Single nucleotide polymorphisms (SNPs) are widely used for genetic diversity analysis, linkage mapping, genome-wide association studies (GWAS), and molecular marker development in crop plants. Genotyping-by-sequencing (GBS) enables cost-effective SNP discovery; however, achieving sufficient marker density in polyploid crops remains challenging because of complex genome structures, high sequence similarity among homologous chromosomes, and repetitive genomic regions. In this study, we optimized a GBS-based bioinformatics pipeline for polyploid kiwifruit (Actinidia spp.) by evaluating restriction enzyme combinations through in silico digestion analysis and comparing the SNP detection efficiency of three variant-calling tools, namely freebayes, bcftools, and Genome Analysis Tool Kit (GATK). The methylation-sensitive ApeKI/TfiI combination generated the highest proportion of DNA fragments within the target size range (200–500 bp) in the kiwifruit reference genome cv. Hongyang (A. chinensis). Using GATK, 828,257 SNPs were identified, approximately 22-fold higher than those detected using freebayes and bcftools, with a comparable transition/transversion (Ts/Tv) ratio. GATK also identified substantially higher absolute numbers of SNPs in genic regions, while the proportion of genic-region SNPs was similar across all three tools. Notably, only the GATK-derived SNP dataset exceeded the estimated marker density discussed in this study for high-density genomic coverage of the kiwifruit genome. These results demonstrate that the combination of methylation-sensitive restriction enzymes and GATK-based variant calling generated a high-density SNP dataset for mixed-ploidy kiwifruit germplasm. Because independent validation of SNP accuracy was beyond the scope of this study, the observed differences should be interpreted as differences in SNP discovery rather than comparative variant-calling accuracy.

Cytoplasmic male sterility (CMS) refers to the failure of flowering plants to produce viable pollen grains during anther development. CMS and the restorer-of-fertility (Rf) system have been documented in Pyrus. Male sterility has been reported in the Asian pear ‘Niitaka’ (P. pyrifolia) and its F1 progeny. However, Rf genes conferring pollen fertility for ‘Niitaka’ cytoplasm in pears remains to be elucidated. Therefore, this study aimed at precisely mapping Rf genes involved in pollen fertility restoration and exploring candidate genes. We analyzed F1 progenies derived from female ‘'Whangkeumbae' (P. pyrifolia) possessing ‘Niitaka’ cytoplasm. Two non-linked loci with complementary dominance were hypothesized to account for the observed separation ratio. Genotyping-by-sequencing (GBS) analysis was performed to identify single nucleotide polymorphism (SNP) markers and finely map the Rf loci. Two Rf loci were preliminarily mapped to linkage groups 4 and 5 and named Rf1 and Rf2, respectively. Insertion/deletion (InDel) markers were developed by analyzing the whole genome sequencing data of the parents. Rf1 and Rf2 regions were delimited to 560 kb and 880 kb, respectively, by fine mapping with InDel markers. Four candidate genes encoding pentatricopeptide repeat proteins were identified in Rf1 and Rf2. To the best of our knowledge, this is the first study to map Rf genes responsible for cytoplasmic male sterility in pears, which advances our understanding of male sterility.

Seed vigor is critical for uniform germination and emergence, directly influencing subsequent seedling development. This is especially important under both normal and stress conditions that may arise post-sowing. Chilling stress during emergence and early growth poses significant challenges for tomato seedlings, potentially leading to uneven emergence, abnormal growth, and higher seedling mortality. This study evaluated the effectiveness of combining drum-priming with melatonin to alleviate chilling stress during these two critical stages. Tomato seeds were primed with melatonin at various concentrations and compared to untreated controls under chilling stress conditions. Higher concentrations of melatonin significantly accelerated emergence and improved early growth under stress. These stress-mitigating effects were evidenced by reductions in oxidative stress markers, such as malondialdehyde and hydrogen peroxide, along with increased total polyphenol and flavonoid contents. Furthermore, melatonin priming preserved photosynthetic efficiency, typically reduced by chilling stress, and enhanced the activities of antioxidant enzymes, including catalase and peroxidase. These biochemical changes reduced oxidative damage and promoted stress resilience. Melatonin also accelerated the expression of genes within the C-repeat binding factor pathway, which is crucial for cold acclimation. This suggests that melatonin priming enabled quicker adaptation to chilling stress following sowing and bolstered seedling resilience during subsequent growth stages. Overall, our results demonstrate that melatonin priming not only enhances germination but also significantly supports seedling growth under adverse conditions. The findings highlight melatonin as a promising tool in crop management strategies to improve resilience against sudden chilling stress.

