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.

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.

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.

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.

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

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.

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.

Glacial Refugia of Ginkgo biloba and Human Impact on Its Genetic Diversity:Evidence from Chloroplast DNA

Variations in the trnK region of chloroplast DNA were investigated in the present study using polymerase chain reactionrestriction fragment length polymorphism to detect the genetic structure and to infer the possible glacial refugia of Ginkgo biloba L. in China. In total, 220 individuals from 12 populations in China and three populations outside China were analyzed, representing the largest number of populations studied by molecular markers to date. Nineteen haplotypes were produced and haplotype A was found in all populations. Populations in south-western China, including WC, JF, PX, and SP, contained 14 of the 19 haplotypes and their genetic diversity ranged from 0.771 4 to 0.867 6. The TM population from China also showed a high genetic diversity （H = 0.848 5）. Most of the genetic variation existed within populations and the differentiation among populations was low （GsT = 0.2）. According to haplotype distribution and the historical record, we suggest that populations of G. biloba have been subjected to extensive human impact, which has compounded our attempt to infer glacial refugia for Ginkgo. Nevertheless, the present results suggest that the center of genetic diversity of Ginkgo is mainly in south-western China and in situ conservation is needed to protect and preserve the genetic resources.

Chloroplast DNA Underwent Independent Selection from Nuclear Genes during Soybean Domestication and Improvement

The chloroplast is one of the most important organs in plants because of its essential role in photosynthesis.Studies have shown that the chloroplast was once a free-living cyanobacteria and was integrated into the host species through endosymbiosis（Goksoyr.1967）.after which a large number of its genes had been donated to the host nuclear genome（Heins and Soll, 1998）.

Chloroplast DNA reveals genetic population structure in Sinomenium acutum in subtropical China

Objective:The population density and diversity of Sinomenium acutum(Menispermaceae)have been greatly reduced recently by overharvesting for medicinal purposes in China.Therefore,it is urgent that the remaining populations are investigated,and that strategies for the utilization and conservation of this species are developed.This study aimed to find the possible glacial refugia and define the genetic diversity of S.acutum for its proper utilization and conservation.Methods:A total of 77 S.acutum samples were collected from four locations,Qinling Mountains,Daba Mountains,Dalou Mountains,and Xuefeng Mountains,in subtropical China.Genetic diversity among and between these populations were phylogenetically analyzed using four chloroplast DNA molecular markers(atpI-atpH,trnQ-50 rps16,trnH-psbA and trnL-trnF).Results:A total of 14 haplotypes(C1 to C14)were found in collected samples.Haplotypes C1 and C3 were shared among all populations,with C3 as the ancestral haplotype.Haplotypes C11 and C12 diverged the most from C3 and other haplotypes.No obvious phylogeographic structure was found in four locations using the GST/NST test.There is no evidence of rapid demographic expansion in S.acutum based on the mismatch distribution,and the results of Tajima’s D test,and Fu’s FS test.Our analyses of molecular variance revealed a high level of genetic variation within populations.In contrast,the genetic differentiation among S.acutum populations was low,indicating frequent gene flow.Conclusion:Xuefeng,Dalou,and Daba Mountains were possible glacial refugia for the populations of S.acutum.C1,C3,C11 and C12 haplotypes of S.acutum should be carefully preserved and managed for their genetic value.

IMPROVEMENT OF THE PURIFICATION OF CHLOROPLAST DNA FROM HIGHER PLANTS

For the study of molecular biology of photosynthesis in higher plants, the isolation and purification of chloroplast DNA (ctDNA)is very important. Among the present methods for isolation of ctDNA from higher plants, the first step is to isolate pure chloroplasts in order to avoid the contamination of nuclear and mitochondrial DNA. Afterwards, the chloroplasts are cleaved to release the ctDNA. For isolation and purification of ctDNA, the methods frequently used are DNaseI method, sucrose density gradient

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。

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.

Construction of Double Cross-over Expression Vector for Chloroplast Multicistron in Brassica napus L.

