

Identification and Characterization of Simple Sequence Repeats (SSR) Markers Associated with Downy Mildew Resistance Locus “<i>RPV1</i>” in Grapes

The cultivation of grapes is severely impacted by the emergence of downy mildew (DM) disease which negatively affects quality and yield possibly resulting in heavy losses. Due to certain shortcomings in the usage of fungicides and the development of new cultivars by plant breeding, marker assisted selection (MAS) will be an efficient alternative method to introduce desired genes into the cultivated varieties in a short time period. The Simple sequence repeats (SSR) markers seem to be the most popular genetic marker of choice for MAS. In the present study, we identified 14 new SSR markers in RPV1 locus that are associated with downy mildew resistance in grapes. The characterization of the identified markers was carried out on the basis of various parameters such as types of repeat motifs, number of repeats, different classes and structure of microsatellites. Additionally, SSR genotyping in 56 different grape accessions was done to determine the susceptibility or resistance of these accessions to DM.

SSRgenotyper: A simple sequence repeat genotyping application for whole-genome resequencing and reduced representational sequencing projects.

PREMISEMany programs can identify simple sequence repeat (SSR) motifs in genomic data. SSRgenotyper extends SSR identification to en masse genotyping from resequencing data for diversity panels and linkage mapping populations.METHODS AND RESULTSSSRgenotyper will find and genotype SSRs from SAM files and an SSR reference FASTA. Several outputs are possible, including a simple table with the SSR marker name, position, and SSR alleles, defined by the repeat number of the repeat motif. Specific output files include a GENEPOP‐formatted file for downstream genetic diversity analyses and a traditional A, H, B mapping file output that is phased to the parents of the population for biparental linkage map construction. Linkage maps produced using SSRgenotyper genotypes were highly collinear with physical maps and correctly inferred known phylogenies.CONCLUSIONSSSRgenotyper provides an easy‐to‐use, accurate, and scalable SSR genotyping platform for whole‐genome resequencing data. SSRgenotyper is freely available at https://github.com/dlewis27/SSRgenotyper.

Genotypic diversity of the Asiatic black truffle, Tuber himalayense, collected in spontaneous and highly productive truffle grounds

Tuber himalayense is a species of edible Asiatic black truffle which is sister to Tuber indicum. Local populations contribute significantly to the reproduction of truffles, but fundamental data of fine-scale genetic structure is lacking in Asiatic truffle species. In this study, we provide the first report on the genotypic diversity and spatial fruiting pattern of an Asiatic truffle T. himalayense, in two spontaneous productive truffle grounds 140 m apart from one another in Yamanashi, Japan. Ascocarps were collected from 2004 to 2009 and in 2011. The spatial distribution of samples was recorded and genotypic diversity of the fungal individuals was examined using 15 newly developed and 4 existing simple sequence repeat (SSR) markers. The spatial distribution analyses suggested non-random, year-specific aggregation patterns of ascocarps in both plots. However, no consistent tendency was observed across the entire sampling period, with fruiting positions showing neither an obvious expansion nor a steady displacement. Although only a single SSR genotype, representing one gleba-forming maternal individual, was detected, the genotype was further divided into two different mating types, suggesting multiple, closely related individuals that were indistinguishable using the SSR analysis. The results indicated strong founding effects along with high inbreeding within populations, likely due to the limited dispersal opportunities into these sites. Despite low genetic diversity in the truffle grounds, the plots remained productive over the sampling years, indicating that low genetic diversity does not necessarily have an adverse effect on truffle production, at least not during a period of several years.

A new one-tube reaction kit for the SSR genotyping of apple (Malus × domestica Borkh.)

Though apple genotyping is mainly used for scientific and breeding purposes, it can also be adopted by national authorities to control the authenticity of apple cultivars. To facilitate the introduction of routine apple genotyping into practice, a new apple simple sequence repeat (SSR) genotyping kit was developed (called the Ap17 in. SSR Genotyping Kit). The kit combines 17 SSR markers including those recommended by the Working Group of the European Cooperative Programme for Plant Genetic Resources (ECPGR), covering all apple linkage groups in a one-tube reaction format, using a fragment analysis method to simplify the genotyping procedure. The kit was successfully tested using 880 unique diploid apple germplasm accessions; the kit can also readily discriminate triploid and tetraploid samples. The total probability of identity for the kit and the sample collection used was calculated to be 1.73 × 10-22. Tables for converting results to enable genotype comparisons between currently-used genotyping systems and the Ap17 in. kit are provided. The kit is ideally suited for validation in laboratories genotyping a large number of apple samples, saving time, costs, and labor, while minimizing technical and human errors.

