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Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

The fruit skin types of pear (

Phytophthora crown rot (PhCR) caused by P. cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently applied to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

The available haplotype-resolved allo-octoploid strawberry (Fragaria × ananassa Duch.) (2n = 8x = 56) genomes were assembled with the trio-binning pipeline, supplied with parental short-reads. We report here a high-quality, haplotype-phased genome assembly of a short-day cultivar, ‘Florida Brilliance’ (FaFB2) without the use of parental sequences. Using Pacific Biosciences (PacBio) long reads and high-throughput chromatic capture (Hi-C) data, we completed telomere-to-telomere phased genome assemblies of both haplotypes. The N50 continuity of the two haploid assemblies were 23.7 Mb and 26.6 Mb before scaffolding and gap-filling. All 56 pseudochromosomes from the phased-1 and phased-2 assemblies contained putative telomere sequences at the 5’ and/or 3’ ends. A high level of collinearity between the haplotypes was confirmed by high-density genetic linkage mapping with 10,269 SNPs, and a high level of collinearity with the ‘Royal Royce’ FaRR1 reference genome was observed. Genome completeness was further confirmed by consensus quality. The LTR assembly Index score for entire genome assembly was 19.72. Moreover, the BUSCO analysis detected over 99% of conserved genes in the combined phased-1 and phased-2 assembly. Both haploid assemblies were annotated using Iso-Seq data from six different ‘Florida Brilliance’ tissues and RNA-Seq data representing various F. × ananassa tissues from the NCBI sequence read archive, resulting in a total of 104,099 genes. This telomere-to-telomere reference genome of ‘Florida Brilliance’ will advance our knowledge of strawberry genome evolution and gene functions, and facilitate the development of new breeding tools and approaches.

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.

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.

Colletotrichum crown rot (CCR) caused by Colletotrichum gloeosporioides is a serious threat to the cultivated strawberry (Fragaria × ananassa). Our previous study reported that a major locus, FaRCg1, increases resistance. However, the genomic structure of FaRCg1 and potential candidate genes associated with the resistance remained unknown. Here, we performed comparative transcriptome analyses of resistant ‘Florida Elyana’ and susceptible ‘Strawberry Festival’ after infection and identified candidate genes potentially involved in resistance. In ‘Florida Elyana’, 6,099 genes were differentially expressed in response to C. gloeosporioides. Gene ontology analysis showed that the most upregulated genes were functionally associated with signaling pathways of plant defense responses. Three genes in the genomic region of FaRCg1 were highly upregulated: a von Willebrand Factor A domain-containing protein, a subtilisin-like protease, and a TIFY 11A-like protein. Subgenome-specific markers developed for the candidate genes were tested with a diverse panel of 219 accessions from University of Florida and North Carolina State University breeding programs. Significant and positive associations were found between the high-resolution melting (HRM) marker genotypes and CCR phenotypes. These newly developed subgenome-specific functional markers for FaRCg1 can facilitate development of resistant varieties through marker-assisted selection.

Pears (Pyrus spp.) are temperate trees and their fruits are consumed throughout the world, but their genetic relationships are difficult to identify due to low morphological diversity. We estimated the genetic relationships and population structure of 231 pear accessions using 10,186 genome-wide single nucleotide polymorphisms (SNPs) driven by genotyping-by-sequencing (GBS). In our phylogenetic tree and population structure analysis, the accessions were classified into two major groups (group I and II), which were characterized by European and Asian pears, and two subgroups in group II. Of the subgroups in group II, group II-1 contained P. pyrifolia and group II-2 consisted of P. bretschneideri, P. ussuriensis, and P. betulifolia. Principal component analysis classified the pears more precisely than the phylogenetic tree, and P. betulifolia, which belonged to group II-2, was divided into two additional groups. These results suggest that genome-wide GBS–SNPs are suitable to estimate the genetic diversity of pear germplasms, including Asian and European pears, and are also valuable for understanding the genetic structure and evolution of pears, predicting breeding materials, and genome-wide association studies. Furthermore, the information resulting from this study could be useful for the management of Korean pear germplasms and development of breeding materials.

Integrated linkage maps are a valuable tool for comparative mapping between and within genus and species. Novel single-nucleotide polymorphism (SNP) linkage maps integrated with simple sequence repeats (SSRs) based on pseudo-chromosomes of the Chinese pear reference genome of ‘Dangshansuli’ (Pyrus bretschneideri) were constructed. In total, 4801 qualified SNP markers were obtained using a customized pipeline. The consensus map for ‘Whangkeumbae’ and ‘Minibae’ contained 321 SNP and 30 SSR markers spanning 1511.1 cM with an average genetic distance of 4.3 cM. A total of 30 SSR markers made it possible to compare our consensus map to other pear and apple maps. SSR markers originating from pear and apple maps showed high co-linearity. SNPs coordinated with pseudo-chromosomes, provide information on physical length coverage for 17 corresponding linkage groups, and enable easier genome annotation for genomic regions detected by quantitative trait loci analysis. Genotyping-by-sequencing-based SNP maps integrated with SSRs in the interspecific mapping population illustrate the genomic structure of Korean pear resources and will be used as our reference maps for tribe Maleae.