[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.

地黄属植物的DNA条形码研究

地黄属（Rehmannia）为玄参科（Scrophulariaceae）药用植物,广泛分布于中国中东部及北部地区。由于地黄属植物经历了快速成种,导致其属内物种间形态性状差异较小,运用传统的形态学分类方法已难以准确地鉴定物种,近年来迅速发展起来的DNA条形码技术为快速、准确地鉴别物种提供了新思路。本研究选用3个叶绿体DNA非编码区片段（trn L-trn F、trn M-trn V和trn S-trn G）及核基因ITS片段,运用PWG-distance和TreeBuilding两种方法对地黄属5个物种75个个体进行了DNA条形码分析。结果表明：单个叶绿体DNA片段或核基因ITS片段对地黄属物种的鉴别率较低（0%~20%）,组合的叶绿体DNA片段分辨能力虽然高于单个DNA片段,但并不能将地黄属5个物种完全区分开;trn S-trn G＋ITS片段组合的分辨率可达100%,能够将地黄属5个物种准确区分,与所有叶绿体DNA片段和核基因ITS片段组合（trn L-trn F＋trn M-trn V＋trn S-trn G＋ITS）的辨别率相同,因此推荐trn S-trn G＋ITS作为地黄属植物的标准条形码。此外,利用DNA条形码鉴别物种时,可采用叶绿体DNA片段和核DNA片段组合的方法来提高物种鉴定的成功率。

江淮流域杂草稻叶绿体DNA的籼粳分化

为了探明江淮流域杂草稻的细胞质来源,根据水稻叶绿体DNA ORF100(Open Reading Frame 100)序列在籼粳间存在69 bp差异的特征,设计了InDel标记cpDNA69。利用该标记对2个分别以籼稻和粳稻为母本的F2群体、22份栽培稻(Oryza sativaL.)、3份普通野生稻(O.rufipogonGriff.)进行了分析验证。PCR结果可以获得缺失和非缺失两种带型,缺失带型与以籼稻为母本的F2群体、10份籼稻材料完全对应;非缺失带型与以粳稻为母本的F2群体、11份粳稻材料完全对应,3份广西普通野生稻为非缺失带型,属粳型,1份以粳稻为母本的籼粳杂交材料为粳型。因此,cpDNA69可以用作叶绿体籼粳鉴定标记。利用该标记对22份杂草稻的叶绿体DNA鉴定的结果表明,7份早年发现的杂草稻,即江苏省连云港穭稻和安徽省怀远、来安、全椒、肥东的塘稻的叶绿体DNA为非缺失带型,属粳型,而近年来在江苏省扬中、高邮、灌云、洪泽、盐都、兴化、如皋等地直播稻田发现的15份红米杂草稻的叶绿体DNA为缺失带型,属籼型。这为进一步研究江淮流域杂草稻的来源提供了依据。

桑树叶绿体基因组DNA的提取及部分序列分析

改进Milligan法 ,提取了桑树叶绿体基因组DNA(cpDNA)。利用特异性引物 ,从cpDNA基因组扩增出tRNL tRNF基因。再用随机引物对同一种桑基因组DNA及cpDNA进行RAPD分析 ,表明提取的DNA是具有相当纯度的cpDNA。进一步用XbaⅠ和HindⅢ进行了cpDNA的酶切和克隆。通过酶切鉴定 ,已克隆到 5个片段 ,对其中 1个片段的 70 0bp序列进行了分析 ,推测克隆的片段可能为trnK基因的内含子。

水稻叶绿体DNA提取和纯化方法优化

改进了水稻叶绿体DNA的提取和纯化方法,获得的高纯度的水稻叶绿体DNA适于PCR扩增等分子操作.本方法要点为:匀浆液2 300 r.min-1离心去核,上清液4 300 r.min-1离心,沉淀用于DNA提取,提取后的叶绿体DNA用RNase和蛋白酶K纯化.