Double flower and hortensia (mophead) hydrangea (Hydrangea macrophylla (Thunb.) Ser.) traits are recessively inherited. Cross breeding of these traits in hydrangea is difficult because it takes about two years from crossing to flowering. In this study, we aimed to obtain DNA linkage markers that would allow accelerated selection of these traits. We used next-generation sequencing to comprehensively collect DNA sequences from the 'Kirakiraboshi' with a double flower and lacecap inflorescence and the 'Frau Yoshimi' with a single flower and hortensia inflorescence, and designed simple sequence repeat (SSR) primer pairs for map construction. We screened 768 SSR primer pairs in 93 F 2 progeny derived from 'Kirakiraboshi' and 'Frau Yoshimi'. We identified 147 loci, which were expanded to 18 linkage groups with a total map length of 980 cM. Linkage analysis identified that both the double flower trait from 'Kirakiraboshi' (d Kira ) and the hortensia trait from 'Frau Yoshimi' (h Frau ) were located on linkage group KF_4. Detailed linkage analysis using 351 F 2 progeny revealed a 34.8 cM map length between the two loci and identified two tightly linked SSR markers, STAB045 for d Kira and HS071 for h Frau . Genetic analysis suggested that double flower and hortensia traits are each controlled by a single recessive gene. Together, the linkage map, SSR markers, and genetic information obtained in this study will be useful for future hydrangea breeding.

Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea macrophylla) is one of its most important traits. In this study, genome sequence information was obtained to explore effective DNA markers and the causative genes for double flower production in hydrangea. Single-molecule real-time sequencing data followed by a Hi-C analysis was employed. Two haplotype-phased sequences were obtained from the heterozygous genome of hydrangea. One assembly consisted of 3,779 scaffolds (2.256 Gb in length and N50 of 1.5 Mb), the other also contained 3,779 scaffolds (2.227 Gb in length, and N50 of 1.4 Mb). A total of 36,930 genes were predicted in the sequences, of which 32,205 and 32,222 were found in each haplotype. A pair of 18 pseudomolecules was constructed along with a high-density SNP genetic linkage map. Using the genome sequence data, and two F2 populations, the SNPs linked to double flower loci (djo and dsu) were discovered. DNA markers linked to djo and dsu were developed, and these could distinguish the recessive double flower allele for each locus, respectively. The LEAFY gene is a very likely candidate as the causative gene for dsu, since frameshift was specifically observed in the double flower accession with dsu.

25Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea 26 macrophylla) is one of its most important traits. In this study, genome sequence information was 27 obtained to explore effective DNA markers and the causative genes for double flower production in 28 hydrangea. Single molecule real-time sequencing data followed by a HiC analysis was employed. The 29 resultant haplotype-phased sequences consisted of 3,779 sequences (2.256 Gb in length and N50 of 30 1.5 Mb), and 18 pseudomolecules comprising 1.08 Gb scaffold sequences along with a high-density 31 SNP genetic linkage map. Using the genome sequence data obtained from two breeding populations, 32the SNPs linked to double flower loci (D jo and D su ), were discovered for each breeding population. 33DNA markers J01 linked to D jo and S01 linked to D su were developed, and these could be used 34 successfully to distinguish the recessive double flower allele for each locus respectively. The LEAFY 35 gene was suggested as the causative gene for D su, since frameshift was specifically observed in double 36 flower accession with d su . The genome information obtained in this study will facilitate a wide range 37 of genomic studies on hydrangea in the future. 38 39 Keywords: 40 Hydrangea, double flower, de novo genome sequencing, DNA marker 41 42 43 44 45 46 47 48 constructed the genomic DNA sequence, obtained SNPs information, and performed gene prediction. 97We also developed DNA markers linked to D jo using SNP information obtained by double digest 98 restriction site associated DNA sequence (ddRAD-Seq) analysis of breeding population 12GM1, 99 which segregated double flower phenotypes of D jo . In addition, we attempted to identify the causative 100 genes for D jo and D su . 101 102 2. Materials and Methods 103 2.