莲叶绿体基因组图谱的构建及其系统进化分析
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摘要
莲属现存两个种：中国莲(Nelumbo nucifera Gaertn.)和美洲黄莲(Nelumbo lutea Willd.).长期以来人们一直认为莲属于睡莲目,其系统进化地位一直存在争议。对莲藕的基因组进行研究,可以为莲藕系统进化分析和莲藕育种提供理论基础。本研究围绕莲藕的基因组进行了三方面的工作：莲藕叶绿体全序列测定以及比较分析；莲藕系统进化分析；莲藕基因组纯合度筛选及基因组测序调查。
主要研究结果如下：
1.以中国莲和美洲黄莲为材料,利用鸟枪法测定了中国莲和美洲黄莲的叶绿体全序列。结果表明：中国莲叶绿体DNA全长163307bp,而美洲黄莲叶绿体DNA全长163206bp。两者GC含量基本相同,中国莲叶绿体GC含量为37.99%而美洲黄莲叶绿体GC含量为38.01%。莲属叶绿体一共编码了113个不同的基因,包括4个rRNA基因(16S,23S,4.5S和5S),30个tRNA基因和79个不同的蛋白质编码基因。其中4个rRNA基因,7个tRNA基因和6个蛋白质编码基因分布在IR区,其基因总数为130。莲属叶绿体有55.66%的序列是基因编码区,其中,48.43%(79086 bp)的序列编码蛋白质；5.53%(9026)的序列编码核糖体RNA(rRNA)；剩下的1.71%(2788bp)编码转运RNA(tRNA)。非编码区占整个叶绿体的44.34%,其中包括基因间隔区(58078 bp；占全叶绿体35.56%)和内含子区(14329 bp；占全叶绿体8.77%)。对两个叶绿体全序列进行比对后,共检测到398个SNP位点和161个INDEL位点。
2.利用中国莲和美洲黄莲的叶绿体全序列,结合其他80个物种的叶绿体全序列,对莲属的系统进化地位进行了分析。截取这82个叶绿体基因组中的78个蛋白质编码基因序列,制作成为DNA矩阵,利用MP,ML和BI三种算法进行系统进化分析。结果表明,莲属为双子叶植物,莲科应该归类到山龙眼目(分类名称)中。利用r8s 1.71软件进行了分子进化钟计算,我们估计莲科有大约110百万年的进化历史,莲属仅存的两个种—中国莲和美洲黄莲的分离时间大致是2.41百万年前。
3.进行莲藕,芡实和茭白等水生作物的SSR分子标记开发,得到一批多态性的SSR分子标记,为后续基因组研究奠定基础。利用莲藕SSR分子标记对莲藕种质进行鉴定,从242份莲藕材料中,选出纯合度最高的中间湖莲为测序材料,在华大基因对其进行全基因组de novo测序初步结果的基础上,通过基因组调查,对中间湖莲基因组大小,基因组杂合度,重复序列含量和GC含量进行了分析。初步分析结果表明,中间湖莲基因组大小约为879Mb,其基因组杂合度非常低,重复序列较少。中间湖莲基因组的平均GC含量约为37%,大部分基因组片段的GC含量在34%与40%之间。这些工作为开展构建莲基因组物理图谱和遗传图谱等工作定奠了基础。
There are two surviving Nelumbo species now:Nelumbo nucifera Gaertn. and Nelumbo lutea Willd. Nelumbo was considered as a member of Nymphaeales (water lilies) for a long time, the systematic position of the genus Nelumbo has long remained unsettled. The genome research is essential to understand the systematic position of Nelumbo, and to improve the lotus breeding. We report the complete plastid genome sequences of the Nelumbo, the phylogence of Nelumbo was inferred and are discussed, and we sequensed the Nelumbo genome.
