Genomic variation across distribution of Micro-Tom, a model cultivar of tomato (Solanum lycopersicum).

Micro-Tom is a cultivar of tomato (Solanum lycopersicum), which is known as a major crop and model plant in Solanaceae. Micro-Tom has phenotypic traits such as dwarfism, and substantial EMS-mutagenized lines have been reported. After Micro-Tom was generated in Florida, USA, it was distributed to research institutes worldwide and used as a genetic resource. In Japan, the Micro-Tom lines have been genetically fixed; currently three lines have been re-distributed from three institutes, but many phenotypes among the lines have been observed. We have determined the genome sequence de novo of the Micro-Tom KDRI line, one of the Micro-Tom lines distributed from Kazusa DNA Research Institute (KDRI) in Japan, and have built chromosome-scale pseudomolecules. Genotypes among six Micro-Tom lines, including three in Japan, one in the United States, one in France, and one in Brazil showed phenotypic alternation. Here, we unveiled the swift emergence of genetic diversity in both phenotypes and genotypes within the Micro-Tom genome sequence during its propagation. These findings offer valuable insights crucial for the management of bioresources.

Different control of resistance to two Colletotrichum orbiculare pathogenic races 0 and 1 in cucumber (Cucumis sativus L.).

KEY MESSAGE: Quantitative trait locus analysis identified independent novel loci in cucumbers responsible for resistance to races 0 and 1 of the anthracnose fungal pathogen Colletotrichum orbiculare. Cucumbers have been reported to be vulnerable to Colletotrichum orbiculare, causing anthracnose disease with significant yield loss under favorable conditions. The deployment of a single recessive Cssgr gene in cucumber breeding for anthracnose resistance was effective until a recent report on high-virulent strains infecting cucumbers in Japan conquering the resistance. QTL mapping was conducted to identify the resistance loci in the cucumber accession Ban Kyuri (G100) against C. orbiculare strains 104-T and CcM-1 of pathogenic races 0 and 1, respectively. A single dominant locus An5 was detected in the disease resistance hotspot on chromosome 5 for resistance to 104-T. Resistance to CcM-1 was governed by three loci with additive effects located on chromosomes 2 (An2) and 1 (An1.1 and An1.2). Molecular markers were developed based on variant calling between the corresponding QTL regions in the de novo assembly of the G100 genome and the publicly available cucumber genomes. Multiple backcrossed populations were deployed to fine-map An5 locus and narrow the region to approximately 222 kbp. Accumulation of An2 and An1.1 alleles displayed an adequate resistance to CcM-1 strain. This study provides functional molecular markers for pyramiding resistance loci that confer sufficient resistance against anthracnose in cucumbers.

A self-compatible pear mutant derived from γ-irradiated pollen carries an 11-Mb duplication in chromosome 17.

Self-compatibility is a highly desirable trait for pear breeding programs. Our breeding program previously developed a novel self-compatible pollen-part Japanese pear mutant (Pyrus pyrifolia Nakai), '415-1', by using γ-irradiated pollen. '415-1' carries the S-genotype S4dS5S5, with "d" indicating a duplication of S 5 responsible for breakdown of self-incompatibility. Until now, the size and inheritance of the duplicated segment was undetermined, and a reliable detection method was lacking. Here, we examined genome duplications and their inheritance in 140 F1 seedlings resulting from a cross between '515-20' (S1S3) and '415-1'. Amplicon sequencing of S-RNase and SFBB18 clearly detected S-haplotype duplications in the seedlings. Intriguingly, 30 partially triploid seedlings including genotypes S1S4dS5, S3S4dS5, S1S5dS5, S3S5dS5, and S3S4dS4 were detected among the 140 seedlings. Depth-of-coverage analysis using ddRAD-seq showed that the duplications in those individuals were limited to chromosome 17. Further analysis through resequencing confirmed an 11-Mb chromosome duplication spanning the middle to the end of chromosome 17. The duplicated segment remained consistent in size across generations. The presence of an S3S4dS4 seedling provided evidence for recombination between the duplicated S5 segment and the original S4haplotype, suggesting that the duplicated segment can pair with other parts of chromosome 17. This research provides valuable insights for improving pear breeding programs using partially triploid individuals.

Assessing the Potential for Genome-Assisted Breeding in Red Perilla Using Quantitative Trait Locus Analysis and Genomic Prediction.

