败酱草及其近缘种叶绿体全基因组分析和DNA条形码构建

目的:基于败酱类药材基原植物的叶绿体基因组序列分析开发特定的DNA条形码,准确鉴别败酱草及其近缘种。方法:采用Illumina高通量测序技术对败酱科败酱属植物白花败酱、黄花败酱和异叶败酱的叶绿体基因组进行测序;采用生物信息学软件对基因组进行组装注释、特征分析、序列比较和系统发育分析;基于叶绿体基因组高突变区构建特异性DNA条形码进行验证。结果:3种败酱属植物叶绿体基因组呈四分体结构,全长分别为159585、158919、158919 bp,编码的基因数分别为130、132、132,包含8个rRNA基因和37个tRNA基因,蛋白质编码基因数分别为85、87、87。ccsA、ndhG、rpl23、ndhF序列作为特异性DNA条形码成功扩增16份样品,黄花败酱、白花败酱和少蕊败酱聚类在同一支,异叶败酱和糙叶败酱聚类在另一支。通过ccsA、ndhG、ndhF序列可进一步鉴别糙叶败酱和异叶败酱。结论:ccsA、ndhG、rpl23、ndhF序列可作为补充特异性DNA条形码区分败酱草及其近缘种。

Inheritance of Chloroplast and Mitochondrial DNA in Chinese Fir (Cunninghamia lanceolata)

The inheritance of mitochondrial (mt) DNA and chloroplast (cp) DNA was investigated in intergeneric hybrids from crossing between Cunninghamia lanceolata (Lamb.) Hook. and Cryptomeria fortunei Hooibrenk. The chloroplast trnL trnF region and one intra genic segment of the mitochondrial gene, Cox Ⅲ, were amplified from those of the parents and hybrids by PCR using gene specific primers. Cp and mtDNA polymorphisms of the amplified regions were detected between the parents after restriction digestions. Restriction fragment length polymorphism (RFLP) analysis revealed that all the F 1 individuals possessed Cox Ⅲ restriction fragment patterns (characteristic of the paternal parent Cryptomeria fortunei ) and the trnL trnF region (identical to the maternal parent Cunninghamia lanceolata ) showing that a different mode of inheritance for organelle DNA has occurred in the hybrids. Furthermore, the maternal inheritance of chloroplast DNA is reported here for the first time in coniferophyta.

SDS-蛋白酶法分离棉花cpDNA及psbA基因启动子、终止子克隆

采用SDS-蛋白酶法提取了棉花的叶绿体DNA。琼脂糖电泳及紫外分光光度分析表明,所提取的叶绿体DNA质量高。用所提取的叶绿体DNA作为模板进行RAPD扩增,获得了清晰的扩增图谱。同时以所提取的叶绿体DNA作为模板,以psbA基因启动子、终止子序列设计引物,成功地扩增出预期为312bp和382bp的目标片段。将目标片段克隆测序,经Blantn分析,结果表明上述片段正是psbA基因启动子、终止子序列。该方法与传统的提取方法相比省时、高效、无污染,为进一步开展棉花叶绿体基因工程奠定了良好基础。

珙桐cpDNA非编码序列引物反应条件优化与筛选

实验采用改良CTAB方法提取总DNA,以珙桐及其近缘种基因组DNA为材料,优化cpDNA非编码序列PCR扩增条件,优据优化的PCR扩增条件,首次筛选出适合于珙桐分子谱系地理学研究的cpDNA非编码序列和引物。结果表明:25μL PCR反应体系中含有50 ng DNA,10×Buffer,2.5 mmol MgCl2,0.4 mmol dNTPs,0.2μmolpri mer1,0.2μmol pri mer2,1.25 U Pfu DNA Polymerase。适宜的PCR反应程序为94℃5 min,94℃1 min,退火时间45 s,退火温度和72℃延伸时间依不同的引物和产物而不同,35个循环,72℃总延伸7 min。稳定性好的、扩增条带清晰且单一的trnSGCU和trnGUUC、F71和R1516、rps16—F和rps16—R、atpB—49和rbcL—188四对引物可作为进一步珙桐分子谱系地理学研究的引物。

