中国农业科学 ›› 2010, Vol. 43 ›› Issue (9): 1771-1781 .doi: 10.3864/j.issn.0578-1752.2010.09.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

桑种质资源ITS序列与系统进化分析

陈仁芳,余茂德,刘秀群,陈龙清

  

  1. (华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室)
  • 收稿日期:2009-10-16 修回日期:2010-01-28 出版日期:2010-05-01 发布日期:2010-05-01
  • 通讯作者: 陈龙清

Analysis on the Internal Transcribed Spacers (ITS) Sequences and Phylogenetics of Mulberry (Morus)

CHEN Ren-fang, YU Mao-de, LIU Xiu-qun, CHEN Long-qing
  

  1. (华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室)
  • Received:2009-10-16 Revised:2010-01-28 Online:2010-05-01 Published:2010-05-01
  • Contact: CHEN Long-qing

摘要:

【目的】分析桑ITS序列,为桑系统位置、进化关系、DNA指纹鉴别、育种亲本选择提供分子生物学依据。【方法】利用收集的73份桑资源,总DNA提取,PCR扩增,测序,并从GenBank下载桑科Moraceae,桑属Morus ITS序列,软件分析ITS长度、变异位点、G+C含量、遗传分歧、同源性百分比差异、系统位置与进化关系。【结果】获得ITS完整序列,基本序列全长576 bp,G+C含量59.55%。(ITS1:174 bp,G+C含量61.49%,ITS2:302 bp,G+C含量62.25%,5.8S:100 bp,G+C含量48.00%)。序列比对表明,共166个变异位点,(ITS1:100,ITS2:66,5.8S:0),一些变异位点有明显的种性特征,可作为特异DNA指纹鉴别位点。最大遗传分歧4.0,山桑(M. bombycis)与其它栽培种遗传分歧0.9—1.4。基于ITS桑系统位置,在非洲硬木树(Milicia excelsa,MEU93585)与新西兰鹊肾树(Streblus glaber,DQ499105)之间,与非洲硬木树(MEU93585)亲缘关系最近。Mrbayes分析,新疆黑桑(M. nigra),北美默里桑(M. murrayana,FJ605515)最原始,进化顺序为新疆黑桑,北美默里桑→白桑(M. alba)→华桑(M. cathayana)→长穗桑(M. wittiorum)。【结论】ITS长度、G+C含量、变异位点均可作桑种质资源DNA特异指纹鉴别的依据,特别是碱基变异位点。桑属劳亚古陆(Laurasia)高纬度起源,由北向南迁移。桑属可由地理分布间接反映系统进化关系。

关键词: 桑, ITS, 指纹鉴别, 进化关系

Abstract:

【Objective】 The phylogeny of mulberry (Morus) was investigated by using ITS sequences and it will provide useful information for mulberry breeding, germplasm DNA fingerprint identification, classification and evolution. 【Method】 Seventy-three mulberry (Morus) resources were collected from different localities in China and sequenced ITS for all samples by sanger dideoxy method. Furthermore, other ITS sequences of Moraceae and Morus were also downloaded from GenBank and aligned with the sequences obtained in this study by Clustalx1.83c software. Then, the G+C content, divergence and similarity among sequences were analyzed by DNAstar software. Finally, based on the ITS sequences, phylogenetic trees were reconstructed by PAUP Version4.0b10 and Mrbayes softwares. 【Result】 Seventy-three complete internal transcribed spacer (ITS) sequences of Morus were obtained. The length of Morus ITS sequences is 576 bp, and its G + C content is 59.55% (thereinto, ITS1:174 bp, G+C:61.49%, ITS2:302 bp, G+C:62.25%, 5.8S:100 bp, G+C:48.00%). It has 166 variable sites in total (thereinto, ITS1: 100, ITS2: 66, 5.8S: 0), some variable sites are species-specific and can be used as DNA molecular markers of mulberry (Morus) resources. The largest divergence is 4.0. Compared with the other cultivated species, the variation of M. bombycis is bigger, which has differences ranging from 0.9 to 1.4 to the others. Based on the ITS system position of Morus, M. bombycis is between Milicia excelsa (MEU93585) and Streblus glaber (DQ499105) and mulberry (Morus) and Milicia excelsa (MEU93585) have a closest relationship. In addition, Xinjiang M. nigra and North America M. murrayana (FJ605515) are the most primordial species. The evolutionary order of Morus is as M. nigra and M. murrayana→M. alba→M. cathayana →M. wittiorum. 【Conclusion】 ITS is suitable for phylogenetic analysis of Morus. The results of the length of ITS, the content of G+C%, and the variation sites have provide a molecular biological basis for the classification, phylogeny, DNA fingerprinting and cross breeding parentage choice of mulberry. Morus derived from Laurasia move from north to south. The phylogeny of Morus can be reflected indirectly by geographical distribution.

Key words: mulberry (Morus), ITS, fingerprint identification, phylogeny