Red perilla is an important medicinal plant used in Kampo medicine. The development of elite varieties of this species is urgently required. Medicinal compounds are generally considered target traits in medicinal plant breeding; however, selection based on compound phenotypes (i.e., conventional selection) is expensive and time consuming. Here, we propose genomic selection (GS) and marker-assisted selection (MAS), which use marker information for selection, as suitable selection methods for medicinal plants, and we evaluate the effectiveness of these methods in perilla breeding. Three breeding populations generated from crosses between one red and three green perilla genotypes were used to elucidate the genetic mechanisms underlying the production of major medicinal compounds using quantitative trait locus analysis and evaluating the accuracy of genomic prediction (GP). We found that GP had a sufficiently high accuracy for all traits, confirming that GS is an effective method for perilla breeding. Moreover, the three populations showed varying degrees of segregation, suggesting that using these populations in breeding may simultaneously enhance multiple target traits. This study contributes to research on the genetic mechanisms of the major medicinal compounds of red perilla, as well as the breeding efficiency of this medicinal plant.

Telomere-to-telomere genome assembly of an allotetraploid pernicious weed, Echinochloa phyllopogon.

Echinochloa phyllopogon is an allotetraploid pernicious weed species found in rice fields worldwide that often exhibit resistance to multiple herbicides. An accurate genome sequence is essential to comprehensively understand the genetic basis underlying the traits of this species. Here, the telomere-to-telomere genome sequence of E. phyllopogon was presented. Eighteen chromosome sequences spanning 1.0 Gb were constructed using the PacBio highly fidelity long technology. Of the 18 chromosomes, 12 sequences were entirely assembled into telomere-to-telomere and gap-free contigs, whereas the remaining six sequences were constructed at the chromosomal level with only eight gaps. The sequences were assigned to the A and B genome with total lengths of 453 and 520 Mb, respectively. Repetitive sequences occupied 42.93% of the A genome and 48.47% of the B genome, although 32,337, and 30,889 high-confidence genes were predicted in the A and B genomes, respectively. This suggested that genome extensions and gene disruptions caused by repeated sequence accumulation often occur in the B genome before polyploidization to establish a tetraploid genome. The highly accurate and comprehensive genome sequence could be a milestone in understanding the molecular mechanisms of the pernicious traits and in developing effective weed control strategies to avoid yield loss in rice production.

From green to red: Urban heat stress drives leaf color evolution.

Prevalence of impervious surface and resulting higher temperatures in urban areas, known as urban heat islands, comprises prominent characteristics in global cities. However, it is not known whether and how urban plants adapt to such heat stress. This study focused on Oxalis corniculata, which has intraspecific polymorphism in leaf color (green and red) and examined whether the leaf color variation is associated with urban heat stress. Field observations revealed that green-leaved plants were dominant in green habitats, and red-leaved individuals were dominant in urban habitats, at local (<500 meters), landscape (<50 kilometers), and global scales. Growth and photosynthesis experiments demonstrated that red-leaved individuals performed better under heat stress, while green-leaved individuals performed better under nonstressful conditions. Genome-wide SNP analysis suggests that the red leaf may have evolved multiple times from the ancestral green leaf. Overall, the results suggest that the red leaves of O. corniculata observed in cities worldwide are evidence of plant adaptive evolution due to urban heat islands.

Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos.

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., 'Nipponbare') regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The natural mutation rate of 1.2 × 10-8 calculated using the variants in the seed-propagated plants was consistent with the values reported previously. The ratio of single nucleotide variants (SNVs) among the variants in the seed-propagated plants was 91.1%, which is higher than 56.1% previously reported, and not significantly different from those in the regenerants. Overall, the ratio of transitions to transversions of SNVs was lower in the regenerants as shown previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

Ongoing Rapid Evolution of a Post-Y Region Revealed by Chromosome-Scale Genome Assembly of a Hexaploid Monoecious Persimmon (Diospyros kaki).