霍山石斛cpDNA全序列微卫星分布及分子鉴别研究

目的:探讨霍山石斛的叶绿体基因组(chloroplast genomic DNA,cpDNA)中微卫星分布规律并设计引物鉴别霍山石斛及其伪品。方法:利用GenBank上已公布的霍山石斛cpDNA全序列,应用分子生物学软件对霍山石斛cpDNA中的微卫星位点统计分析,并设计筛选特异性引物对霍山石斛进行鉴别。结果:霍山石斛cpDNA中微卫星位点共有52个,以单碱基重复序列为主,共有25个,占总位点48%,其次二碱基重复序列有16个,占总位点30.8%,三碱基及三碱基以上重复序列在cpDNA中分布极少。在通过微卫星位点所设计的4对引物中,有2对引物可对霍山石斛及部分混伪品进行有效的鉴别。结论:所设计的引物可以对霍山石斛及其伪混品进行鉴别,且所得数据为霍山石斛的种群遗传和起源分化的研究提供了支持。

Genetic Diversity of Indocalamus Determined by Chloroplast DNA Sequence

[Objective]The aim was to research the relationship and genetic diversity of Indocalamus.[Method]Using 13 samples of Indocalamus and 3 samples of Sasa as materials,the intergenic regions of trnL-trnF gene in chloroplast were amplified by PCR,and sequence analysis and phylogenetic trees construction were carried out.[Result]Using the universal primer,the intergenic regions of trnL-trnF were amplified,the lengths of the segments varied from 1 008 bp to 1 103 bp,of which 940 bp was compared.The dendrogram of trnL-trnF sequences showed that Indocalamus and Sasa were clustered together and they were homologous by 99%.All the samples were divided into five groups,the first group included 12 samples such as Indosalamus pedalis,I.pumilus,I.victorialis,I.longiauritus,I.tessellatus,Sasa sinica,Sasa pygmaea,I.barbatus,I.guangdongensis,I.herklotsii,I.Hirtivaginatus and S.fortunei.I.decorus,I.lacunosus,I.Latifolius and I.Migoi were respectively divided into four groups.[Conclusion]The high homology of all samples showed the low evolution speed and little information sites which suggested that the phylogeny of Indocalamus could not be well resolved by the intergenic region of trnL-trnF.

基于cpDNA序列分析不同生境芦苇种群的遗传结构

芦苇(Phragmites australis)是禾本科多年生植物,是生态幅极广的世界性物种之一。在长期适应不同生境的过程中,从形态到遗传产生了高度分化,形成种内丰富的生态型。芦苇自然种群遗传结构的研究有助于丰富进化遗传学理论并推动其在物种保护和生态恢复方面的应用。基于3个叶绿体DNA(cp DNA)的片断(rpl16,mat K和trn L-F)对我国东北、西南和黄河中上游地区的水生和旱生芦苇的7个种群共96个个体进行遗传结构分析。结果表明旱生芦苇种群的遗传多样性为0.702,基因流为0.904;水生芦苇种群的遗传多样性为0.826,基因流为0.431。种群水平的遗传多样性为0.814,其中有49.0%的变异存在于种群之间,相对比较高;芦苇种群的基因流为0.520。虽然种群水平和物种水平的基因流都小于1.0,但这并不影响其物种的稳定性和遗传多样性,推测可能与芦苇的繁殖方式和种群数量庞大有关。

利用SSR标记研究甘蓝型油菜叶绿体基因组DNA多态性

叶绿体基因组具有母性遗传、单倍性、高度的保守性和明显的种内差异等特点。研究不同品种叶绿体基因多样化程度,可为甘蓝型油菜的收集与引种及遗传改良提供指导。利用15对特异性叶绿体SSR标记对287份国内外甘蓝型油菜叶绿体基因组多样性进行了分析,结果显示,在15对叶绿体SSR特异引物中,5对引物扩增产生多态性条带合计19条,平均每对引物检测出3.8条多态性条带;287份材料共划分成14个单倍型,优势单倍型H01占比74.91%、H02占比13.59%、H03占比4.88%,其余11种单倍型(H04-H14)占比合计6.62%;H02为波里马、陕2A胞质类型,为中国特有单倍型;国外甘蓝型油菜中,俄罗斯与瑞典的单倍型较丰富,引进俄罗斯与瑞典的资源能更高效拓宽中国甘蓝型油菜的遗传多样性本底。本研究还获得2对引物(MF-4和ccmp2)得到特异条带,并为此建立了快速鉴定波里马、陕2A胞质类型的方法,为波里马与陕2A细胞质类型材料的鉴定与保护提供了方法。