Cytoplasmic male sterility (CMS) caused by mitochondrial genome alterations in flowering plants plays a crucial role in hybrid breeding systems. In pear (Pyrus spp.), pollenless phenotypes have been consistently observed in progeny. However, the genetic basis and inheritance mechanisms of male sterility in pears remain poorly understood. To investigate the inheritance mode, we performed segregation analysis in four F₁ populations derived from crosses using a cultivar carrying CMS-type cytoplasm as the maternal parent. The observed male sterility segregation ratios confirmed a maternal inheritance pattern consistent with the CMS model and suggested differential effects of nuclear fertility restorer genes from various pollen parents. We analyzed whole-genome sequencing data from four pear accessions, identifying an 860 bp mitochondrial DNA sequence associated with male-sterile individuals. This sequence was located near cox3 and apt8, commonly co-located with CMS loci in other plant species. Within this region, we identified orf206, a chimeric open reading frame composed of 113 bp from nad3 in Pyrus betulifolia and 403 bp from atp9-1 of Malus × domestica. The predicted protein encoded by orf206 contained three transmembrane domains, which are typical features of CMS-associated proteins. Our results demonstrate that male sterility in pears is maternally inherited and support orf206 as a strong candidate gene for CMS induction. Furthermore, we developed an InDel marker (CBpMtid03 and CBpMtid07) targeting the CMS-specific mitochondrial sequence enabling the efficient identification of CMS individuals in breeding programs. These findings provide insights into the molecular mechanisms underlying pollen sterility in pears and facilitate marker-assisted selection in pear breeding.

Genetic linkage maps can be used to identify genetic loci associated with traits of interest. Apple fruit characteristics are influenced by environmental factors and are controlled by quantitative trait loci (QTL). Here, we constructed single parental and integrated bi-parental genetic linkage maps of apples using genotyping-by-sequencing (GBS)-derived single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) to show the importance of single parental maps and to identify QTLs associated with fruit skin color and acidity. The 'Gala' map consisted of 725 GBS-SNPs and 40 SSRs across 1,400.8 cM with a marker density of 1.96 cM. In the 'Jonathan' map, 698 GBS-SNPs and 40 SSRs were anchored in 1,635.7 cM and the marker density was 2.36 cM. The 17 linkage groups of these two parental genetic linkage maps could represent 17 apple chromosomes and covered approximately 69% of the reference genome. The resolutions of the two single parental maps and the integrated bi-parental map were similar. QTL analysis revealed QTLs associated with fruit skin color in linkage groups (LGs) 9, 10, and 13 of the 'Jonathan' map. In addition, a fruit acidity-related QTL was found in LG13 of the 'Jonathan' map. Because the GBS-SNPs represented the physical location of the apple genome, SSRs displayed co-linearity, and the genetic linkage maps of 'Gala' and 'Jonathan' have sufficient genomic information, they could be used as reference maps for QTL analysis in apples.

Agastache is a genus of perennial herbaceous plants belonging to the mint family, Lamiaceae. Several Agastache species are commercially cultivated and used as medicinal, culinary, and ornamental plants. However, information on the genetic diversity and population structure of the species remains unclear. In the present study, genetic diversity within Agastache species was analyzed using simple sequence repeat (SSR) markers. In this study, 249,746 SSRs were identified in the A. rugosa genome and primer pairs were designed for 56,675 SSRs. The majority of SSR repeat types were dinucleotides (60.65%), followed by trinucleotides (12.38%), and pentanucleotides (12.10%). PCR conditions were established for 250 primer pairs, 111 of which were found to be polymorphic in A. rugosa germplasm. The number of alleles (NA) ranged from 2 to 19, major allele frequency (MAF) ranged from 0.11 to 0.95, observed heterozygosity (HO) ranged from 0 to 0.89, and polymorphic information content (PIC) ranged from 0.09 to 0.92. Cross-species amplification of SSRs markers in other Agastache species showed amplification rates of 82.6% for A. foeniculum and 78.1% in A. urticifolia, with an average of 80.37%. Cluster analysis of the 19 A. rugosa accessions using SSRs markers revealed four major clusters, and population STRUCTURE analysis using 79 SSRs markers revealed three groups and three subgroups among the A. rugosa populations. The SSRs markers developed can contribute to applications such as varietal identification, genetic diversity analysis, and population structure analysis of A. rugosa germplasm.

