乳白石蒜rDNA-ITS序列分析及种内系统发育研究

Analysis on rDNA-ITS sequence and research of intra-specific phylogeny of Lycoris albiflora

对来源于不同产地的11个乳白石蒜(Lycoris albiflora Koidz.)种源的rDNA-ITS序列进行了扩增、纯化、克隆、酶切和测序,并对各种源的rDNA-ITS序列长度、G+C含量、碱基差异和遗传距离进行了比较分析,构建了系统发育树。结果表明,乳白石蒜11个种源的rDNA-ITS序列具有较高的同源性,但不同种源间的ITS序列长度和碱基变异较大;乳白石蒜rDNA-ITS序列总长度约为700 bp,共有318个变异位点;ITS1、ITS2和5.8S rDNA片段长度分别为222～245、240～252和163 bp;ITS序列中的G+C含量均明显高于A+T含量,ITS1和ITS2片段中的G+C含量分别为53.8%～70.5%和63.4%～73.4%;碱基变异类型多为颠换、转换、插入和缺失。种源间的遗传距离差异较大,其中浙江天目山种源(TM3)与浙江宁波种源(NB2)的遗传距离最小(0.000),其他种源间遗传距离为0.067～0.323。基于ITS序列分析结果可将11个乳白石蒜种源聚为3大类,第1类包括采自浙江天目山(TM1和TM2)和浙江宁波(NB3)的3个种源,花和花丝多为白色;第2类包含来源于不同产地的7个种源,花多为乳白色或乳黄色;第3类仅有采自浙江兰溪(LX)的1个种源,花色变异较大。研究结果表明,乳白石蒜种内有丰富的遗传变异,种源间的ITS序列差异与花的特征变化一致,但与地理分布并不相关;rDNA-ITS序列分析可用于乳白石蒜的亲缘关系、物种鉴别和遗传多样性研究。

The rDNA-ITS sequence of eleven provenances of Lycoris albiflora Koidz. from different locations was amplified, purified, cloned, enzyme digested and sequenced. Sequence length, G ＋ C content, base difference and genetic distance of rDNA-ITS sequence of different provenances of L. albiflora were comparatively analyzed, and the phylogenetic dendrogram was constructed, too. The results show that the rDNA-ITS sequence of L. albiflora is great homologous, but variations in total length of rDNA-ITS sequence and base among different provenances are greater. Total length of rDNA-ITS sequence is about 700 bp and there are 318 variable sites. The fragment length of ITS1, ITS2 and 5.8S rDNA is 222 -245, 240 -252 and 163 bp, respectively. G ＋ C content in rDNA-ITS sequence is higher than A ＋ T content, and G ＋ C content in ITS1 and ITS2 fragments is 53.8% -70.5% and 63.4% - 73. 4%, respectively. Most of nucleotide variations of rDNA-ITS sequence of L. albiflora are transversion, conversion, insertion and deletion. The difference of genetic distance among provenances is greater, in which the genetic distance between TM3 from Tianmu Mountain of Zhejiang and NB2 from Ningbo of Zhejiang is 0. 000, and that among other provenances is 0. 067 - 0. 323. The eleven provenances are clustered into three groups based on analysis result of rDNA-ITS sequence. The first group contains three provenances （TM1 and TM2 from Tianmu Mountain of Zhejiang and NB3 from Ningbo of Zhejiang ）, their flowers and filaments are mostly white. The second group contains seven provenances from different locations, their flowers are mostly milky white and buff. The third grouo only contains one provenance （ LX from Lanxi of Zhejiang）, and the variation of its flower color is greater. It is suggested that there are rich intra-specific genetic variations in L. albiflora, and the difference of rDNA-ITS sequence is identical with the change of flower character, but is uncorrelated with the geographical distribution of these provenances. It is concluded that the rDNA-ITS sequence analysis can be used in researches of genetic relationship, species identification and genetic diversity of L. albiflora.