The main results are as follows:
1. We sequenced the two complete plastid genome sequences of N. lutea and N. nucifera using Shot-Gun sequencing. The sizes of the N. lutea and N. nucifera plastid genomes are 163,206 and 163,307 bp, respectively. The overall GC contents are 38.01%(N. lutea) and 37.99%(N. nucifera), similar to the currently available plastid genomes of stem dicots. The Nelumbo plastome contains 113 different genes, including four distinct rRNAs (16S,23S,4.5S and 5S),30 distinct tRNAs and 79 distinct peptide-coding genes. Four rRNA, seven tRNA and six peptide-coding genes (including rps12) are duplicated in the IR region, giving a total of 130 genes.55.66% of the plastid is covered by coding regions, including peptide-coding genes (79,086 bp; 48.43%), ribosomal RNA genes (9,026 bp; 5.53%) and transfer RNA genes (2,788bp; 1.71%). The remaining 44.34% consists of intergenic regions (58,078 bp; 35.56%) and introns (14,329 bp; 8.77%). We detected 398 SNP (single nucleotide polymorphism) sites and 161 INDEL (insert/delete) sites between the N. lutea and N nucifera plastid genomes.
2. To resolve the long-standing controversies about the phylogeny of Nelumbo, we report here the two complete plastid genome sequences of N. lutea and N. nucifera. Simultaneously, a DNA matrix containing 78 protein-coding genes from four gymnosperms and 78 angiosperms was analyzed using three phylogenetic methods (maximum parsimony, MP; maximum likelihood, ML; and Bayesian inference, BI). The results implied that Nelumbo is a stem endicot and should be placed into the Proteales. Nelumbonaceae have an age of 110 million years, and the splitting between N. lutea and N. nucifera is estimated to have occurred about 2.41 Mya.
3. We report the development and characterization of microsatellite primers designed for Nelumbo nucifera, Euryale ferox and Zizania latifolia.Using these polymorphic microsatellite markers, we select the ZJH wild lotus as the best sequencing material. We did the Nelumbo genome survey using about 18G sequencing data. The genome size of Nelumbo is about 879Mp and the heterozygous rate in this genome is very low. There are some repeat regions in this genome, according to the distribution of GC depth, we can infer that the GC content of Nelumbo genome is about 37%. This work will be useful in nelumbo linkge map constraction and physical map constraction.
主要研究结果如下：
1.以中国莲和美洲黄莲为材料,利用鸟枪法测定了中国莲和美洲黄莲的叶绿体全序列。结果表明：中国莲叶绿体DNA全长163307bp,而美洲黄莲叶绿体DNA全长163206bp。两者GC含量基本相同,中国莲叶绿体GC含量为37.99%而美洲黄莲叶绿体GC含量为38.01%。莲属叶绿体一共编码了113个不同的基因,包括4个rRNA基因(16S,23S,4.5S和5S),30个tRNA基因和79个不同的蛋白质编码基因。其中4个rRNA基因,7个tRNA基因和6个蛋白质编码基因分布在IR区,其基因总数为130。莲属叶绿体有55.66%的序列是基因编码区,其中,48.43%(79086 bp)的序列编码蛋白质；5.53%(9026)的序列编码核糖体RNA(rRNA)；剩下的1.71%(2788bp)编码转运RNA(tRNA)。非编码区占整个叶绿体的44.34%,其中包括基因间隔区(58078 bp；占全叶绿体35.56%)和内含子区(14329 bp；占全叶绿体8.77%)。对两个叶绿体全序列进行比对后,共检测到398个SNP位点和161个INDEL位点。
2.利用中国莲和美洲黄莲的叶绿体全序列,结合其他80个物种的叶绿体全序列,对莲属的系统进化地位进行了分析。截取这82个叶绿体基因组中的78个蛋白质编码基因序列,制作成为DNA矩阵,利用MP,ML和BI三种算法进行系统进化分析。结果表明,莲属为双子叶植物,莲科应该归类到山龙眼目(分类名称)中。利用r8s 1.71软件进行了分子进化钟计算,我们估计莲科有大约110百万年的进化历史,莲属仅存的两个种—中国莲和美洲黄莲的分离时间大致是2.41百万年前。
