Development Of Single Nucleotide Polymorphism Markers Research Articles

Development of Cost-Effective SNP Markers for Genetic Variation Analysis and Variety Identification in Cultivated Pears (Pyrus spp.).

Pear (Pyrus spp.) is a major fruit crop in the Rosaceae family, and extensive efforts have been undertaken to develop elite varieties. With advances in genome sequencing technologies, single-nucleotide polymorphisms (SNPs) are commonly used as DNA markers in crop species. In this study, a large-scale discovery of SNPs was conducted using genotyping by sequencing in a collection of 48 cultivated pear accessions. A total of 256,538 confident SNPs were found on 17 chromosomes, and 288 SNPs were filtered based on polymorphic information content, heterozygosity rate, and genome distribution. This subset of SNPs was used to genotype an additional 144 accessions, consisting of P. pyrifolia (53), P. ussuriensis (27), P. bretschneideri (19), P. communis (26), interspecific hybrids (14), and others (5). The 232 SNPs with reliable polymorphisms revealed genetic variations between and within species in the 192 pear accessions. The Asian species (P. pyrifolia, P. ussuriensis, and P. bretschneideri) and interspecific hybrids were genetically differentiated from the European species (P. communis). Furthermore, the P. pyrifolia population showed higher genetic diversity relative to the other populations. The 232 SNPs and four subsets (192, 96, 48, and 24 SNPs) were assessed for variety identification. The 192 SNP subset identified 173 (90.1%) of 192 accessions, which was comparable to 175 (91.1%) from the 232 SNPs. The other three subsets showed 81.8% (24 SNPs) to 87.5% (96 SNPs) identification rates. The resulting SNPs will be a useful resource to investigate genetic variations and develop an efficient DNA barcoding system for variety identification in cultivated pears.

Development of Single-Nucleotide Polymorphism Markers and Population Genetic Analysis of the Hadal Amphipod Alicella gigantea across the Mariana and New Britain Trenches

Alicella gigantea, the largest amphipod scavengers found to date, play key roles in the food web of the hadal ecosystem. However, the genetic structure of A. gigantea populations among different trenches has not been reported yet. In this study, SNP (single-nucleotide polymorphism) markers were developed for three A. gigantea geographic populations collected from the southern Mariana Trench (SMT), the central New Britain Trench (CNBT), and the eastern New Britain Trench (ENBT), based on the SLAF-seq (specific locus amplified fragment sequencing) technology. A total of 570,168 filtered SNPs were screened out for subsequent population genetic analysis. Results showed that the inbreeding levels across the three geographic populations were relatively low, and the genomic inbreeding coefficients of the three populations were similar in magnitude. Based on the results of phylogenetic analysis, population structure analysis, and principal component analysis, it is believed that the three A. gigantea geographic populations belong to the same population, and the kinship relationship between the ENBT and CNBT populations is close. Moreover, the differential candidate adaptive sites on the SNPs suggest that there may be variations in metabolic rates among the three geographic populations, possibly linked to differences in food availability and sources in different trenches, ultimately resulting in different survival strategies in A. gigantea populations within distinct trenches. Compared with the Mariana Trench, the New Britain Trench has a richer organic matter input, and it is speculated that the A. gigantea Mariana Trench population may adopt a lower metabolic rate to cope with the harsher environment of nutrient deficiency.

A Scalable and Robust Chloroplast Genotyping Solution: Development and Application of SNP and InDel Markers in the Maize Chloroplast Genome.

Maize(Zea mays. L) is a globally important crop, and understanding its genetic diversity is crucial for plant breeding phylogenetic analyses and comparative genetics. While nuclear markers have been extensively used for mapping agriculturally important genes, they are limited in recognizing characteristics, such as cytoplasmic male sterility and reciprocal cross hybrids. In this study, we performed next-generation sequencing of 176samples, and the maize cultivars represented five distinct groups. A total of 89 single nucleotide polymorphisms (SNPs) and 11 insertion/deletion polymorphisms (InDels) were identified. To enable high-throughput detection, we successfully amplified and confirmed 49 SNP and InDel markers, which were defined as a Varietal Chloroplast Panel (VCP) using the Kompetitive Allele Specific PCR (KASP). The specific markers provided a valuable tool for identifying chloroplast groups. The verification experiment, focusing on the identification of reciprocal cross hybrids and cytoplasmic male sterility hybrids, demonstrated the significant advantages of VCP markers in maternal inheritance characterization. Furthermore, only a small subset of these markers is needed to provide useful information, showcasing the effectiveness of these markers in elucidating the artificial selection process of elite maize lines.