Gene-based markers are valuable tools in breeding programs due to their direct linkage to traits of interest. In dioecious plants, such as kiwifruit (Actinidia spp.), sex-discriminating markers can shorten the breeding cycle by enabling the selection of preferred sexes at the juvenile stage. To develop a gene-based sex-discriminating marker, resequencing was conducted on female and male A. arguta accessions, and insertion and deletion (InDel) variations within sex-related genes were explored. A total of 203,116 InDels were detected between female and male A. arguta accessions, and 118,865 InDels were heterozygous between the two accessions. Sequence similarity between thirty-seven sex-related genes from seven dioecious species and the kiwifruit reference genome was investigated, revealing that ten genes exhibited similarities ranging from 59 to 79%. Among the 118,865 InDels, seven InDels were located on four sex-related genes encoding agamous-like MADS-box genes and hypothetical proteins. A 20 bp insertion in male A. arguta located in the agamous-like MADS-box gene was converted into an InDel marker, which clearly discriminates female and male A. arguta accessions and the interspecific hybrid cultivar. The InDel marker was designated CBk25id01 and produced approximately 350 bp amplicon only in the male A. arguta. The CBk25id01 linked to the agamous-like MADS-box gene involved in floral organ development may help understand sex differentiation and accelerate the breeding of kiwifruits.

Differentially expressed genes (DEGs) detected in the comparison between the scab-susceptible cultivar 'Whasan' and the scab-resistant cultivar 'Greensis' at 0 hours after inoculation with Venturia nashicola. The abbreviations 'W' and 'G' denote 'Whasan' and 'Greensis', respectively, and the numerical value following the abbreviations represents the time in hours after inoculation with V. nashicola. The expression levels of each DEG were quantifed as fragments per kilobase of exon per million mapped fragments (FPKM) and log2-fold change (FC). All the DEGs were statistically significant at a significant level of p < 0.05 and q < 0.05.

Cryopreservation is an important technique used in the conservation of various plant tissues. This study proposes a cryopreservation method for the long-term conservation of eastern bracken fern gametophytes ( Pteridium aquilinum var. latiusculum ). Encapsulation–dehydration of the gametophytes was performed, and the exogenous sucrose and abscisic acid (ABA) preculture conditions were investigated. Gametophytes are sensitive to dehydration and drying, and the following treatment conditions were applied: encapsulation by alginate containing 0.75 M sucrose, 18-h loading treatment with 0.75 M sucrose, and 6-h drying treatment. The survival rate following cryopreservation was determined. The water content of < 27.5% in the alginate beads after dehydration and drying was found to be appropriate for ensuring survival. Additionally, performing an exogenous sucrose and ABA preculture was essential before encapsulation to achieve a survival of ≥ 90%. The high stress induced by cryopreservation and exogenous preculture regulated the expression of PaSuSy , PaLEA14 , and PaABI1b and the endogenous ABA content. In eastern bracken gametophytes, ABI1 appears to be a negative regulator of ABA signaling. These results indicate that the encapsulation–dehydration method is effective for the long-term conservation of eastern bracken fern gametophytes, and exogenous preculture alleviates abiotic stress and increases the survival rate.

Fruit shape is one of the important quantitative traits in pear ( Pyrus spp.) breeding program, thus genetic study related to fruit shape could be beneficial to pear breeding. Quantitative trait loci (QTL) analysis was carried out using ‘Whangkeumbae’ ( P. pyrifolia , round) × ‘Yali’ ( P. bretschneideri , pyriform) and high-resolution melting (HRM) markers were developed. The genetic linkage map of ‘Whangkeumbae’ × ‘Yali’ was constructed using single nucleotide polymorphisms (SNPs) derived from Axiom Pear 70 K Genotyping Array and simple sequence repeats. The integrated genetic linkage map of ‘Whangkeumbae’ × ‘Yali’ showed ~ 90% of genome coverage, a total genetic distance of 998.2 cM, and a marker density of 1.6 cM. F 1 progenies of ‘Whangkeumbae’ × ‘Yali’ showed normal distribution of fruit length (L), diameter (D), and L/D ratio. Three QTLs located in linkage group (LG) 6, 7, and 12 were identified with LOD thresholds of 2.8–3.0. Six HRM markers were developed using array-SNPs anchored in the QTLs and predicted fruit shape with 28.6–65.3% accuracy. Notably, accuracy was increased by ~ 90% using an HRM marker combination consisting of CBp06sn02, CBp07sn01, and CBp12sn03. These results could provide a better understanding of the genetic mechanism of fruit shape development and reducing pear breeding period.