cpDNA标记在马铃薯杂交亲本选配中的应用

亲本选配是马铃薯杂交育种工作的重点和难点。利用叶绿体DNA（cpDNA）标记方法，对两个马铃薯杂交组合（Shepody×CIP 004和PB06×CIP 004）的亲本、及其杂交实生种子后代群体的细胞质遗传背景进行了分析。结果显示：Shepody拥有T-型cpDNA标记，属于普通栽培亚种（Solanum tuberosum ssp．tuberosum）；CIP004和PB06拥有A-型cpDNA标记，均属于安第斯栽培亚种（Solanum tuberosum ssp．andigena）；在cpDNA两个标记位点（H1和H2）上，2个杂交组合实生种子后代群体的细胞质遗传背景分别与其母本完全一致，平均生育期分别与其母本接近。上述结果表明，cpDNA标记技术在马铃薯杂交亲本选配和早世代选育中具有重要的应用价值。

栽培草莓叶绿体分离与cpDNA的提取方法

以栽培草莓‘红颊’(Fragaria×ananassa Duch.‘Benihoppe’)新鲜幼嫩叶片为材料,利用高盐-低pH的缓冲液结合Percoll密度梯度离心分离纯化叶绿体,再用SDS-蛋白酶K法提取叶绿体DNA(cpDNA)。经荧光显微镜、琼脂糖凝胶电泳和PCR扩增检测,结果表明:该方法分离的草莓叶绿体完整性高,叶绿体DNA质量好,纯度高,能够满足后续基因组测序等实验的基本要求,为草莓属及其他草本植物cpDNA的提取提供参考。

Chloroplast DNA-based genetic variation of Rosa roxburghii in Southwest China:Phylogeography and conservation implications

Rosa roxburghii Tratt.is a well-known commercial horticultural crop in China with nutritional and medicinal value.Wild germplasms of this species are mainly distributed in Southwest China but the population is decreasing due to continuous exploitation,habitat destruction,and fragmentation.Therefore,assessing the genetic diversity and phylogeography is essential for efficient conservation.Herein,two chloroplast intergenic spacers(trnL-trnF and accD-psaI)were investigated in 255 individuals from 29 R.roxburghii populations and 18 haplotypes(H1–H18)were identified.High levels of haplotype diversity(Hd=0.829)and nucleotide diversity(π=1.3×10^(−3))were detected in these populations.Also,the genetic variation representing 86.4%of the total variation was detected by an analysis of molecular variance.A significant correlation was established between genetic divergence and geographic distance by the Mantel test(r=0.204,P=0.04,9999 permutations),suggesting the isolation-by-distance model.A significantly higher Nst than Gst(Nst=0.257,Gst=0.136,P<0.05)indicated the phylogeographic structure of R.roxburghii.Further phylogeographic analysis revealed rapid range expansion in the population,probably between 647073 and 217848 years ago.The primary processes shaping the genetic patterns of the R.roxburghii populations included restricted gene flow with isolation distance within clades 1-8,2-3,and overall,contiguous expansion within clades 1-3 and 3-2,past fragmentation,and/or long-distance colonization within clades 1-9 and 2-2.Conservation priority should be given to the core populations GZ,FQ,DF,DS,xy,AL,LC,PB,and XY in the Yunnan-Guizhou Plateau,NZ and MX in the Qingling-Bashan mountains,and MN in the Hengduan mountains,where an in situ preservation and management strategy should be applied.

江华苦茶不同单株间亲缘关系的cpDNA序列分析

DNA序列分析在物种的系统进化、分类和鉴定等方面展示出了强大的生命力,其中cpDNA序列分析因具有基因组小、进化速率适中、母系遗传等特点,已被大量用于系统进化研究。本研究采用4对cpDNA引物对收集来的32份江华苦茶资源进行了亲缘关系研究,其中有3对测序成功。扩增序列长度分别为634(1F-724R)、499(rbcla-rev)、532(rbcla-ajf634)。变异位点数量分别为5(1F-724R)、4(rbcla-rev)、4(rbcla-ajf634)。变异率从高到低分别是rbcla-rev(0.80%)、1F-724R(0.79%)、rbcla-ajf634(0.75%)。将3对引物测序得到的序列拼接,按照MP法构建了分子系统树,将参试的32份茶树资源分为4大类。本研究为江华苦茶亲缘关系研究开辟了一条新途径,为江华苦茶资源的应用和保护研究提供理论参考。

A Simple Method for Isolating Chloroplast DNA and Mitochondria DNA from the Same Rapeseed Green Leaf Tissue

In the study, we present a fast, simple and inexpensive protocol for isolating chloroplast and mitochondrial DNA from one rapeseed leaf tissue sample. The chloroplast and mitochondria were separated from the same green leaf tissue by differential centrifugations. The protocol is the first report that isolates plant chloroplast DNA （cpDNA） and mitochondrial DNA （mtDNA） from the same sample homogenate. The organelle DNA yield is 2-10 micrograms per gram of tissue; the DNA was fully restrictable and was successfully used for sequencing.

