Using SSR markers to evaluate the genetic diversity of Lentinula edodes’ natural germplasm in China

In this study, microsatellite markers were utilized to reveal the genetic diversity of 56 strains of Lentinula edodes grown in China. A total of 224 DNA bands were detected through 25 primer pairs, of which, 223 bands (99.6%) were polymorphic between two or more strains. The variation in SSR (Simple Sequence Repeat) DNA band patterns and average genetic similarity among the wild strains of L. edodes obtained from the same region uncovered a vast genetic diversity in the natural germplasm found in China. Compared with L. edodes strains from other areas, the genetic diversity of those from the Yunnan Plateau, Hengduanshan mountains, Taiwan, and south China was significantly greater. Based on cluster analysis and principal coordinate analysis, the results indicated that all L. edodes strains could be divided into three major groups. These results effectively displayed the differences between the strains from North and South China, and those from the same or adjoining regions could cluster preferentially into small groups in most cases, suggesting the positive correlation between the cluster results and the geographical origin for the natural germplasm of Chinese L. edodes. To our knowledge, this is a pioneering report on the utilization of the SSR marker technique in analyzing L. edodes’ genetic diversity.     

Development and characterization of microsatellite markers in sawih tree (Duabanga moluccana Blume) using ISSR-suppression PCR techniques

Duabanga moluccana (or locally known as sawih) is an indigenous fast growing tropical tree species that confers various advantages for the timber industry and for planted forests development. In this paper, we isolated and characterized 8 polymorphic microsatellite markers from the D. moluccana genome using ISSR-suppression PCR techniques. The number of alleles and PIC values ranged from 3 to 8 alleles per locus and from 0.488 to 0.792, respectively. Three microsatellite loci were deviated from Hardy-Weinberg equilibrium (P < 0.05). The transferability rate ranged from 24 to 100 % among the three indigenous tree species tested. This indicates that the newly developed microsatellite markers would be useful tools for population genetic studies on D. moluccana and other indigenous tree species.     

The commercial exploitation of the white rot fungus Lentinula edodes (shiitake)

The Japanese wood mushroom, Lentinula edodes, can be cultivated under UK climatic conditions using extensive cultivation systems adapted from traditional growing methods developed in Japan and Taiwan. As well as producing a desirable and high value food product, cultivation offers a viable alternative enterprise for farmers. The growing methods described could provide incentives for the management of broad-leaved woodland in Britain with consequent conservation benefits. Four different strains of Lentinula edodes were grown on oak logs under natural environmental conditions below the woodland canopy and fruited in polyethylene tunnels. Yields ranged from 0.5–0.9 kg wet weight shiitake per cordwood log per year over a three year fruiting period.     

First microsatellite markers developed and applied for the genetic diversity study and population structure of Didymella pisi associated with ascochyta blight of dry pea in Montana

Didymella pisi is the predominant causal pathogen of ascochyta blight of dry pea causing yield losses in Montana, where 415 000 acres were planted to dry pea in 2018. Thirty-three microsatellite markers were developed for dry pea pathogenic fungus, Didymella pisi, these markers were used to analyze genetic diversity and population structure of 205 isolates from four different geographical regions of Montana. These loci produced a total of 216 alleles with an average of 1.63 alleles per microsatellite marker. The polymorphic information content values ranged from 0.020 to 0.990 with an average of 0.323. The average observed heterozygosity across all loci varied from 0.000 to 0.018. The gene diversity among the loci ranged from 0.003 to 0.461. Unweighted Neighbor-joining and population structure analysis grouped these 205 isolates into two major sub-groups. The clusters did not match the geographic origin of the isolates. Analysis of molecular variance showed 85 % of the total variation within populations and only 15 % among populations. There was moderate genetic variation in the total populations (PhiPT = 0.153). Information obtained from this study could be useful as a base to design strategies for improved management such as breeding for resistance to ascochyta blight of dry pea in Montana.     

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (F_ST = 0.049) as evidenced by high level of gene flow estimate (N_m = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.     

Genetic architecture of resistance to Septoria tritici blotch (Mycosphaerella graminicola) in European winter wheat

Genome-wide marker–trait associations (MTA) were established in a population of 358 European winter wheat cultivars and 14 spring wheat cultivars (Triticum aestivum L.) for resistance to Septoria tritici blotch caused by the fungal pathogen Mycosphaerella graminicola. The MTA were based on field data in two consecutive years and genotypic data on 732 microsatellite markers. Best linear unbiased estimations (BLUEs) for resistance were calculated across the trials and ranged from 0.67 (most resistant) to 19.63 (most susceptible) with an average value of 4.93. A total of 115 MTA relating to 68 molecular markers was discovered for the two trials and BLUEs by using a mixed linear model corrected by a kinship matrix. In addition, two candidate genes, Ppd-D1 for photoperiodism and the dwarfing gene Rht-D1, were significantly associated with resistance to Septoria tritici blotch. Several MTA co-located with known resistance genes, e.g. Stb1, 3, 4, 6 and 8, while multiple additional MTA were discovered on several chromosomes, such as 2A, 2D, 3A, 5B, 7A and 7D. The results provide proof of concept for the method of genome-wide association analysis and indicate the presence of further Stb resistance genes in the European winter wheat pool.     