Development of SNP and InDel markers by genome resequencing and transcriptome sequencing in radish (Raphanus sativus L.)

BackgroundSingle nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the most abundant genetic variations and widely distribute across the genomes in plant. Development of SNP and InDel markers is a valuable tool for genetics and genomic research in radish (Raphanus sativus L.).ResultsIn this study, a total of 366,679 single nucleotide polymorphisms (SNPs) and 97,973 insertion-deletion (InDel) markers were identified based on genome resequencing between ‘YZH’ and ‘XHT’. In all, 53,343 SNPs and 4,257 InDels were detected in two cultivars by transcriptome sequencing. Among the InDel variations, 85 genomic and 15 transcriptomic InDels were newly developed and validated PCR. The 100 polymorphic InDels markers generated 207 alleles among 200 Chinese radish germplasm, with an average 2.07 of the number of alleles (Na) and with an average 0.33 of the polymorphism information content (PIC). Population structure and phylogenetic relationship revealed that the radish cultivars from northern China were clustered together and the southwest China cultivars were clustered together. RNA-Seq analysis revealed that 11,003 differentially expressed genes (DEGs) were identified between the two cultivars, of which 5,020 were upregulated and 5,983 were downregulated. In total, 145 flowering time-related DGEs were detected, most of which were involved in flowering time integrator, circadian clock/photoperiod autonomous, and vernalization pathways. In flowering time-related DGEs region, 150 transcriptomic SNPs and 9 InDels were obtained.ConclusionsThe large amount of SNPs and InDels identified in this study will provide a valuable marker resource for radish genetic and genomic studies. The SNPs and InDels within flowering time-related DGEs provide fundamental insight into for dissecting molecular mechanism of bolting and flowering in radish.

Development of SNP markers for Cucurbita species discrimination

There are five domesticated species in the genus Cucurbita. These are Cucurbita. pepo, Cucurbita maxima, Cucurbita moschata, Cucurbita ficifolia, and Cucurbita argyrosperma. Cucurbita has a large genetic diversity, with various growth forms, sizes, shapes, colors, compositions, tastes, and resistance to biotic and abiotic factors. Maintaining the biological diversity of genetic resources and managing the right historical information are important first steps in the development of new varieties with desirable traits. This study presents the classification of Cucurbita species using DNA barcodes and the development of powerful single nucleotide polymorphism (SNP) markers that can distinguish the five Cucurbita species. A total of 54 accessions from five Cucurbita species (C. pepo, C. maxima, C. moschata, C. ficifolia, and C. argyrosperma) were used for SNP marker development. The chloroplast DNA was extracted, and the three universal barcoding regions (matK, trnH-psbA, and rbcL) were sequenced. Neighbor-joining (NJ) trees were constructed using the nucleotide sequence of the cpDNA barcodes. The rbcL and trnH-psbA barcodes failed to differentiate between C. moschata and C. argyrosperma, whereas the matK region efficiently differentiated with 87% bootstrap support. Moreover, the matK region showed a species identification rate of 100%, making it an efficient marker for identifying the five domesticated Cucurbita species. Multiplex SNaPShot (mini sequencing) analysis of four SNPs in the matK region revealed four species-specific SNPs that effectively classify Cucurbita species into five groups. When all four markers are used together, they are effective. These four species specific SNP markers were validated using known Cucurbita germplasm (111 accessions) and applied for the species identification of unknown Cucurbita germplasm (370 accessions) available at the RDA gene bank in the Republic of Korea. These markers were 100% effective for identifying the five Cucurbita species. Therefore, these SNP markers can be used for effective management of Cucurbita genetic resources in genebanks around the world.