A study of the genetic diversity in the world soybean collection using microsatellite markers associated with fungal disease resistance

Background.Soybean (Glycine max (L.) Merr.) gradually becomes one of the leading legume crops in Kazakhstan. The area under soybeans in the country has been increasing annually and requires the development of adapted cultivars with a higher yield, improved quality characters, and resistance to emerging fungal diseases. The enlargement of the crop’s gene pool also suggests the need to study and document local soybean accessions to meet the standards of the available world soybean collection by using reliable and informative types of DNA markers.Materials and methods. In this study, the soybean collection consisting of 288 accessions from different countries, including 36 cultivars and promising lines from Kazakhstan, was studied. The molecular genetic analysis was performed using nine polymorphic SSR (simple sequence repeats) markers, seven of which (Satt244, Satt565, Satt038, Satt309, Satt371, Satt570 and Sat_308) were associated with resistance to three main fungal diseases of soybean – frogeye leaf spot, fusarium root rot, and purple seed stain.Results. The average PIC (polymorphism information content) value of the analyzed SSR markers constituted 0.66 ± 0.07, confirming their highlevel polymorphism. The principal coordinate analysis suggested that the local accessions were genetically most close to the accessions from East Asia. As the collection showed a robust resistance to three studied fungal diseases in Almaty Region during 2018–2019, the distribution of the studied SSR markers in the population was not significantly associated with resistance to the analyzed diseases under field conditions.Conclusion. SSR genotyping of the soybean collection helped to identify accessions that potentially possess resistance-associated alleles of fungal disease resistance genes. The data obtained can be further used for the development of DNA documentation and the breeding the promising cultivars and lines of soybean.

Molecular mapping of QTLs for yield related traits in recombinant inbred line (RIL) population derived from the popular rice hybrid KRH-2 and their validation through SNP genotyping

The study was undertaken to identify the quantitative trait loci (QTLs) governing yield and its related traits using a recombinant inbred line (RIL) population derived from the popular rice hybrid, KRH-2 (IR58025A/KMR3R). A genetic map spanning 294.2 cM was constructed with 126 simple sequence repeats (SSR) loci uniformly distributed across the rice genome. QTL analysis using phenotyping and genotyping information identified a total of 22 QTLs. Of these, five major effect QTLs were identified for the following traits: total grain yield/plant (qYLD3-1), panicle weight (qPW3-1), plant height (qPH12-1), flag leaf width (qFLW4-1) and panicle length (qPL3-1), explaining 20.23–22.76% of the phenotypic variance with LOD scores range of 6.5–10.59. Few genomic regions controlling several traits (QTL hotspot) were identified on chromosome 3 for total grain yield/plant (qYLD3-1) and panicle length (qPL3-1). Significant epistatic interactions were also observed for total grain yield per plant (YLD) and panicle length (PL). While most of these QTLs were observed to be co-localized with the previously reported QTL regions, a novel, major QTL associated with panicle length (qPL3-1) was also identified. SNP genotyping of selected high and low yielding RILs and their QTL mapping with 1,082 SNPs validated most of the QTLs identified through SSR genotyping. This facilitated the identification of novel major effect QTLs with much better resolution and precision. In-silico analysis of novel QTLs revealed the biological functions of the putative candidate gene (s) associated with selected traits. Most of the high-yielding RILs possessing the major yield related QTLs were identified to be complete restorers, indicating their possible utilization in development of superior rice hybrids.

The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.).

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.

Studies on genetic variability of Capsicum frutescens var. Cakra Hijau induced by ethyl methane sulphonate (EMS) using SSR marker

Genetic variability of wild type and EMS induced mutant cayenne peppers (Capsicum frutescens) var. Cakra Hijau is studied morphological traits and simple sequence repeat (SSR) markers. Phenotypic characters were investigated using standard pepper descriptors. The polymorphic SSR marker Ca19, Ca26, Ca52, Ca56, and Ca96were analyzed using unweighted pair group methods with arithmetic means (UPGMA) with Jaccard’s similarity index. The Polymorphic information content (PIC) value ranging from 0-0.228 with the highest index on Ca96. The Morphological traits showed of clusters (6 clades) with a lower cut-off value (0.2568) than SSR-based dendrogram did (4 clades; 0,5108). Therefore, genetic variability induced by EMS mutant were differentiated using morphological and SSR genotyping.