Background Vicia bungei is an economically important forage crop in South Korea and China. Although detailed genetic and genomic data can improve population genetic studies, conservation efforts, and improved breeding of crops, few such data are available for Vicia species in general and none at all for V. bungei . Therefore, the main objectives of this study were to sequence, assemble, and annotate V. bungei chloroplast genome and to identify simple sequence repeats (SSRs) as polymorphic genetic markers. Results The whole-genome sequence of V. bungei was generated using an Illumina MiSeq platform. De novo assembly of complete chloroplast genome sequences was performed for the low-coverage sequence using CLC Genome Assembler with a 200–600-bp overlap size. Vicia bungei chloroplast genome was 130,796-bp long. The genome lacked an inverted repeat unit and thus resembled those of species in the inverted repeat-lacking clade within Fabaceae. Genome annotation using Dual OrganellarGenoMe Annotator (DOGMA) identified 107 genes, comprising 75 protein-coding, 28 transfer RNA, and 4 ribosomal RNA genes. In total, 432 SSRs were detected in V. bungei chloroplast genome, including 64 mononucleotides, 14 dinucleotides, 5 trinucleotides, 4 tetranucleotides, 233 pentanucleotides, 90 hexanucleotides, and 14 complex repeated motifs. These were used to develop 232 novel chloroplast SSR markers, 39 of which were chosen at random to test amplification and genetic diversity in Vicia species (20 accessions from seven species). The unweighted pair group method with arithmetic mean cluster analysis identified seven clusters at the interspecies level and intraspecific differences within clusters. Conclusion The complete chloroplast genome sequence of V. bungei was determined. This reference genome should facilitate chloroplast resequencing and future searches for additional genetic markers using population samples. The novel chloroplast genome resources and SSR markers will greatly contribute to the conservation of the genus Vicia and facilitate genetic and evolutionary studies of this genus and of other higher plants.

The fruit skin types of pear (Pyrus spp.) are divided into russet, smooth, and intermediate. One of the important traits in pear breeding programs is russet on pear fruit skin because it affects the commercial value. In the present study, a high-density genetic linkage map of ‘Whangkeumbae’ (smooth) × ‘Minibae’ (russet) was constructed. In addition, quantitative trait loci (QTL) analysis was performed to identify russet related QTL and develop a cleaved amplified polymorphism sequence (CAPS) marker. Together with SNPs derived from Axiom Pear 70K Genotyping Array and genotyping-by-sequencing derived SNPs and SSRs generated in previous study, an integrated genetic linkage map of ‘Whangkeumbae’ × ‘Minibae’ was constructed. A total of 1263 markers were anchored in 17 linkage groups (LGs) with a total genetic distance of 1894.02 cM and an average marker density of 1.48 cM. The chromosome coverage of ‘Whangkeumbae’ × ‘Minibae’ map was improved because the SNPs derived from Axiom Pear 70K Genotyping Array were anchored. QTL analysis was performed using previous russet phenotype data evaluated with russet coverage and Hunter a. As a result of QTL analysis, russet coverage- and Hunter a-related QTLs were identified in LG8 of the ‘Whangkeumbae’ × ‘Minibae’ map, and SNPs located in the QTL region were heterozygous in the ‘Minibae’. Although the russet coverage- and Hunter a-related QTLs were commonly detected in LG8, the logarithm of odds values of SNPs in the QTL region were higher in QTL related to russet coverage than to Hunter a. The CAPS marker (CBp08ca01) was developed using an array SNP located in the russet coverage related QTL, and the genotype of CBp08ca01 showed a 1:1 ratio in ‘Whangkeumbae’ × ‘Minibae’ (χ2 = 0.65, p > 0.05). ‘Whangkeumbae’ and ‘Minibae’ were thought to have rr and Rr genotypes, respectively, and the genetic factors controlling the russet formation might be located in chromosome 8. The CBp08ca01 was able to select F1 individuals with less than 30% russet coverage. Thus, it will be a useful tool for marker-assisted selection in pears.

Actinidia arguta is a Korean native kiwifruit with valuable traits for kiwifruit breeding, such as cold hardiness, disease tolerance, and high nutritional value. Early sex discrimination is a priority in kiwifruit breeding programs because Actinidia species are dioecious vines. A cleaved amplified polymorphic sequence (CAPS) marker discriminating sex was developed using genotyping-bysequencing (GBS)-derived single nucleotide polymorphisms (SNPs). Previously obtained GBS reads were re-aligned to the kiwifruit reference genome organized at the pseudo-chromosomal level. These GBS-SNPs could divide A. arguta accessions according to sex. Candidate CAPS markers were designed using these SNPs located on sex chromosomes. Among them, only the CAPS marker designed using the SNP located at 10,236,410 bp on sex chromosome was able to discriminate the sex of A. arguta accessions. This might be because the SNP located at 10,236,410 bp had higher read depth than other candidate SNPs. This CAPS marker, named CBk25ca01, produced DNA fragments of 300 and 147/153 bp in female and male A. arguta accessions, respectively. The CBk25ca01 marker will be useful to improve kiwifruit breeding programs by reducing the time needed to discriminate sex.

