Table1_Development and Application of EST-SSR Markers in Cephalotaxus oliveri From Transcriptome Sequences.DOCX

Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3′UTR, and 5′UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune, Ametotaxus argotaenia, and Pseudotaxus chienii, respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species.

Cephalotaxus oliveri，中国特有的一种针叶植物，兼具药用和观赏价值。然而，鉴于该物种分子标记和遗传信息的有限性，不足以支持其进一步的遗传学研究。在本研究中，我们首次对转录组序列进行了特征化分析，并开发了EST-SSRs。结果显示，从36446个非冗余基因中鉴定出共计5089个SSR，其密度为每11.1 kb有一个SSR。最常见的类型为三核苷酸重复，排除单核苷酸重复，其次为二核苷酸重复。在三核苷酸和二核苷酸重复中，AAG/CTT和AT/AT分别表现出最高的频率。在所鉴定出的SSR中，分别有671个、1125个和1958个位于编码序列（CDS）、3′非翻译区（3′UTR）和5′非翻译区（5′UTR）。功能注释显示，含有SSR的非冗余基因涉及生长与发育，并具有多种生物学功能。在成功设计的引物对中，随机选取了238对引物进行EST-SSR标记的扩增和验证，并识别出47对引物表现出多态性。最终，选用了28对高多态性引物进行遗传分析，揭示了中等程度的遗传多样性。七个自然采集的C. oliveri位点被划分为两个遗传组。此外，这28个EST-SSRs在Cephalotaxus fortune、Ametotaxus argotaenia和Pseudotaxus chienii中的迁移率分别为96.43%、71.43%和78.57%。本研究开发的这些标记为C. oliveri及其相关物种的进一步遗传和适应性进化研究奠定了基础。