Linkage mapping of rat Chromosome 5 markers generated from chromosome-specific libraries

Seventy-six novel microsatellite markers with various simple sequence repeat (SSR) motifs are reported in this paper. They were generated on the basis of non-radioactive library screening procedures from flow-sorted rat Chromosome (Chr) 5-specific DNA, and were mapped in three rat backcross populations. Fifty-four of these markers mapped to Chr 5, while the other 22 mapped to other chromosomes of the rat genome. The marker D3Uwm8 is a new microsatellite marker for the rat syndecan 4 (ryudocan) gene. A genotyping protocol based on agarose gel electrophoresis is also provided in this paper. Received: 17 December 1998 / Accepted: 17 February 1999     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Characterization of microsatellite loci in the western tarnished plant bug,Lygus hesperus Knight (Hemiptera: Miridae)

A microsatellite‐enriched partial genomic DNA library of Lygus hesperus was generated and screened by sequencing. Ten polymorphic microsatellite marker loci were characterized by genotyping 92 insect samples. The observed number of alleles ranged from three to seven with an average of 4.5 (SE ± 0.45), while the effective number of alleles ranged from 1.21 to 3.02 with an average of 2.14 (SE ± 0.20). Significant deviations from Hardy–Weinberg expectations were detected at three loci. Significant linkage disequilibrium was also detected between the loci LhMS2‐54 and LhMS3‐32. Seven of the L. hesperus markers could be transferred to Lygus lineolaris.     

Determination of the minimum number of microsatellite markers for individual genotyping in wild boar (Sus scrofa) using a test with close relatives

In the context of developing a noninvasive, practicable method for population size estimation in wild boar, we present a stepwise procedure to reduce the number of required microsatellite markers for individual genotyping. Step1: an initial marker set of 12 microsatellite loci was tested for species specificity with nontarget DNA and resulted in an exclusion of two markers. Step 2: a variability test regarding heterozygosity and deviations from Hardy–Weinberg equilibrium led to the rejection of two further markers. Step 3: the remaining eight markers were tested for transferability across populations with three separate wild boar sample sets. Step 4: on the basis of probability of identity values, a reduction from eight to five markers was possible. Step 5: a novel test using tissue samples from female wild boars and their embryos provided evidence that four variable microsatellite markers and one sex marker are sufficient for individual identification of close relatives. Step 6: feces samples were finally used to estimate PCR (PS) and genotyping success (GS). In conclusion, we recommend a specific four-marker combination with both PS and GS >50% for a reliable individual identification in noninvasive population size estimation of wild boar.     

跨种扩增筛选大鳄龟微卫星标记及遗传多样性分析

跨种扩增是一种能够快速、有效地获得物种微卫星标记的方法。本研究利用在近缘种中已发表的微卫星DNA引物,对大鳄龟（Macroclemys temminckii）进行跨种PCR扩增,在合成的69对引物中获得8对具有多态性的微卫星位点。对PCR扩增产物进行统计,得出观测杂合度（H_o）的范围是0.041 7~0.954 5,平均为0.384 8;期望杂合度（H_E）的范围为0.041 7~0.811 8,平均为0.479 1;多态信息含量范围为0.040 0~0.759 2,平均为0.423 2;经过卡方检验后,部分微卫星位点符合哈迪-温伯格平衡。总体来说,这些位点是研究大鳄龟遗传结构的良好分子标记。     

Non-destructive analysis of the moisture content in shiitake mushrooms (Lentinula edodes) using near-infrared imaging at 1450 nm

Moisture content (MC) is an important factor in the freshness of shiitake mushrooms (Lentinula edodes). In this study, we developed a simple prediction method for MC of shiitake mushrooms using near-infrared (NIR) imaging at 1450 nm, as the band at 1450 nm is strongly correlated with OH groups. First, a NIR image was captured by an InGaAs sensor with a bandpass filter of 1450 nm; the captured image was then calibrated using an image taken at 1100 nm to remove shade and shape effects of mushrooms. The actual MC of shiitake mushrooms was measured using an oven-drying method, and a calibration curve was obtained for the cap surface and the gill side, respectively. The correlation coefficient reached more than 0.85, and the standard error of calibration ranged from 3.1% to 8.2%. Non-destructive MC images were quantified using the constructed calibration curves, and the experimental results showed that differences in the water-holding property can be visualized over 6 h for each side using our method.     