Polymorphism and heredity of cpDNA and mtDNA in the Section Leuce of Populus

The chloroplast DNA （cpDNA） and mitochondrial DNA （mtDNA） of 16 Populus species （Section Leuce） and their F1 generation were detected using PCR-RFLP technique. The results show that cpDNA in the F1 generation of 22 hybrid combinations was inherited maternally, which supported the conclusions of the study of plasmid cytology. The mtDNA fragments amplified by PCR were consistent with the restriction maps in all hybrid combinations and no polymorphism was detected, indicating that the Section Leuce is highly conserved in mitochondrial gene sequences. These results provided direct evidence of maternal chloroplast inheritance in Populus tomentosa, P. bolleana, P. davidiana, P. adenopoda, P. tomentosa × P. bolleana, P. alba × P. glandulosa and P. alba × P. tomentosa.

A hybrid swarm population of Pinus densiflora × P. sylvestris inferred from sequence analysis of chloroplast DNA and morphological characters

To confirm a hybrid swarm population of Pinus densiflora × P. sylvestris in Jilin, China, we used needles and seeds from P. densiflora, P. sylvestris, and P. densiflora × P. sylvestris collected from natural stands or experimental stations to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker （cpDNA SSRs）. Total genomic DNA was extracted and subjected to sequence analysis of the pine cpDNA SSR marker Pt15169. Results show that morphological characters from 4-year old seedlings did not correlate with sequence variants of this marker. Marker haplotypes from all P. sylvestris trees had a CTAT element that was absent from all sampled P. densiflora trees. However, both haplotype classes involving this insertion/deletion element were found in a P. densiflora × P. sylvestris population and its seedling progeny. It was concluded that the P. densiflora × P. sylvestris accessions sampled from Jilin, China resulted from bi-directional crosses, as evidenced by both species’ cpDNA haplotypes within the hybrid swarm population.

Interspecific Hybridization between <i>Arisaema sikokianum</i>and <i>A. serratum</i>(Araceae) Confirmed through Nuclear and Chloroplast DNA Comparisons

A morphologically intermediate plant between Arisaema sikokianum Franch. et Sav. and A. serratum (Thunb.) Schott has been newly found in Kochi Prefecture, Shikoku, Japan. The putative hybrid has the intermediate morphological characteristics of the parental species. Molecular analysis using PCR-RFLP of internal transcribed spacer (ITS) in nuclear DNA (nrDNA) indicates that the putative hybrid has a combined pattern of the two putative parent species. Moreover, the sequence result of chloroplast DNA (cpDNA) of the putative hybrid was identical to that of A. sikokianum. These results suggest that the putative hybrid is a hybrid between A. sikokianum and A. serratum and that it was formed by interactive gene exchanging via pollens from A. serratum to A. sikokianum. It is the first record of a hybrid between A. sikokianum and A. serratum.

Chloroplast DNA Polymorphism in Rice (<i>Oryza sativa</i>L.)

We analyzed the sequence alignment on 25 AA rice and 24 non-AA rice chloroplasts using two length diversity markers (ORF 100 and ORF29-TrnCGCA) and four sequence markers existed in introns of rps16 gene and TrnTUGU-TrnLUAA spacer to explore the chloroplast diversity of different types of rice using PCR amplification and sequencing. Results showed that in terms of the length of ORF100 and ORF29-TrnCGCA, chloroplast DNA (cp DNA) of Hainan ordinary wild rice, Dongxiang ordinary wild rice, Hepu ordinary wild rice and three-line cytoplasmic male sterile wild rice were indica-type, Chaling ordinary wild rice, Fusui ordinary wild rice, Niwara wild rice, Brazilian upland rice and Lemont were japonica-type among in AA genome. Besides, all non-AA wild rice was japonica-type. There were 4 indica-japonica markers utilizing introns of rps16 gene and TrnTUGU-TrnLUAA. We found that all the ordinary wild rice in Chaling and Fusui of AA genome presented as japonica specific sites, while the others owned two indica and japonica specific sites, respectively. There were two indica-japonica sites separately and a 6-base specific fragment in three-line cytoplasmic male sterile materials except Yuetai A, simultaneously, 2-base difference from Hainan wild rice. Moreover, Brazilian upland rice and Lemont were entire japonica specific sites. Result of three markers indicated that the cp DNA of non-AA wild rice was japonica-type and result of one marker showed indica-type. Sequencing results also suggested that wild rice existed many polymorphic base sites, CCDD genome, wart wild rice and malay wild rice had their own specific sites. In conclusion, significant differentiation trend of indica-japonica exhibits in chloroplast of ordinary wild rice, and non-AA wild rice is generally japonica-type. The cytoplasmic polymorphism level of three-line sterile lines is low. It is worth considering whether the cytoplasm of Honglian-type sterile line Yuetai A comes from Hainan ordinary wild rice. Furthermore, genetic polymorphisms in wild rice are far more than in cultivar.