Plants have evolved sex chromosomes independently in many lineages, and loss of separate sexes can also occur. In this study, we assembled a monoecious recently hexaploidized persimmon (Diospyros kaki), in which the Y chromosome has lost the maleness-determining function. Comparative genomic analysis of D. kaki and its dioecious relatives uncovered the evolutionary process by which the nonfunctional Y chromosome (or Ymonoecy) was derived, which involved silencing of the sex-determining gene, OGI, approximately 2 million years ago. Analyses of the entire X and Ymonoecy chromosomes suggested that D. kaki's nonfunctional male-specific region of the Y chromosome (MSY), which we call a post-MSY, has conserved some characteristics of the original functional MSY. Specifically, comparing the functional MSY in Diospyros lotus and the nonfunctional "post-MSY" in D. kaki indicated that both have been rapidly rearranged, mainly via ongoing transposable element bursts, resembling structural changes often detected in Y-linked regions, some of which can enlarge the nonrecombining regions. The recent evolution of the post-MSY (and possibly also MSYs in dioecious Diospyros species) therefore probably reflects these regions' ancestral location in a pericentromeric region, rather than the presence of male-determining genes and/or genes controlling sexually dimorphic traits.

Genetic basis of lineage-specific evolution of fruit traits in hexaploid persimmon.

Frequent polyploidization events in plants have led to the establishment of many lineage-specific traits representing each species. Little is known about the genetic bases for these specific traits in polyploids, presumably due to plant genomic complexity and their difficulties in applying genetic approaches. Hexaploid Oriental persimmon (Diospyros kaki) has evolved specific fruit characteristics, including wide variations in fruit shapes and astringency. In this study, using whole-genome diploidized/quantitative genotypes from ddRAD-Seq data of 173 persimmon cultivars, we examined their population structures and potential correlations between their structural transitions and variations in nine fruit traits. The population structures of persimmon cultivars were highly randomized and not substantially correlated with the representative fruit traits focused on in this study, except for fruit astringency. With genome-wide association analytic tools considering polyploid alleles, we identified the loci associated with the nine fruit traits; we mainly focused on fruit-shape variations, which have been numerically characterized by principal component analysis of elliptic Fourier descriptors. The genomic regions that putatively underwent selective sweep exhibited no overlap with the loci associated with these persimmon-specific fruit traits. These insights will contribute to understanding the genetic mechanisms by which fruit traits are independently established, possibly due to polyploidization events.

Polyploid GWAS reveals the basis of molecular marker development for complex breeding traits including starch content in the storage roots of sweet potato.

Given the importance of prioritizing genome-based breeding of sweet potato to enable the promotion of food and nutritional security for future human societies, here, we aimed to dissect the genetic basis of storage root starch content (SC) when associated with a complex set of breeding traits including dry matter (DM) rate, storage root fresh weight (SRFW), and anthocyanin (AN) content in a mapping population containing purple-fleshed sweet potato. A polyploid genome-wide association study (GWAS) was extensively exploited using 90,222 single-nucleotide polymorphisms (SNPs) obtained from a bi-parental 204 F1 population between 'Konaishin' (having high SC but no AN) and 'Akemurasaki' (having high AN content but moderate SC). Through the comparison of polyploid GWAS on the whole set of the 204 F1, 93 high-AN-containing F1, and 111 low-AN-containing F1 populations, a total of two (consists of six SNPs), two (14 SNPs), four (eight SNPs), and nine (214 SNPs) significantly associated signals were identified for the variations of SC, DM, SRFW, and the relative AN content, respectively. Of them, a novel signal associated with SC, which was most consistent in 2019 and 2020 in both the 204 F1 and 111 low-AN-containing F1 populations, was identified in homologous group 15. The five SNP markers associated with homologous group 15 could affect SC improvement with a degree of positive effect (~4.33) and screen high-starch-containing lines with higher efficiency (~68%). In a database search of 62 genes involved in starch metabolism, five genes including enzyme genes granule-bound starch synthase I (IbGBSSI), α-amylase 1D, α-amylase 1E, and α-amylase 3, and one transporter gene ATP/ADP-transporter were located on homologous group 15. In an extensive qRT-PCR of these genes using the storage roots harvested at 2, 3, and 4 months after field transplantation in 2022, IbGBSSI, which encodes the starch synthase isozyme that catalyzes the biosynthesis of amylose molecule, was most consistently elevated during starch accumulation in sweet potato. These results would enhance our understanding of the underlying genetic basis of a complex set of breeding traits in the starchy roots of sweet potato, and the molecular information, particularly for SC, would be a potential platform for molecular marker development for this trait.