Development of diagnostic SNP markers for identification of rye 1RS translocations in wheat

AbstractWheat (Triticum aestivum L.) wild relatives are rich genetic resources for genetic improvement of wheat. The short arm of rye (Secale cereale L.) chromosome 1 (1RS) contains many beneficial genes for resistance to drought, insects, and diseases, therefore the 1RS wheat translocations have been widely used in wheat breeding programs worldwide. To facilitate marker‐assisted identification of 1RS in wheat breeding, we designed a set of Kompetitive allele‐specific polymerase chain reaction (KASP) markers to detect the translocations of 1RS with long arm from wheat chromosome 1A (1RS.1AL) and 1RS with long arm from wheat chromosome 1B (1RS.1BL) based on the sequence flanking single nucleotide polymorphisms (SNPs) generated by genotyping‐by‐sequencing (GBS) between wheat and rye panels. We identified a set of three SNP markers for diagnosis and differentiation of the two 1RS translocations in wheat. Among the three KASP markers, 1AS‐05056 is 1RS specific, and clearly separated the genotypes with 1RS (1RS.1AL and 1RS.1BL) from those without the two 1RS translocations; whereas 1AS‐29875 clearly identified the 1RS.1AL translocation and 1BS‐16654 separated 1RS.1BL from 1RS.1AL. The three new KASP markers together accurately identified the presence of 1RS translocation on either 1A or 1B chromosome in a panel of 95 representative U.S. winter wheat accessions, which were further validated in two additional U.S. hard winter wheat panels. Therefore, they can be used for routine screening of 1RS.1BL and 1RS.1AL translocation lines in wheat breeding programs.

Development of SNP (Single Nucleotide Polymorphism) Markers and Evaluation of Marker Combinations for Identification of Korean Wheat Cultivars

Development of SNP (Single Nucleotide Polymorphism) Markers and Evaluation of Marker Combinations for Identification of Korean Wheat Cultivars

Population structure of Caspian Kutum (Rutilus frisii, Nordmann, 1840) in the southern coast of Caspian Sea using Genome-wide single nucleotide polymorphism markers

The Caspian kutum (Rutilus frisii, Nordmann, 1840) is one of the most important semi-anadromous endemic fish species in the southern part of the Caspian Sea. The fisheries from wild populations of R. frisii are declined drastically due to variability of sea levels, overfishing, and illegal fishing. For this regard, genetic studies is very noticed to maintain of remained this endangered species. Here, we used restriction site-associated DNA sequencing (RAD-seq) approach to explore population structure, genetic variability, and single nucleotide polymorphisms (SNPs) from 146 individuals from eight rivers in Iran. In total 86,274 SNP loci were determined, and after filtering, 16,193 high-quality SNP markers were applied in statistical analyses. Twenty-eight percent of selected SNPs were located in genic regions and about 65% of these SNPs were in the coding sequence. Of the 102 SNP loci, 42 loci were found to be polymorphic and bi-allelic. The minor allele frequency (MAF) of these SNPs varied from 0.156 to 0.471. The observed heterozygosity (Ho) per locus ranged from 0.119 to 0.864, while the expected heterozygosity (He) per locus was from 0.142 to 0.569. Among these SNP loci, 22 SNP loci were significantly deviated from the Hardy–Weinberg equilibrium (P < 0.01) after Bonferroni correction. Both Principal coordinate analysis (PCA) and Bayesian clustering satisfyingly showed population genomic structure in Caspian kutum populations, indicating the description of distinct population segments. The first development of SNP markers in this study provides the better monitoring of population genetic variation in Caspian kutum at a more detailed level and in the scarce of genetic information about this species.

