节瓜及近缘葫芦科作物种质资源rDNA-ITS序列分析与系统进化研究

Analysis on the Internal Transcribed Spacers( ITS) Sequences and Phylogenetics of Chieh-qua and its Affinis Cucurbitaceae Germplasm Resources

通过PCR扩增获得了56份节瓜种质资源及7份相关瓜类种质资源的rDNA-ITS序列,测序后结合GenBank中34份葫芦科作物的ITS序列,利用生物信息学软件分析序列长度、变异位点、G＋C含量、遗传分歧、同源性百分比差异、系统进化关系。节瓜种质资源ITS基本序列全长612-617 bp,G＋C含量59.97%-61.07%,供试材料间共96个变异位点,其中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。基于ITS序列差异,56份节瓜材料聚为5大类,其中12-2-3材料为单独一个分支,黑毛节瓜H2251为第二分支,H5FA为第三分支,剩余节瓜材料聚为第四、五分支。节瓜材料12-2-3与其他材料遗传分歧最大,农艺性状与抗性也差异较大,可用作节瓜杂交育种的亲本以扩大选择范围。节瓜的系统位置排列在Indomelothria blumei（GU799496）与Dactyliandra welwitschii（HQ201973）之间,与Indomelothria blumei亲缘关系最近;Mrbayes软件分析表明,葫芦科作物Zehneria thwaitesii（AM981145）、Ctenolepis cerasiformis（AM981142）、Cucumis melo（AM36377）、Dactyliandra welwitschii（HQ201973）、Trochomeria macrocarpa（AM981141）最原始,系统进化顺序为甜瓜→南瓜、栝楼、苦瓜、丝瓜、西瓜、蒲瓜→冬瓜、节瓜→黄瓜。本研究通过分析节瓜及近缘葫芦科作物种质资源的ITS序列,为其系统进化、DNA指纹鉴别、育种亲本选择及比较组学分析等提供了分子生物学依据。

56 chieh-qua and 7 other cucurbits germplasm resources were collected from south China,subsequently rDNA-ITS amplication and sequencing were performed. Combining ITS sequences of several cucurbitaceous plants downloaded from GenBank,the lengths,variable sites,G ＋ C content,genetic divergences,homologies,phylogenetics,and system position were analyzed by bioinformatic softwares. ITS complete sequences of chieh-qua and its affinis cucurbitaceae germplasm resources were obtained. The full-length of chieh-qua ITS was612- 617 bp,G ＋ C content were 59. 97%- 61. 07%. The sequence alignment results showed that there were 94 variable sites（ ITS1： 47,ITS2： 40,5. 8S： 7） among chieh-qua germplasm resources,some of which had significant species-specific characteristics and could be used as DNA fingerprint marker. 56 chieh-qua germplasm materials were clustered into 3 groups based on ITS sequences variance,12-2-3 was ranked into the first branch,＇Heimaojiegua H2251＇ and ＇ H5FA＇ were classified into the second and third branch respectively,and the rest materials were grouped into the fourth and fifth group. Compared with the other cultivated species,the genetic divergence of chieh-qua material 12-2-3 was the biggest ranging from 3. 3 to 4. 6,and there are large differences on agronomic traits and resistance between 12-2-3 and others,leading to be used as crossbreeding parent. The system position of chieh-qua was between Indomelothria blumei（ GU799496） and Dactyliandra welwitschii（ HQ201973）,and had the closest relationship with Indomelothria blumei. Mrbayes software analysis showed that Zehneria thwaitesii（ AM981145）,Ctenolepis cerasiformis（ AM981142）,Cucumis melo（ AM36377）,Dactyliandra welwitschii（ HQ20 1973）,Trochomeria macrocarpa（ AM981141） were the most primordial species,the system evolution order was Cucumis melo（ AM36377） → Cucurbita moschata,Trichosanthes yunnanensis,Momordica charantia,Luffa aegyptiaca,Citrullus lanatus（ FJ915098）,Lagenaria siceraria → Benincasa hispida,Benincasa hispida var. chieh-qua→ Cucumis sativus. The phylogeny of chieh-qua and its affinis cucurbitaceae germplasm resources was investigated by using ITS sequences and it would provide useful information for their breeding,germplasm DNA fingerprint identification,classification and evolution and comparative analysis,etc.