Identification of novel sex determination loci in Japanese weedy melon.

KEY MESSAGE: Japanese weedy melon exhibits unique sex expression with interactions between previously reported sex determination genes and two novel loci. Sex expression contributes to fruit quality and yield in the Cucurbitaceae. In melon, orchestrated regulation by sex determination genes explains the mechanism of sex expression, resulting in a great variety of sexual morphologies. In this study, we examined the Japanese weedy melon UT1, which does not follow the reported model of sex expression. We conducted QTL analysis using F2 plants for flower sex on the main stem and the lateral branch and mapped "occurrence of pistil-bearing flower on the main stem" locus on Chr. 3 (Opbf3.1) and "type of pistil-bearing flower" (female or bisexual) loci on Chr. 2 (tpbf2.1) and Chr. 8 (tpbf8.1). The Opbf3.1 included the known sex determination gene CmACS11. Sequence comparison of CmACS11 between parental lines revealed three nonsynonymous SNPs. A CAPS marker developed from one of the SNPs was closely linked to the occurrence of pistil-bearing flowers on the main stem in two F2 populations with different genetic backgrounds. The UT1 allele on Opbf3.1 was dominant in F1 lines from crosses between UT1 and diverse cultivars and breeding lines. This study suggests that Opbf3.1 and tpbf8.1 may promote the development of pistil and stamen primordia by inhibiting CmWIP1 and CmACS-7 functions, respectively, making the UT1 plants hermaphrodite. The results of this study provide new insights into the molecular mechanisms of sex determination in melons and considerations for the application of femaleness in melon breeding.

Telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake).

Here, we report the first telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake), which consists of 13 sequences (spanning 161.0 Mb) and a 76 kb circular mitochondrial genome. All the 13 sequences were supported with telomeric repeats at the ends. GC-rich regions are located at the middle of the sequences and are enriched with long interspersed nuclear elements (LINEs). Repetitive sequences including long-terminal repeats (LTRs) and LINEs occupy 71.6% of the genome. A total of 21,887 potential protein-coding genes were predicted. The genomic data reported in this study served not only matsutake gene sequences but also genome structures and intergenic sequences. The information gained would be a great reference for exploring the genetics, genomics, and evolutionary study of matsutake in the future, and ultimately facilitate the conservation of this vulnerable genetic resource.

An improved reference genome for Trifolium subterraneum L. provides insight into molecular diversity and intra-specific phylogeny.

Subterranean clover (Trifolium subterraneum L., Ts) is a geocarpic, self-fertile annual forage legume with a compact diploid genome (n = x = 8, 544 Mb/1C). Its resilience and climate adaptivity have made it an economically important species in Mediterranean and temperate zones. Using the cultivar Daliak, we generated higher resolution sequence data, created a new genome assembly TSUd_3.0, and conducted molecular diversity analysis for copy number variant (CNV) and single-nucleotide polymorphism (SNP) among 36 cultivars. TSUd_3.0 substantively improves prior genome assemblies with new Hi-C and long-read sequence data, covering 531 Mb, containing 41,979 annotated genes and generating a 94.4% BUSCO score. Comparative genomic analysis among select members of the tribe Trifolieae indicated TSUd 3.0 corrects six assembly-error inversion/duplications and confirmed phylogenetic relationships. Its synteny with T. pratense, T. repens, Medicago truncatula and Lotus japonicus genomes were assessed, with the more distantly related T. repens and M. truncatula showing higher levels of co-linearity with Ts than between Ts and its close relative T. pratense. Resequencing of 36 cultivars discovered 7,789,537 SNPs subsequently used for genomic diversity assessment and sequence-based clustering. Heterozygosity estimates ranged from 1% to 21% within the 36 cultivars and may be influenced by admixture. Phylogenetic analysis supported subspecific genetic structure, although it indicates four or five groups, rather than the three recognized subspecies. Furthermore, there were incidences where cultivars characterized as belonging to a particular subspecies clustered with another subspecies when using genomic data. These outcomes suggest that further investigation of Ts sub-specific classification using molecular and morpho-physiological data is needed to clarify these relationships. This upgraded reference genome, complemented with comprehensive sequence diversity analysis of 36 cultivars, provides a platform for future gene functional analysis of key traits, and genome-based breeding strategies for climate adaptation and agronomic performance. Pangenome analysis, more in-depth intra-specific phylogenomic analysis using the Ts core collection, and functional genetic and genomic studies are needed to further augment knowledge of Trifolium genomes.