Assessment of genetic diversity and SNP marker development within peanut germplasm in Taiwan by RAD-seq

The cultivated peanut (Arachis hypogaea L.) is an important oil crop but has a narrow genetic diversity. Molecular markers can be used to probe the genetic diversity of various germplasm. In this study, the restriction site associated DNA (RAD) approach was utilized to sequence 31 accessions of Taiwanese peanut germplasm, leading to the identification of a total of 17,610 single nucleotide polymorphisms (SNPs). When we grouped these 31 accessions into two subsets according to origin, we found that the “global” subset (n = 17) was more genetically diverse than the “local” subset (n = 14). Concerning botanical varieties, the var. fastigiata subset had greater genetic diversity than the other two subsets of var. vulgaris and var. hypogaea, suggesting that novel genetic resources should be introduced into breeding programs to enhance genetic diversity. Principal component analysis (PCA) of genotyping data separated the 31 accessions into three clusters largely according to the botanical varieties, consistent with the PCA result for 282 accessions genotyped by 14 kompetitive allele-specific PCR (KASP) markers developed in this study. The SNP markers identified in this work not only revealed the genetic relationship and population structure of current germplasm in Taiwan, but also offer an efficient tool for breeding and further genetic applications.

Development of SSR and SNP markers for identifying opium poppy.

Papaver somniferum L. (opium poppy) is the original plant of heroin, which is a major narcotic drug, and this plant has brought great harm to human health. However, the ban on opium poppy cultivation and trafficking is facing great challenges because of abnormal profits. Therefore, rapid and accurate identification is important to address the abovementioned problems. In this study, eleven simple sequence repeats (SSR) markers and two single nucleotide polymorphism (SNP) markers were mined to distinguish opium poppy from other six Papaver species. These molecular markers were further verified through a large number of plant materials of these seven Papaver species. An excellent multiplex polymerase chain reaction (PCR) system that simultaneously amplifies the three of eleven SSR markers was developed, which effectively improves the efficiency and speed of identification. The present research is of great implication for identifying and investigating the illegal cultivation and trafficking of opium poppy.

Development and Validation of SNP and InDel Markers for Pod-Shattering Tolerance in Soybean.

Pod-shattering causes a significant yield loss in many soybean cultivars. Shattering-tolerant cultivars provide the most effective approach to minimizing this loss. We developed molecular markers for pod-shattering and validated them in soybeans with diverse genetic backgrounds. The genes Glyma.16g141200, Glyma.16g141500, and Glyma.16g076600, identified in our previous study by quantitative trait locus (QTL) mapping and whole-genome resequencing, were selected for marker development. The whole-genome resequencing of three parental lines (one shattering-tolerant and two shattering-susceptible) identified single nucleotide polymorphism (SNP) and/or insertion/deletion (InDel) regions within or near the selected genes. Two SNPs and one InDel were converted to Kompetitive Allele-Specific PCR (KASP) and InDel markers, respectively. The accuracy of the markers was examined in the two recombinant inbred line populations used for the QTL mapping, as well as the 120 varieties and elite lines, through allelic discrimination and phenotyping by the oven-drying method. Both types of markers successfully discriminated the pod shattering-tolerant and shattering-susceptible genotypes. The prediction accuracy, which was as high as 90.9% for the RILs and was 100% for the varieties and elite lines, also supported the accuracy and usefulness of these markers. Thus, the markers can be used effectively for genetic and genomic studies and the marker-assisted selection for pod-shattering tolerance in soybean.

SNP Development in Penaeus vannamei via Next-Generation Sequencing and DNA Pool Sequencing

Next-generation sequencing and pool sequencing have been widely used in SNP (single-nucleotide polymorphism) detection and population genetics research; however, there are few reports on SNPs related to the growth of Penaeus vannamei. The purpose of this study was to call SNPs from rapid-growing (RG) and slow-growing (SG) individuals’ transcriptomes and use DNA pool sequencing to assess the reliability of SNPs. Two parameters were applied to detect SNPs. One parameter was the p-values generated using Fisher’s exact test, which were used to calculate the significance of allele frequency differences between RG and SG. The other one was the AFI (minor allele frequency imbalance), which was defined to highlight the fold changes in MAF (minor allele frequency) values between RG and SG. There were 216,015 hypothetical SNPs, which were obtained based on the transcriptome data. Finally, 104 high-quality SNPs and 96,819 low-quality SNPs were predicted. Then, 18 high-quality SNPs and 17 low-quality SNPs were selected to assess the reliability of the detection process. Here, 72.22% (13/18) accuracy was achieved for high-quality SNPs, while only 52.94% (9/17) accuracy was achieved for low-quality SNPs. These SNPs enrich the data for population genetics studies of P. vannamei and may play a role in the development of SNP markers for future breeding studies.