Phenological shifts and genetic differentiation between sympatric populations of Sargassum horneri (Fucales, Phaeophyceae) in Japan

Marked seasonality, especially in sexual reproduction, is common among seaweed species along temperate coasts and increases the possibility of successful fertilization in outcrossing species. A phenological shift in reproductive seasons, therefore, could be an effective isolation barrier between conspecific seasonal populations, although its power has not been verified in algae.Sargassum horneri, a major component of seaweed beds along the temperate coast of Japan, is known for variability in its reproductive phenology. To understand the significance of phenological shift as an isolation barrier in seaweed species, phenological investigations ofS. horneriseasonal populations on the Sea of Japan coast of central Honshu, Japan, were combined with Bayesian cluster analysis based on a nuclear simple sequence repeat genotype. Results from these analyses concordantly suggest a genetic differentiation between the seasonal populations, although almost 20% of field-collected plants were estimated to be hybrids or have a hybrid origin based on results of Bayesian cluster analyses using experimental hybrids. A collapse of seasonal isolation was also detected at the site of the field investigation, and a high percentage of putative hybrids in the following generation at the site (41%) suggested significant seasonal isolation in the differentiation observed in this study.

Genetic authentication of cultivars with flower-variant types using SSR markers in spring orchid, Cymbidium goeringii

Spring orchid, Cymbidium goeringii, is one of the most important and popular species in the orchid family in Northeast Asia. The purpose of this study was to determine how closely the simple sequence repeat (SSR) genotypes match between individual plants assumed to be the same cultivar in C. goeringii. For this study, we collected 1566 samples from 17 C. goeringii cultivars of the flower-variant types and determined the frequencies and distribution of the combined genotypes of 13 SSR loci by the multiplex PCR method. The phylogenetic distance matrix and the principal coordinate analysis revealed that the correlation between horticultural phenotypes and genetic distance was loosely matched, but two cultivars of Japanese origin were clearly separated from the Korean cultivars. The results showed that the ratio of the most frequent combined genotypes in 10 cultivar groups is approximately 60%, which suggests that almost 40% of C. goeringii sold may not be genuine. This is the first forensic attempt to authenticate whether each plant is genetically genuine for the corresponding cultivars in spring orchid. We suggest preparation of a guideline for DNA deposit and profiling of the SSR genotypes for the newly registered C. goeringii cultivars and believe that this study will be helpful for establishing a molecular genetic method for forensic authentication of these cultivars.

First Report of Race TTRTF of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in Eritrea