Russet, one of the fruit skin colors of Asian pears, affects the fruit’s commercial value. A precise phenotyping method is required because fruit skin colors occur irregularly in various pear cultivars. Here, we propose a bi-dimensional image analysis to evaluate the russet of pear skin accurately and effectively by comparing the results of image analysis with the Hunter a value. The fruits of ‘Whangkeumbae’ (non-russet), ‘Minibae’ (russet), and their F1 individuals were used for russet evaluation. The Hunter a value was measured using a color-difference meter. The russet coverage (%) was calculated using Photoshop software. In F1 individuals derived from ‘Whangkeumbae’ × ‘Minibae’, the Hunter a value represented normal distribution, and the russet coverage distributed variously. The positive correlation coefficients between the Hunter a value and russet coverage was confirmed (r > 0.7). Although several F1 individuals showed abundant russet formation, they showed a negative Hunter a value. Therefore, the bi-dimensional image analysis more accurately evaluated russet in pear fruit skin than did the Hunter a value. The bi-dimensional image analysis could be used for high-throughput phenotyping in pears.

To identify the genetic characteristics of Asian pears, phylogenetic relationship and population structure were analyzed in 43 pear accessions using single nucleotide polymorphisms (SNPs) derived from genotyping-by-sequencing (GBS). The phylogenetic tree showed that the Asian pear accessions could be divided into 5 groups, representing P. betulifolia, P. ussuriensis, P. bretschneideri, P. pyrifolia and unclassified accessions, respectively. Among 5 unclassified accessions, 'Najuseji' showed close genetic relationship with P. betulifolia. The accession 'Gamiri' was included in the P. pyrifolia group. These results were consistent with those of a principal component analysis and population structure. In the population structure, most of Korean native pears have mixed genome structure with P. ussuriensis and P. pyrifolia. Although, 'Sunchangingyeri' and 'Hangangbae' showed mixed ancestry, they could be classified as P. pyrifolia. Using SNPs derived from GBS, we classified Asian pear germplasms in detail. These results could help the management of Asian pear germplasms.

Asian pear scab is a fungal disease caused by Venturia nashicola. The identification of genes conferring scab resistance could facilitate the breeding of disease-resistant cultivars. Therefore, the present study aimed to identify a scab-resistance gene using an interspecific hybrid population ((Pyrus pyrifolia × P. communis) × P. pyrifolia). Artificial inoculation of V. nashicola was carried out for two years. The segregation ratio (1:1) of resistant to susceptible individuals indicated that resistance to V. nashicola was inherited from P. communis and controlled by a single dominant gene. Based on two years phenotypic data with the Kruskal–Wallis test and interval mapping, 12 common markers were significantly associated with scab resistance. A novel scab resistance gene, Rvn3, was mapped in linkage group 6 of the interspecific hybrid pear, and co-linearity between Rvn3 and one of the apple scab resistance genes, Rvi14, was confirmed. Notably, an insertion in pseudo-chromosome 6 of the interspecific hybrid cultivar showed homology with apple scab resistance genes. Hence, the newly discovered Rvn3 was considered an ortholog of the apple scab resistance gene. Since the mapping population used in the present study is a pseudo-BC1 population, pyramiding of multiple resistance genes to pseudo-BC1 could facilitate the breeding of pear cultivars with durable resistance.

We compared genetic information of GBS-SNPs produced by aligning sequencing reads to scaffold and pseudo-chromosome level of the reference genome to identify more suitable SNPs information according to levels of the reference genome. Two SNP matrixes were obtained by aligning raw sequencing reads to scaffold and pseudo-chromosome levels of the reference genome, respectively. Four criteria were used for SNP filtering: (1) bi-allele; (2) missing value <10%; (3) minor allele frequency (MAF) >5%; and (4) polymorphism information content (PIC) value >= 0.3. As a result, the number of filtered SNPs was 4,987 and 6,505 in the scaffold and pseudo-chromosome level, respectively. The values of expected heterozygosity and PIC in scaffold and pseudo-chromosome level were similar. The averages of MAF and observed heterozygosity (Ho) values, which were 0.361 and 0.360, in the pseudo-chromosome level were larger than those which were 0.350 and 0.328, in scaffold level. Development of genetic markers based on a pseudo-chromosome level will be appropriate for genetic analysis since the number of SNPs and values of MAF and Ho were higher than the scaffold level.