Production of haploid gynogens to inform genomic resource development in the paleotetraploid pallid sturgeon (Scaphirhynchus albus)

Order Acipenseriformes (sturgeons and paddlefishes) is an ancient lineage of osteichthyan fishes (>200 million years old) with most extant species at conservation risk. A relatively basal species, the pallid sturgeon, Scaphirhynchus albus, is a federally endangered species native to the Mississippi and Missouri River basins. Hybridization with sympatric shovelnose sturgeon, S. platorynchus, is one of several threats to pallid sturgeon. Current molecular markers cannot reliably distinguish among pure species and multigenerational backcrosses. This information is critical for implementation of management strategies to increase populations through natural reproduction and artificial propagation. Genotypes from a large panel of unlinked single-nucleotide polymorphisms (SNPs) may provide greater resolution of the two species; however, paralogous sequence variants (PSVs) within individuals resulting from an ancient whole genome duplication event confound SNP development. The aim of this study was to produce pallid sturgeon gynogens that contain 100% homozygous DNA contributed by only the maternal parent and have enough DNA for future SNP marker development. When homozygous gynogens are sequenced, heterozygosity at a locus within an individual indicates the presence of incorrectly aligned sequences that contain PSVs; accurate identification of these multi-locus contigs can facilitate their exclusion when developing disomic markers. In this study, we attempted to produce two types of pallid sturgeon gynogens: a) haploid gynogens produced from the activation of pallid sturgeon eggs with ultraviolet-irradiated sperm from the distantly related paddlefish (Polyodon spathula), and b) doubled haploids produced from the activation of pallid sturgeon eggs with irradiated paddlefish milt followed by thermal shock to suppress the first mitotic division. Production of doubled haploids, gynogens with 100% homozygous DNA and double the genome content of haploid gynogens, was pursued because it was originally unknown if haploid gyongens would survive long enough to attain enough genetic material for SNP marker development. We performed flow cytometry and microsatellite genotyping on the specimens in order to confirm haploid and doubled haploid status. Our study was unable to successfully yield doubled haploids; however, we successfully produced haploid gynogens that contained enough nuclear DNA for our future SNP marker development study. Interestingly, this study also produced paddlefish × pallid sturgeon hybrids in the control groups in two separate years; this is the first study to report viable offspring between the paddlefish and a Scaphirhynchus sturgeon species and reflects on the malleability of the genomes of the species in this order.

Development of single-nucleotide polymorphism markers of salinity tolerance for Tunisian durum wheat using RNA sequencing

ABSTRACT Salinity stress is a critical abiotic stress that reduces crop productivity. Durum wheat (Triticum turgidum ssp. durum) is an essential cereal in Tunisia, a country threatened by salinity stress. In this study, 26 accessions of Tunisian durum wheat were subjected to salinity stress induced by 500 mM NaCl, and their salinity tolerance was evaluated by examining phenotypic parameters and calculating the salinity tolerance trait index and salinity tolerance index. Furthermore, two Tunisian durum wheat cultivars, namely ‘Om Rabia’ (salinity-tolerant) and ‘Mahmoudi’ (salinity-susceptible), were subjected to RNA sequencing (RNA-Seq) analysis, and single-nucleotide polymorphisms (SNPs) were detected from the RNA-Seq data of these two cultivars. The impacts of all SNP variants on genes were predicted, and 157 candidate SNP primer pairs were designed preferentially using high-impact transcripts. Following confirmation using polymerase chain reaction (PCR) analysis of data from ‘Om Rabia’ and ‘Mahmoudi’, 17 SNP markers were developed and applied to assess the remaining 26 Tunisian durum wheat lines. Three developed SNP markers (KUCMB_TRIDC7AG078450.6_12, KUCMB_TRIDC2BG061830.1_05, and KUCMB_TRIDC2BG061830.1_08) showed PCR bands only in salinity-tolerant lines but not in moderately tolerant or susceptible lines. Therefore, the developed SNP markers may be used to screen the salinity tolerance of various durum wheat accessions.

Genotyping by sequencing for SNP marker development in onion.