Recurrent neo-sex chromosome evolution in kiwifruit.

Sex chromosome evolution is thought to be tightly associated with the acquisition and maintenance of sexual dimorphisms. Plant sex chromosomes have evolved independently in many lineages1,2 and can provide a powerful comparative framework to study this. We assembled and annotated genome sequences of three kiwifruit species (genus Actinidia) and uncovered recurrent sex chromosome turnovers in multiple lineages. Specifically, we observed structural evolution of the neo-Y chromosomes, which was driven via rapid bursts of transposable element insertions. Surprisingly, sexual dimorphisms were conserved in the different species studied, despite the fact that the partially sex-linked genes differ between them. Using gene editing in kiwifruit, we demonstrated that one of the two Y-chromosome-encoded sex-determining genes, Shy Girl, shows pleiotropic effects that can explain the conserved sexual dimorphisms. These plant sex chromosomes therefore maintain sexual dimorphisms through the conservation of a single gene, without a process involving interactions between separate sex-determining genes and genes for sexually dimorphic traits.

Genome Sequence and Analysis of Nicotiana benthamiana, the Model Plant for Interactions between Organisms.

Nicotiana benthamiana is widely used as a model plant for dicotyledonous angiosperms. In fact, the strains used in research are highly susceptible to a wide range of viruses. Accordingly, these strains are subject to plant pathology and plant-microbe interactions. In terms of plant-plant interactions, N. benthamiana is one of the plants that exhibit grafting affinity with plants from different families. Thus, N. benthamiana is a good model for plant biology and has been the subject of genome sequencing analyses for many years. However, N. benthamiana has a complex allopolyploid genome, and its previous reference genome is fragmented into 141,000 scaffolds. As a result, molecular genetic analysis is difficult to perform. To improve this effort, de novo whole-genome assembly was performed in N. benthamiana with Hifi reads, and 1,668 contigs were generated with a total length of 3.1 Gb. The 21 longest scaffolds, regarded as pseudomolecules, contained a 2.8-Gb sequence, occupying 95.6% of the assembled genome. A total of 57,583 high-confidence gene sequences were predicted. Based on a comparison of the genome structures between N. benthamiana and N. tabacum, N. benthamiana was found to have more complex chromosomal rearrangements, reflecting the age of interspecific hybridization. To verify the accuracy of the annotations, the cell wall modification genes involved in grafting were analyzed, which revealed not only the previously indeterminate untranslated region, intron and open reading frame sequences but also the genomic locations of their family genes. Owing to improved genome assembly and annotation, N. benthamiana would increasingly be more widely accessible.

Chromosome-scale genome assembly of Eustoma grandiflorum, the first complete genome sequence in the genus Eustoma.

Eustoma grandiflorum (Raf.) Shinn. is an annual herbaceous plant native to the southern United States, Mexico, and the Greater Antilles. It has a large flower with a variety of colors and is an important flower crop. In this study, we established a chromosome-scale de novo assembly of E. grandiflorum genome sequences by integrating four genomic and genetic approaches: (1) Pacific Biosciences (PacBio) Sequel deep sequencing, (2) error correction of the assembly by Illumina short reads, (3) scaffolding by chromatin conformation capture sequencing (Hi-C), and (4) genetic linkage maps derived from an F2 mapping population. Thirty-six pseudomolecules and 64 unplaced scaffolds were created, with a total length of 1,324.8 Mb. A total of 36,619 genes were predicted on the genome as high-confidence genes. A comparison of genome structure between E. grandiflorum and C. canephora or O. pumila suggested whole-genome duplication after the divergence between the families Gentianaceae and Rubiaceae. Phylogenetic analysis with single-copy genes suggested that the divergence time between Gentianaceae and Rubiaceae was 74.94 MYA. Genetic diversity analysis was performed for nine commercial E. grandiflorum varieties bred in Japan, from which 254,205 variants were identified. This first report on the construction of a reference genome sequence in the genus Eustoma is expected to contribute to genetic and genomic studies in this genus and in the family Gentianaceae.