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (H_O?=?0.312; H_E?=?0.436) and B. subterranea presented the highest values (H_O?=?0.687; H_E?=?0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q?≥?0.976) and the low combined probability of identity (I?≤?9.91?×?10^–6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

     

Analysis of microsatellite loci in tree of heaven (<Emphasis Type="Italic">Ailanthus altissima</Emphasis> (Mill.) Swingle) using SSR-GBS

Microsatellite markers are still the marker of choice for many research questions in the field of forest genetics. However, the number of available markers is often low for species that have not been studied intensively like the tree of heaven (Ailanthus altissima). During the last decade, next-generation sequencing (NGS) has offered advanced techniques for efficiently identifying microsatellite markers and accurately genotyping samples. Here, we identify new microsatellite markers for the tree of heaven by applying an NGS-based method using the Illumina MiSeq platform. NGS technology was proved to be an effective method for fast and cost-efficient identification of microsatellite markers by implementing a genotyping-by-sequencing approach based on Illumina amplicon sequencing (SSR-GBS). We screened three populations from Eastern Austria for genetic variation at 19 newly identified microsatellite loci. We tested two different genotyping approaches: (1) considering only allele lengths (forming a so-called “allele length dataset”), (2) taking also single nucleotide polymorphisms (SNPs) within the amplified fragments into account (forming a so-called “SNP dataset”). The results revealed higher values for all genetic diversity parameters, as well as a better resolution of genetic assignment, when the latter approach was followed. Thus, by taking advantage of sequence information which is provided by SSR-GBS, one may achieve considerable gains in performance using the same marker set. The developed markers provide a cost-efficient tool for genotyping populations of tree of heaven and the approach presented here promises to be of high value for medium throughput genotyping applications in non-model forest tree species. We will use this method to widen the perspectives for further population genetic investigations of the tree of heaven.     

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.     

Construction of a linkage map of Lentinula edodes (shiitake) with the HEGS (high-efficiency genome scanning) system: use of versatile AFLP and PCR-based gene markers

An improved linkage map of Lentinula edodes (shiitake) was constructed with an HEGS (high-efficiency genome scanning) system. Two hundred twenty-one HEGS-derived amplified fragment length polymorphism (AFLP-H) markers and 21 gene markers were developed and combined with 203 previously developed sequencer-derived AFLP markers (AFLP-S markers) and 3 mating factor loci (A, Bα, and Bβ) to construct a comprehensive linkage analysis. As a result, a novel linkage map with 166 markers including 2 mating factors (A and B), 10 HEGS-derived gene markers, 72 AFLP-H markers, and 82 AFLP-S markers was obtained. Of the total 448 markers, 273 could not be located on a linear map and thus were assigned to linkage groups as accessory markers. The map covers a total length of 1398.4 centimorgans (cM) with an average marker interval distance of 8.4 cM. The map consists of 11 linkage groups (LGs) in agreement with our previous map, and 7 LGs among them were found to contain branched linkages, which may be the result of reciprocal translocations representing dynamic reorganization of the shiitake genome. The previously reported linkage map was improved in terms of number of markers, marker density, linear order of markers, and total map length. Contribution no. 384 of the Tottori Mycological Institute     

Linkage map of human chromosome 9 microsatellite polymorphisms.

Ten microsatellite markers composed of polymorphic (CA)n or (AAAT)n repeats were mapped to chromosome 9. PIC values for these markers ranged from 0.46 to 0.82. The marker at the D9S54 locus was localized to 9pter-p22 by means of a somatic cell hybrid; another marker at D9S103 was similarly localized to 9q34-qter. Two-point lod scores and individual meiotic recombination events were used to position the 10 markers relative to each other. The best order resulting from these analyses was D9S54-D9S104-[D9S52-D9S43-D9S50]-D9S53+ ++- [D9S106-D9S105]-D9S51-D9S103, with order of the loci within brackets uncertain. Two-point linkage analysis was also used to approximate the positions of the microsatellite markers relative to those of 33 markers contained in the public CEPH database (v.3) and to one other available microsatellite marker at the D9S15 locus.     

Genetic characteristics of 32 autosomal STR loci and its application for identifying a locus in hereditary hearing loss in the Korean population

Microsatellites, short tandem repeats, are useful markers for genetic analysis because of their high frequency of occurrence over the genome, high information content due to variable repeat lengths, and ease of typing. To establish a panel of microsatellite markers useful for genetic studies for hereditary hearing loss in the Korean population, the allele frequencies and heterozygosities of 32 microsatellite markers in 172 unrelated Korean individuals were examined. The heterozygosity values for these markers ranged from 48 to 87%. All the markers except D6S1038 and D14S1034 marker showed PIC values over 0.5. This indicates these markers have a high degree of polymorphism and are randomly distributed in the Korean population. Therefore, the combinations of these STR loci provide a powerful tool to find the candidate loci of a causative gens for non-syndromic hearing loss in the Korean population.     

Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values <2800 from those with OCHU values >2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.