Onion (Allium cepa) is not highly tractable for development of molecular markers due to its large (16 gigabases per 1C) nuclear genome. Single nucleotide polymorphisms (SNPs) are useful for genetic characterization and marker-aided selection of onion because of codominance and common occurrence in elite germplasm. We completed genotyping by sequencing (GBS) to identify SNPs in onion using 46 F2 plants, parents of the F2 plants (Ailsa Craig 43 and Brigham Yellow Globe 15-23), two doubled haploid (DH) lines (DH2107 and DH2110), and plants from 94 accessions in the USDA National Plant Germplasm System (NPGS). SNPs were called using the TASSEL 3.0 Universal Network Enabled Analysis (UNEAK) bioinformatics pipeline. Sequences from the F2 and DH plants were used to construct a pseudo-reference genome against which genotypes from all accessions were scored. Quality filters were used to identify a set of 284 high quality SNPs, which were placed onto an existing genetic map for the F2 family. Accessions showed a moderate level of diversity (mean He = 0.341) and evidence of inbreeding (mean F = 0.592). GBS is promising for SNP discovery in onion, although lack of a reference genome required extensive custom scripts for bioinformatics analyses to identify high quality markers.

Development of diagnostic SNP markers for quality assurance and control in sweetpotato [Ipomoea batatas (L.) Lam.] breeding programs.

Quality assurance and control (QA/QC) is an essential element of a breeding program's optimization efforts towards increased genetic gains. Due to auto-hexaploid genome complexity, a low-cost marker platform for routine QA/QC in sweetpotato breeding programs is still unavailable. We used 662 parents of the International Potato Center (CIP)'s global breeding program spanning Peru, Uganda, Mozambique and Ghana, to develop a low-density highly informative single nucleotide polymorphism (SNP) marker set to be deployed for routine QA/QC. Segregation of the selected 30 SNPs (two SNPs per base chromosome) in a recombined breeding population was evaluated using 282 progeny from some of the parents above. The progeny were replicated from in-vitro, screenhouse and field, and the selected SNP-set was confirmed to identify relatively similar mislabeling error rates as a high density SNP-set of 10,159 markers. Six additional trait-specific markers were added to the selected SNP set from previous quantitative trait loci mapping studies. The 36-SNP set will be deployed for QA/QC in breeding pipelines and in fingerprinting of advanced clones or released varieties to monitor genetic gains in famers' fields. The study also enabled evaluation of CIP's global breeding population structure and the effect of some of the most devastating stresses like sweetpotato virus disease on genetic variation management. These results will inform future deployment of genomic selection in sweetpotato.

Development of powdery mildew race 5-specific SNP markers in Cucumis melo L. using whole-genome resequencing

Melon (Cucumis melo L.), belonging to the Cucurbitaceae family, is cultivated worldwide and is highly valued for its fruit quality. However, this important crop is negatively affected by several races of powdery mildew (PM) fungus, Podosphaera xanthii. Hence, exploration of PM race-specific resistant markers would be an effective strategy to develop race-specific melon cultivars. Young leaves from four melon genotypes, including susceptible SCNU1154 and race-specific-resistant Edisto47, PMR5, and MR1 lines, were sequenced by next-generation sequencing, specifically whole-genome resequencing (WGR), to detect race-specific single nucleotide polymorphism (SNP) markers. Including putative resistance gene (R-gene) SNPs, 168, 83, and 122 race 5-specific SNPs with the exact variation were identified on PM-related chromosome 2, 5, and 12, respectively, for distinguishing race-specific lines by comparing the WGR data in the C. melo genome. Based on proximity to the PM resistance quantitative trait loci (QTLs) in physical maps, 43, 37, and 48 SNPs were screened on chromosome 2, 5, and 12, respectively. Using a derived cleaved amplified polymorphic sequence (dCAPS) method, polymorphisms were only found in three SNPs (SNPR5_119, SNPR5_120, and SNPR5_121) out of 48 against race 5 on chromosome 12. Among these three, a putative R-gene, an NBS-LRR-type SNP, SNPR5_119, displayed similar genotypic and phenotypic variation to race 5-specific susceptible (SCNU1154, PMR45, WMR29, and Edisto47) and resistant (PI414723, PMR5, and MR1) lines in C. melo except for PI124112. Importantly, two other intergenic SNPs, SNPR5_120 and SNPR5_121, showed genotypic and phenotypic variation between susceptible and resistant lines tested. High-resolution melting (HRM) analysis was used to further validate the same expression patterns of SNPR5_120 as a dCAPS method in all lines tested. Therefore, the identified PM race 5-specific candidate markers described here might be useful for a marker-assisted selection breeding program in C. melo.