Chromosome-scale genome assembly of a Japanese chili pepper landrace, Capsicum annuum 'Takanotsume'.

Here, we report the genome sequence of a popular Japanese chili pepper landrace, Capsicum annuum 'Takanotsume'. We used long-read sequencing and optical mapping, together with the genetic mapping technique, to obtain the chromosome-scale genome assembly of 'Takanotsume'. The assembly consists of 12 pseudomolecules, which corresponds to the basic chromosome number of C. annuum, and is 3,058.5 Mb in size, spanning 97.0% of the estimated genome size. A total of 34,324 high-confidence genes were predicted in the genome, and 83.4% of the genome assembly was occupied by repetitive sequences. Comparative genomics of linked-read sequencing-derived de novo genome assemblies of two Capsicum chinense lines and whole-genome resequencing analysis of Capsicum species revealed not only nucleotide sequence variations but also genome structure variations (i.e. chromosomal rearrangements and transposon-insertion polymorphisms) between 'Takanotsume' and its relatives. Overall, the genome sequence data generated in this study will accelerate the pan-genomics and breeding of Capsicum, and facilitate the dissection of genetic mechanisms underlying the agronomically important traits of 'Takanotsume'.

Chromosome-scale genome assembly of Glycyrrhiza uralensis revealed metabolic gene cluster centred specialized metabolites biosynthesis.

A high-quality genome assembly is imperative to explore the evolutionary basis of characteristic attributes that define chemotype and provide essential resources for a molecular breeding strategy for enhanced production of medicinal metabolites. Here, using single-molecule high-fidelity (HiFi) sequencing reads, we report chromosome-scale genome assembly for Chinese licorice (Glycyrrhiza uralensis), a widely used herbal and natural medicine. The entire genome assembly was achieved in eight chromosomes, with contig and scaffold N50 as 36.02 and 60.2 Mb, respectively. With only 17 assembly gaps and half of the chromosomes having no or one assembly gap, the presented genome assembly is among the best plant genomes to date. Our results showed an advantage of using highly accurate long-read HiFi sequencing data for assembling a highly heterozygous genome including its complexed repeat content. Additionally, our analysis revealed that G. uralensis experienced a recent whole-genome duplication at approximately 59.02 million years ago post a gamma (γ) whole-genome triplication event, which contributed to its present chemotype features. The metabolic gene cluster analysis identified 355 gene clusters, which included the entire biosynthesis pathway of glycyrrhizin. The genome assembly and its annotations provide an essential resource for licorice improvement through molecular breeding and the discovery of valuable genes for engineering bioactive components and understanding the evolution of specialized metabolites biosynthesis.

A genome-wide association and fine-mapping study of white rust resistance in hexaploid chrysanthemum cultivars with a wild diploid reference genome.

White rust caused by Puccinia horiana is one of the most serious diseases of chrysanthemum (Chrysanthemum × morifolium). In this study, we report the DNA markers associated with resistance against P. horiana via a simple approach using the genome of a wild diploid relative, Chrysanthemum seticuspe. First, we identified the important region of the genome in the resistant cultivar "Ariesu" via a genome-wide association study. Simplex single nucleotide polymorphism (SNP) markers mined from ddRAD-Seq were used in a biparental population originating from crosses between resistant "Ariesu" and susceptible "Yellow Queen". The C. seticuspe genome was used as a reference. For the fine mapping of P. horiana resistance locus 2 (Phr2), a comparative whole genome sequencing study was conducted. Although the genome sequences of chrysanthemum cultivars assembled via the short-read approach were fragmented, reliable genome alignments were reconstructed by mapping onto the chromosome level of the C. seticuspe pseudomolecule. Base variants were then identified by comparing the assembled genome sequences of resistant "Ariesu" and susceptible "Yellow Queen". Consequently, SNP markers that were closer to Phr2 compared with ddRAD-Seq markers were obtained. These SNP markers co-segregated with resistance in F1 progenies originating from resistant "Ariesu" and showed robust transferability for detecting Phr2-conferring resistance among chrysanthemum genetic resources. The wild C. seticuspe pseudomolecule, a de facto monoploid genome used for ddRAD-Seq analysis and assembled genome sequence comparison, demonstrated this method's utility as a model for developing DNA markers in hexaploid chrysanthemum cultivars.