HomePlant DiseaseVol. 104, No. 3First Report of Race TTRTF of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in Eritrea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Race TTRTF of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in EritreaMehran Patpour, Annemarie F. Justesen, Asmelash W. Tecle, Mahboobeh Yazdani, Mohsen Yasaie, and Mogens S. HovmøllerMehran Patpour†Corresponding author: M. Patpour; E-mail Address: mehran.patpour@agro.au.dkhttp://orcid.org/0000-0002-4914-2697Global Rust Reference Center, Department of Agroecology, University of Aarhus, DenmarkSearch for more papers by this author, Annemarie F. JustesenGlobal Rust Reference Center, Department of Agroecology, University of Aarhus, DenmarkSearch for more papers by this author, Asmelash W. TecleMinistry of Agriculture, National Agricultural Research Institute, EritreaSearch for more papers by this author, Mahboobeh YazdaniFars Agriculture Organization, IranSearch for more papers by this author, Mohsen YasaieSeed and Plant Improvement Research Dep., Fars Agricultural and Natural Resources Research and Education Center, Shiraz, IranSearch for more papers by this author, and Mogens S. HovmøllerGlobal Rust Reference Center, Department of Agroecology, University of Aarhus, DenmarkSearch for more papers by this author AffiliationsAuthors and Affiliations Mehran Patpour1 † Annemarie F. Justesen1 Asmelash W. Tecle2 Mahboobeh Yazdani3 Mohsen Yasaie4 Mogens S. Hovmøller1 1Global Rust Reference Center, Department of Agroecology, University of Aarhus, Denmark 2Ministry of Agriculture, National Agricultural Research Institute, Eritrea 3Fars Agriculture Organization, Iran 4Seed and Plant Improvement Research Dep., Fars Agricultural and Natural Resources Research and Education Center, Shiraz, Iran Published Online:7 Jan 2020https://doi.org/10.1094/PDIS-10-19-2133-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Race TTRTF of Puccinia graminis f. sp. tritici was first detected in 2016 in a severe outbreak of wheat stem rust on thousands of hectares of durum and bread wheat in Sicily, Italy (Bhattacharya 2017; Patpour et al. 2018). However, a recent study suggested that TTRTF was already present in Georgia in 2014 (Olivera et al. 2019). During 2016 to 2018, 140 samples of rust-infected wheat and barley were collected from field trials and farmers’ fields in Eritrea and submitted to the Global Rust Reference Center (GRRC), Denmark. Fifty-one urediniospore isolates were derived from 62 samples recovered on the susceptible wheat cultivars Morocco and Line E. Race typing was completed for 40 isolates based on 20 North American wheat lines using the standard race-typing procedures and infection type (IT) criteria determining virulence and avirulence (Jin et al. 2008). All isolates except two, representing 24 sampling sites, were diagnosed as TTRTF, whereas two samples collected from farmers’ fields in Metera and Temajila were identified as TKKTF and TTKSK, respectively. In April 2019, two stem rust samples were collected from wheat in Fars province, Iran. After submission to GRRC, two single-pustule isolates per sample were raised and race typed as described above. All isolates were determined to be race TTRTF, previously reported without data documentation (Roohparvar and Omrani 2018). This is the first report of race TTRTF in Eritrea (2016) and southern Iran (2019). Six isolates of TTRTF from Eritrea and two from Iran were tested for additional virulence using 24 supplementary lines and cultivars containing Sr7a, Sr13a, Sr22, Sr25, Sr26, Sr27, Sr31, Sr32, Sr33, Sr35, Sr37, Sr39, Sr40, Sr44, Sr45, Sr47, SrSatu, Sr1RSAmigo, Sr50, Siouxland (Sr24+31), Sisson (Sr31+36), and Triumph 64 (SrTmp). Durum wheat Iumillo (Sr9g,12,+) and Leeds (Sr9e,13b,+) were also included in the experiment. The results were consistent for all isolates across two to three independent experiments. In summary, TTRTF had virulence for 23 Sr genes (IT 3 or higher): Sr5, Sr6, Sr7a, Sr7b, Sr8a, Sr9a, Sr9b, Sr9d, Sr9e, Sr9g, Sr10, Sr11, Sr13b, Sr17, Sr21, Sr35, Sr36, Sr37, Sr38, Sr44, Sr45, SrTmp, and SrMcN. Fed*3/Gabo*51BL.1RS-1-1 (Sr50) showed IT 3-3, which is significantly higher than for typical avirulent isolates. Simple sequence repeat genotyping of TTRTF isolates confirmed that they belonged to clade III-B defined by Olivera et al. (2019). The occurrence of TTRTF in Eritrea and Iran in 2016 and 2019, respectively, extends the known distribution of this race (sampling year in parenthesis): Italy (2016), Georgia (2014), Hungary (2017) (Olivera et al. 2019), and Egypt (2015 to 2016) (Esmail and Szabo 2018). TTRTF is considered a serious threat to wheat productivity in affected areas due to a broad virulence spectrum, including virulence to Sr13b from durum wheat and higher than usual IT for Sr50. Detection of TTRTF in different regions since 2014 stresses the high potential of onward spread to new areas and imminent risks for new epidemic outbreaks of wheat stem rust in many countries, because they are highly dependent on wheat as main source for food and feed. There is an urgent need to sustain wheat rust surveillance efforts globally and to identify effective sources of resistance to stem rust in cereal germplasm.The author(s) declare no conflict of interest.

Genetic Structure and Subclonal Variation of Extant and Recent U.S. Lineages of Phytophthora infestans.

The oomycete Phytophthora infestans is an important plant pathogen on potato and tomato crops. We examined the genetic structure of extant 20th and 21st century U.S. lineages of P. infestans and compared them with populations from South America and Mexico to examine genetic relationships and potential sources of lineages. US-23, currently the most prevalent lineage detected in the United States, shared genetic similarity primarily with the BR-1 lineage identified in the 1990s from Bolivia and Brazil. Lineages US-8, US-14, and US-24, predominantly virulent on potato, formed a cluster distinct from other U.S. lineages. Many of the other U.S. lineages shared significant genetic similarity with Mexican populations. The US-1 lineage, dominant in the mid-20th century, clustered with US-1 lineages from Peru. A survey of the presence of RXLR effector PiAVR2 revealed that some lineages carried PiAVR2, its resistance-breaking variant PiAVR2-like, or both. Minimum spanning networks developed from simple sequence repeat genotype datasets from USABlight outbreaks clearly showed the expansion of US-23 over a 6-year time period and geographic substructuring of some lineages in the western United States. Many clonal lineages of P. infestans in the United States have come from introductions from Mexico, but the US-23 and US-1 lineages were most likely introduced from other sources.

Population Genetic Analysis of Phytophthora parasitica From Tobacco in Chongqing, Southwestern China.