Diallelic SNP marker development and genetic linkage map construction in octoploid strawberry (Fragaria × ananassa) through next-generation resequencing and high-resolution melting analysis

The octoploid strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural crops in the world. However, few genomic studies have been performed in this plant due to its polyploidy nature and complex genome. In this work, we developed diallelic single-nucleotide polymorphism (SNP) markers using next-generation resequencing data and high-resolution melting (HRM) analysis. We also constructed a genetic linkage map using an F1 segregating population derived from a cross between two cultivars, F. × ananassa ‘Sulhyang’ and ‘Senga-sengana’. A previous study revealed 20,848 SNP positions that can be developed into HRM markers. In this study, 576 SNP-based and 34 simple sequence repeat (SSR)-based HRM markers were developed using 832 newly designed and 461 previously reported primer sets, respectively. In addition, a strawberry genetic linkage map covering a total genetic distance of 1504.7 cM and consisting of 759 loci on 35 linkage groups was constructed. The linkage groups were assigned to corresponding chromosomes using the informative SSR-based markers and a BLAST search to the diploid strawberry genome, Fragaria vesca ver. 2.0.a1. Furthermore, the genome-wide HRM markers were applied to analyze the homozygosity of the inbred strawberry line ‘Wongyo 3115’, which showed a homozygosity of 96.6%. This marker and map information will be useful for future genomic and genetic research of the octoploid strawberry.

Identification of a SNP and development of a PCR-based allele-specific marker of the sporulation-deficient (sporeless) trait of the Tamogitake 108Y2D mutant using next-generation sequencing.

The mass scattering of basidiospores during the cultivation of edible mushrooms causes serious problems, such as allergic reactions in workers. Sporulation-deficient (sporeless) cultivars would be very useful for preventing these issues. We aimed to identify the single-nucleotide polymorphism (SNP) that is responsible for the single dominant sporeless mutation of the Tamogitake 108Y2D mutant using next-generation sequencing (NGS) and TILLING technology and to develop an allele-specific PCR marker for sporeless breeding. By comparing the sequences of the wild-type and its mutant genomes, we identified 685 mutation loci in gene regions and pinpointed one SNP only consistent with sporeless phenotype for 105 segregants, i.e., a C to T located at position 1,950 of the exonic region of a putative fungal transcription factor that generated a stop codon. We developed an allele-specific marker based on the identified SNP, and its high practicality was validated using tests against progenies from several hybrids and wild isolates from different geographical origins. Thus, the allele-specific PCR marker developed here will be useful for marker-assisted selection in the breeding of the sporeless trait of this mushroom. Furthermore, the technical success of SNP identification and marker development based on NGS genome data can help achieve efficient mutation breeding in mushrooms.

Development of SNP markers for grain yield screening of Brazilian rice cultivars

Abstract: The objective of this work was to identify and validate single-nucleotide polymorphism (SNP) markers related to grain yield in rice (Oryza sativa) core collection. The genome-wide association studies (GWAS) methodology was applied for genotyping of 541 rice accessions by 167,470 SNPs. The grain yield of these accessions was estimated through the joint analysis of nine field experiments carried out in six Brazilian states. Fifteen SNPs were significantly associated with grain yield, and out of the ten SNPs converted to TaqMan assays, four discriminated the most productive accessions. These markers were used for the screening of rice accessions with favorable alleles. The selected accessions were, then, evaluated in field experiments in target environments, in order to select the most productive ones. This screening reduces the number of accessions evaluated experimentally, making it possible to prioritize those with higher productive potential, which allows of the increase of the number of replicates and, consequently, of the experimental accuracy.