3.进行莲藕,芡实和茭白等水生作物的SSR分子标记开发,得到一批多态性的SSR分子标记,为后续基因组研究奠定基础。利用莲藕SSR分子标记对莲藕种质进行鉴定,从242份莲藕材料中,选出纯合度最高的中间湖莲为测序材料,在华大基因对其进行全基因组de novo测序初步结果的基础上,通过基因组调查,对中间湖莲基因组大小,基因组杂合度,重复序列含量和GC含量进行了分析。初步分析结果表明,中间湖莲基因组大小约为879Mb,其基因组杂合度非常低,重复序列较少。中间湖莲基因组的平均GC含量约为37%,大部分基因组片段的GC含量在34%与40%之间。这些工作为开展构建莲基因组物理图谱和遗传图谱等工作定奠了基础。
There are two surviving Nelumbo species now:Nelumbo nucifera Gaertn. and Nelumbo lutea Willd. Nelumbo was considered as a member of Nymphaeales (water lilies) for a long time, the systematic position of the genus Nelumbo has long remained unsettled. The genome research is essential to understand the systematic position of Nelumbo, and to improve the lotus breeding. We report the complete plastid genome sequences of the Nelumbo, the phylogence of Nelumbo was inferred and are discussed, and we sequensed the Nelumbo genome.
The main results are as follows:
1. We sequenced the two complete plastid genome sequences of N. lutea and N. nucifera using Shot-Gun sequencing. The sizes of the N. lutea and N. nucifera plastid genomes are 163,206 and 163,307 bp, respectively. The overall GC contents are 38.01%(N. lutea) and 37.99%(N. nucifera), similar to the currently available plastid genomes of stem dicots. The Nelumbo plastome contains 113 different genes, including four distinct rRNAs (16S,23S,4.5S and 5S),30 distinct tRNAs and 79 distinct peptide-coding genes. Four rRNA, seven tRNA and six peptide-coding genes (including rps12) are duplicated in the IR region, giving a total of 130 genes.55.66% of the plastid is covered by coding regions, including peptide-coding genes (79,086 bp; 48.43%), ribosomal RNA genes (9,026 bp; 5.53%) and transfer RNA genes (2,788bp; 1.71%). The remaining 44.34% consists of intergenic regions (58,078 bp; 35.56%) and introns (14,329 bp; 8.77%). We detected 398 SNP (single nucleotide polymorphism) sites and 161 INDEL (insert/delete) sites between the N. lutea and N nucifera plastid genomes.
2. To resolve the long-standing controversies about the phylogeny of Nelumbo, we report here the two complete plastid genome sequences of N. lutea and N. nucifera. Simultaneously, a DNA matrix containing 78 protein-coding genes from four gymnosperms and 78 angiosperms was analyzed using three phylogenetic methods (maximum parsimony, MP; maximum likelihood, ML; and Bayesian inference, BI). The results implied that Nelumbo is a stem endicot and should be placed into the Proteales. Nelumbonaceae have an age of 110 million years, and the splitting between N. lutea and N. nucifera is estimated to have occurred about 2.41 Mya.
3. We report the development and characterization of microsatellite primers designed for Nelumbo nucifera, Euryale ferox and Zizania latifolia.Using these polymorphic microsatellite markers, we select the ZJH wild lotus as the best sequencing material. We did the Nelumbo genome survey using about 18G sequencing data. The genome size of Nelumbo is about 879Mp and the heterozygous rate in this genome is very low. There are some repeat regions in this genome, according to the distribution of GC depth, we can infer that the GC content of Nelumbo genome is about 37%. This work will be useful in nelumbo linkge map constraction and physical map constraction.
引文
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