背景:日本落叶松(Larixkaempferi)是一种经济上重要的落叶针叶树种,生长在冷温带森林中,是日本特有的。Kuril落叶松(L.gmeliniivar.japonica)是一种自然分布在千岛群岛和萨哈林岛的Dahurian落叶松。杂交落叶松(L.gmeliniivar.粳稻×L.kaempferi)表现出杂种优势,表现为幼体快速生长和对田鼠放牧的高度抗性。由于这些优越的特点已经被林业管理者所重视,杂交落叶松是北海道最重要的人工林树种之一。为了加速这些物种的分子育种,我们收集并比较了全长cDNA同工型(Iso-Seq)和RNA-Seq短读,并将它们合并以构建候选基因作为两种落叶松物种的参考。为了验证结果,从参考序列中筛选与一些开花信号相关基因相关的候选蛋白编码基因(ORFs),并阐明了与密切相关物种的系统发育关系。
结果:使用PacBioRSll的同种型测序和RNA-Seq短读序列的从头组装,我们在日本落叶松和千岛落叶松中确定了50,690和38,684个ORF,分别。日本落叶松和千岛落叶松的BUSCO完整性值分别为90.5%和92.1%,分别。在比较了两种落叶松的ORF后,共有19,813个集群,包括22,571个日本落叶松ORF和22,667个千岛落叶松ORF,包含在维恩图的交点中。此外,我们筛选了几个与开花信号相关的ORF(抑制CO1:SOC1,LEAFY:LFY,开花轨迹T:FT,CONSTANCE:CO)来自两个参考序列,在其他物种中发现的非常相似。
结论:收集的ORF将用作日本和千岛落叶松分子育种的参考序列,以及阐明针叶树基因组的进化和研究功能基因组学。
BACKGROUND: Japanese larch (Larix kaempferi) is an economically important deciduous conifer species that grows in cool-temperate forests and is endemic to Japan. Kuril larch (L. gmelinii var. japonica) is a variety of Dahurian larch that is naturally distributed in the Kuril Islands and Sakhalin. The hybrid larch (L. gmelinii var. japonica × L. kaempferi) exhibits heterosis, which manifests as rapid juvenile growth and high resistance to vole grazing. Since these superior characteristics have been valued by forestry managers, the hybrid larch is one of the most important plantation species in Hokkaido. To accelerate molecular breeding in these species, we collected and compared full-length cDNA isoforms (Iso-Seq) and RNA-Seq short-read, and merged them to construct candidate gene as reference for both Larix species. To validate the results, candidate protein-coding genes (ORFs) related to some flowering signal-related genes were screened from the reference sequences, and the phylogenetic relationship with closely related species was elucidated.
RESULTS: Using the isoform sequencing of PacBio RS ll and the de novo assembly of RNA-Seq short-read sequences, we identified 50,690 and 38,684 ORFs in Japanese larch and Kuril larch, respectively. BUSCO completeness values were 90.5% and 92.1% in the Japanese and Kuril larches, respectively. After comparing the collected ORFs from the two larch species, a total of 19,813 clusters, comprising 22,571 Japanese larch ORFs and 22,667 Kuril larch ORFs, were contained in the intersection of the Venn diagram. In addition, we screened several ORFs related to flowering signals (SUPPRESSER OF OVEREXPRESSION OF CO1: SOC1, LEAFY: LFY, FLOWERING Locus T: FT, CONSTANCE: CO) from both reference sequences, and very similar found in other species.
CONCLUSIONS: The collected ORFs will be useful as reference sequences for molecular breeding of Japanese and Kuril larches, and also for clarifying the evolution of the conifer genome and investigating functional genomics.