Identification of <i>Angelica acutiloba</i>and Related Species by Analysis of Inter- and Intra-Specific Sequence Variations in Chloroplast and Nuclear DNA Sequences

Japanese Angelica Root prepared from Angelica acutiloba var. acutiloba and A. acutiloba var. sugiyamae, known in Japan as “Toki” and “Hokkai Toki”, is an important crude drug used in Kampo medicine (traditional Japanese medicine). However, since these Angelica varieties have recently outcrossed with each other, it is unclear whether Japanese Angelica Root sold for use in Kampo medicine is a pure variety. Here, we describe DNA sequence polymorphisms that can be used to distinguish between A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae. In our analyses, differences in the trnK region of chloroplast DNA distinguished among some A. acutiloba varieties and related species, but not between A. acutiloba var. acutiloba and A. acutiloba var. iwatensis. One geographical strain of A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae showed identical sequences in three regions of chloroplast DNA, but differences in the internal transcribed spacer region of nuclear ribosomal DNA. One strain of A. acutiloba var. iwatensis and A. acutiloba var. sugiyamae had identical sequences in all of the chloroplast and nuclear ribosomal DNA regions examined. These findings show that A. acutiloba var. acutiloba has hybridized with A. acutiloba var. sugiyamae and that the “Hokkai Toki” variety resulted from outcrossing with A. acutiloba var. iwatensis. Molecular authentication based on analyses of chloroplast and nuclear ribosomal DNA sequences of A. acutiloba and related species is an efficient method to authenticate Japanese Angelica Root at the variety level. Therefore, these analyses can determine whether a product is derived from A. acutiloba var. acutiloba or A. acutiloba var. sugiyamae.

Distribution and Phylogeography of Caryopteris incana (Lamiaceae) Based on Chloroplast DNA Sequences in West Kyushu, Japan

Caryopteris incana is a continental plant, transferred to Japan from continental Asia via a land bridge between the Korean Peninsula and Tsushima Islands during a glacial period. It currently grows wild in West Kyushu, Japan. In a previous study, we investigated the distribution of C. incana in the Tsushima Islands and confirmed the genetic structure of populations by using chloroplast DNA sequence analysis, suggesting that different haplotypes were distributed in the same area. Thus, it seemed that populations of C. incana throughout the Tsushima Islands colonized at different times;each haplotype had remained within its population without mixing. In this study, we conducted fieldwork to construct a detailed distribution map in West Kyushu excluding the Tsushima Islands. Additionally, we confirmed genetic structure of the C. incana population in these areas by using chloroplast DNA sequence analysis to study the intraspecific phylogenetic relationship of C. incana in Japan. We confirmed 37 natural populations in 257 locations throughout West Kyushu excluding the 72 natural populations in the Tsushima Islands. We also confirmed a recent decreasing trend in the number of natural populations in the Nagasaki Mainland. Using the leaves of individuals cultivated from seeds collected from each natural population, we analyzed the chloroplast DNA sequence variations. Among the investigated populations, sequence variations were confirmed in six regions of chloroplast DNA, and those haplotypes were mainly classified into two groups distributed in different areas on the phylogenetic tree. This finding revealed that the common ancestor of C. incana in Japan diverged early into two groups, followed by a fragmentation in population distribution for each area. The haplotype network almost reflected the geographical distribution on haplotypes. However, several haplotypes that were distributed in other areas were confirmed in the Nagasaki Mainland, suggesting a complicated distribution formation in the past.

Primers for the Amplification of the Circular Chloroplast DNA from the A-genome Group of Cultivated Cotton

The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application