Colletotrichum crown rot (CCR) caused by Colletotrichum gloeosporioides is a serious threat to the cultivated strawberry (Fragaria × ananassa). Our previous study reported that a major locus, FaRCg1, increases resistance. However, the genomic structure of FaRCg1 and potential candidate genes associated with the resistance remained unknown. Here, we performed comparative transcriptome analyses of resistant ‘Florida Elyana’ and susceptible ‘Strawberry Festival’ after infection and identified candidate genes potentially involved in resistance. In ‘Florida Elyana’, 6,099 genes were differentially expressed in response to C. gloeosporioides. Gene ontology analysis showed that the most upregulated genes were functionally associated with signaling pathways of plant defense responses. Three genes in the genomic region of FaRCg1 were highly upregulated: a von Willebrand Factor A domain-containing protein, a subtilisin-like protease, and a TIFY 11A-like protein. Subgenome-specific markers developed for the candidate genes were tested with a diverse panel of 219 accessions from University of Florida and North Carolina State University breeding programs. Significant and positive associations were found between the high-resolution melting (HRM) marker genotypes and CCR phenotypes. These newly developed subgenome-specific functional markers for FaRCg1 can facilitate development of resistant varieties through marker-assisted selection.

Genotyping-by-sequencing (GBS) was used to investigate the genetic diversity and population structure of kiwifruits (Actinidia spp.). Using single nucleotide polymorphisms detected by GBS, phylogenetic tree and population structure were constructed for 89 kiwifruit accessions including Korean native A. arguta. The kiwifruit accessions were clearly divided into two groups in the phylogenetic tree. These groups were characterized by the presence or absence of hairs on pericarp. In the population structure analysis, the peak of delta K was detected at K = 5, suggesting that the 89 kiwifruit accessions were divided into five clusters. Each cluster represented A. chinensis, A. deliciosa, A. eriantha with wild accessions, female A. arguta, and male A. arguta. The result of the population structure supported the genetic background of each accession. We also performed genetic diversity analysis of A. arguta accessions. Consequently, A. arguta accessions were characterized by sex and 13 A. arguta accessions, occupying 33.3% of the total collection, were selected as a core set for use as germplasm to develop disease and cold stress resistant cultivars in future kiwifruit breeding programs. These results suggest that GBS approach is suitable for genetic diversity analysis of kiwifruits. Moreover, our results could be applied in kiwifruit breeding program to develop disease and cold stress resistant cultivars using Korean native A. arguta. Particularly, the developed Korean native A. arguta core set will be used as breeding materials for crop improvement strategies.

In genotyping-by-sequencing (GBS) library construction, restriction enzyme (RE) can influence the size and number of DNA fragments. The objective of the present study was to improve GBS efficiency in pears (Pyrus spp.) by selecting optimized RE combination. To prove GBS efficiency of selected RE combination, population structure and genetic diversity results of Asian pears were compared in two single nucleotide polymorphisms (SNP) sets derived from different GBS libraries. After in silico digestion, ApeKI, ApeKI/TfiI, and ApeKI/MseI were selected to construct GBS libraries and the number of SNPs obtained from ApeKI/TfiI library were about six times more than that from the ApeKI library. In addition, the SNPs of ApeKI/TfiI library showed high accuracy in classification of Asian pear accessions. Thus, ApeKI/TfiI combination is recommended for construction of GBS library in pears because such RE combination could provide genome-wide and numerous informative SNPs for pear genetic studies.

배나무는 원산지와 분화방향에 따라 유럽, 미국, 호주 등에서 주로 재배되는 서양배와 중국, 일본, 한국 등 동남 아시아 지역을 중심으로 분포 및 재배되고 있는 동양배로 구분된다. 17개의 기본염색체를 가진 배나무는 대부분 이배성(2n=2x=34)이며, 단일 S 유전자좌에 의해 조절되는 자가불화합성과 과수 작물의 주요 특징인 유년성으로 인해 유전 연구 및 정밀한 품종 육성에 큰 제한을 받고 있다. 배나무속 식물의 유전연구는 분자생물학 관련 기술의 발달로 다양한 형태의 분자 표지의 개발이 이루어짐과 동시에 유연관계분석, 유전자지도작성, QTL 분석과 같은 다양한 유전연구에 활발히 이용되었다. 또한 배나무의 유전자지도는 병 저항성이나 다양한 유용형질과 연관된 QTL 확인을 위한 연구로 이어지고 있다. 대량 병렬 반응 및 다중처리를 토대로 획기적인 염기서열 분석 비용의 감소를 이뤄낸 NGS 기술은 대용량, 고효율, 저비용으로 식물 유전체 해독을 가능하게 하여, 중국배 'Danshansuli'와 유럽배 'Bartlett'에서 유전체 분석이 완료되었다. 최근 국내에서는 황금배, 청실리 및 미니배의 resequencing 및 GBS를 통한 SNP 탐색 등의 연구를 통해 화기, 숙기 당도 등 농업적으로 유용형질에 대한 게놈전체 연관분석을 수행하고 있다. 【Based on the place of its origin, pear tree (Pyrus spp.) is largely divided into European pears (P. communis, cultivated mainly in Europe and the U.S.) and Asian pears (P. pyrifolia, P. bretschneideri, and P. ussuriensis, distributed and grown in East Asian countries including China, Japan, and Korea). Most pear trees have 17 chromosomes (diploidy, 2n=2x=34). Their genetic studies and precise cultivar breeding are highly restricted by conditions such as self-incompatibility controlled by S-locus and juvenility as one major character of fruit crops. Genetic studies on Pyrus have been promoted by the development of various molecular markers. These markers are being utilized actively in various genetic studies, including genetic relationship analysis, genetic mapping, and QTL analysis. In addition, research on pear genetic linkage maps has been extended to studies for the identification of QTL for target traits such as disease resistance and genetic loci of useful traits. NGS technology has radically reduced sequencing expenses based on massive parallel reactions to enable high-capacity and high-efficiency. NGS based genome analyses have been completed for Chinese pear 'Danshansuli' and European pear 'Bartlett'. In Korea, GWAS for agricultural valuable traits such as floral structure, ripening, and total soluble contents have been conducted through resequencing. GBS has been performed for 'Whangkeumbae', 'Cheongsilri', and 'Minibae'.】