Tobacco black shank, caused by Phytophthora parasitica, is one of the most notorious tobacco diseases and causes huge economic losses worldwide. Understanding the genetic variation of P. parasitica populations is essential to the development of disease control measures. In this research, 210 simple sequence repeat (SSR) markers for P. parasitica were identified, 10 of which were polymorphic among nine reference strains. We further performed population genetic analysis of 245 P. parasitica isolates randomly collected from tobacco fields in Chongqing for mating type, molecular variation at 14 SSR loci (four of which were identified previously), and sensitivity to the fungicide metalaxyl. The results showed that the A2 mating type was dominant and no A1 mating type isolate was discovered. SSR genotyping distinguished 245 P. parasitica isolates into 46 genotypes, four of which were dominant in the population. Low genotypic diversity and excess heterozygosity were common in nearly all of the populations from Chongqing. Population analysis showed that no differentiation existed among different populations. All isolates tested were highly sensitive to metalaxyl. Taken together, our results showed that the P. parasitica populations from tobacco fields in Chongqing belonged to a clonal lineage and were highly sensitive to metalaxyl.

Target SSR-Seq: A Novel SSR Genotyping Technology Associate With Perfect SSRs in Genetic Analysis of Cucumber Varieties.

Simple sequence repeats (SSR) – also known as microsatellites – have been used extensively in genetic analysis, fine mapping, quantitative trait locus (QTL) mapping, as well as marker-assisted selection (MAS) breeding and other techniques. Despite a plethora of studies reporting that perfect SSRs with stable motifs and flanking sequences are more efficient for genetic research, the lack of a high throughput technology for SSR genotyping has limited their use as genetic targets in many crops. In this study, we developed a technology called Target SSR-seq that combined the multiplexed amplification of perfect SSRs with high throughput sequencing. This method can genotype plenty of SSR loci in hundreds of samples with highly accurate results, due to the substantial coverage afforded by high throughput sequencing. We also detected 844 perfect SSRs based on 182 resequencing datasets in cucumber, of which 91 SSRs were selected for Target SSR-seq. Finally, 122 SSRs, including 31 SSRs for varieties identification, were used to genotype 382 key cucumber varieties readily available in Chinese markets using our Target SSR-seq method. Libraries of PCR products were constructed and then sequenced on the Illumina HiSeq X Ten platform. Bioinformatics analysis revealed that 111 filtered SSRs were accurately genotyped with an average coverage of 1289× at an extremely low cost; furthermore, 398 alleles were observed in 382 cucumber cultivars. Genetic analysis identified four populations: northern China type, southern China type, European type, and Xishuangbanna type. Moreover, we acquired a set of 16 core SSRs for the identification of 382 cucumber varieties, of which 42 were isolated as backbone cucumber varieties. This study demonstrated that Target SSR-seq is a novel and efficient method for genetic research.

Systematics of the Widely Distributed Japanese Clouded Salamander, Hynobius nebulosus (Amphibia: Caudata: Hynobiidae), and Its Closest Relatives

The abundant clouded salamander from western Japan, Hynobius nebulosus, has long been considered a single widespread species, although some authors have suggested the inclusion of several cryptic species. This led to a molecular and morphological analysis of populations from all parts of the known range. Phylogenetic relationships were inferred from complete sequences of the mitochondrial cytochrome b gene, and nuclear genome differentiations were estimated by multiplexed inter simple sequence repeat genotyping by sequencing (MIG-seq). The results suggest that H. nebulosus hitherto recognized consists of at least nine species. We applied existing names to two of them, H. nebulosus (Temminck et Schlegel) and H. vandenburghi Dunn, and described seven others as new species.

Construction of Fingerprint of <i>Michelia</i> Germplasm by Fluorescent SSR Markers

In this study, a fingerprinting system for 16 Michelia germplasms was established using high-throughput genotyping of fluorescent simple sequence repeat (SSR) markers. In total, 102 alleles were detected using 13 pairs of SSR markers from closely related species, and the number of alleles per locus ranged from 5 to 11, with an average of 7.8 alleles. Observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) ranged from 0.1250 to 0.5625 (mean 0.3650), 0.6703 to 0.9113 (mean 0.8099), and 0.5748 to 0.8714 (mean 0.7515), respectively. Among the four selected core primer pairs, the combinations LT106 and SGA5, LT106 and LT58, and SGA5 and MMA51 could unambiguously distinguish 16 Michelia germplasms. A cluster analysis showed the similarity coefficient of the 16 Michelia germplasms to range from 0.70 to 0.90, and different individuals from the same species clustered in the same branch. The fluorescence SSR genotyping system established in this study was efficient, rapid, and accurate; moreover, this approach provides a theoretical basis for identifying germplasms and for the protection of new varieties of the genus Michelia , and it provides a robust foundation for further breeding development of Michelia sp.