Matsumoto A, Tsumura Y
Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan.
Theor Appl Genet. 2004 Dec;110(1):80-91. doi: 10.1007/s00122-004-1754-1. Epub 2004 Nov 10.
We have developed and evaluated sequence-tagged site (STS) primers based on expressed sequence-tag information derived from sugi (Cryptomeria japonica) for use in hinoki (Chamaecyparis obtusa), a species that belongs to a different family (although it appears to be fairly closely related to sugi). Of the 417 C. japonica STS primer pairs we screened, 120 (approximately 30%) were transferable and provided specific PCR amplification products from 16 C. obtusa plus trees. We used haploid megagametophytes to investigate the homology of 80 STS fragments between C. obtusa and C. japonica and to identify orthologous loci. Nearly 90% of the fragments showed high (>70%) degrees of similarity between the species, and 35 STSs indicated homology to entries with the same putative function in a public DNA database. Of the 120 STS fragments amplified, 72 showed restriction fragment length polymorphisms; in addition, the CC2430 primers detected amplicon length polymorphism. We assessed the inheritance pattern of 27 cleaved amplified polymorphic sequence markers, using 20 individuals from the segregation population. All the markers analyzed were consistent with the marker inheritance patterns obtained from the screening panel, and no markers (except CC2716) showed significant (P<0.01) deviation from the expected segregation ratio. In total, 136 polymorphic markers were developed using C. japonica-based STS primers without any sequence modification. In addition, the applicability of STS-based markers developed in one species to other species was found to closely reflect the evolutionary distance between the species, which is roughly concordant with the difference between their rbcL sequences. We plan to use these markers for genetic studies in C. obtusa. Most of the markers should also provide reliable anchor loci for comparative mapping studies of the C. obtusa and C. japonica genomes.
我们基于从日本柳杉（Cryptomeria japonica）获得的表达序列标签信息，开发并评估了序列标签位点（STS）引物，用于扁柏（Chamaecyparis obtusa），扁柏属于不同的科（尽管它似乎与日本柳杉关系相当密切）。在我们筛选的417对日本柳杉STS引物对中，有120对（约30%）可转移，并从16株扁柏优树上获得了特异性PCR扩增产物。我们使用单倍体雌配子体研究了扁柏和日本柳杉之间80个STS片段的同源性，并鉴定直系同源位点。近90%的片段在两个物种间显示出高度（>70%）相似性，35个STS与公共DNA数据库中具有相同推定功能的条目显示出同源性。在扩增的120个STS片段中，72个显示出限制性片段长度多态性；此外，CC2430引物检测到扩增子长度多态性。我们使用分离群体中的20个个体评估了27个酶切扩增多态性序列标记的遗传模式。所有分析的标记都与从筛选群体获得的标记遗传模式一致，并且没有标记（除CC2716外）显示出与预期分离比有显著（P<0.01）偏差。总共使用基于日本柳杉的STS引物开发了136个多态性标记，且未进行任何序列修饰。此外，发现一个物种中开发的基于STS的标记对其他物种的适用性密切反映了物种间的进化距离，这与它们的rbcL序列差异大致一致。我们计划将这些标记用于扁柏的遗传研究。大多数标记也应为扁柏和日本柳杉基因组的比较作图研究提供可靠的锚定位点。
