钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ,COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16SrRNA基因部分长度为413bp,COI基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％;COI基因605％。对位排序比较表明,16SrRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COI片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000,有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011,白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16SrRNA和COI基因在种间存在明显的多态性,证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。     

粤西镇海湾近江牡蛎线粒体16S rRNA基因序列变异分析

采用聚合酶链式反应 (PCR)技术对粤西镇海湾水域的近江牡蛎Crassostrearivularis(Gould)群体 2 7个个体的线粒体DNA16SrRNA基因序列片段进行扩增 ,获得大约 5 0 0bp的扩增产物。PCR产物经纯化后进行序列测定 ,经ClustalX同源排序 ,除去引物及部分端部序列 ,得到4 14bp的核苷酸片段。 2 7个个体共检测到 2个变异位点 ,均为颠换位点 ,没发现碱基位点插入、缺失及转换位点 ,共 3种单倍型 ,每个单倍型只有一个碱基的差异。运用DNASP软件计算得该群体的核苷酸多样性和平均核苷酸差异数分别为 0 .0 0 0 36和 0 .14 815。此结果提示镇海湾近江牡蛎群体遗传多样性已很低 ,很有必要从其他分布区引进近江牡蛎亲贝来扩大该种群的遗传多样性     

黄喉拟水龟体表溃疡病原菌SG_(24)的分类鉴定

从患溃疡的黄喉拟水龟病灶中分离到一株病原菌SG24。对菌株SG24进行了常规生理生化测定和ATBExpression半自动细菌鉴定仪鉴定,并测定16S rRNA序列,分析其与相关细菌相应序列的同源性,构建了系统进化树。普通细菌学方法结果显示菌株SG24为黏质沙雷氏菌(Serratia marcescens)。以沙雷氏菌属的16S rRNA基因序列设计一对引物进行PCR扩增,获得了菌株SG24大小约950 bp的16S rRNA部分基因片段,测序结果显示菌株SG24与黏质沙雷氏菌同类,与已登录的黏质沙雷氏菌(S.marcescensDQ207558)的16S rRNA同源性大于99%。综合以上分类鉴定结果,确定菌株SG24属于沙雷氏菌属的黏质沙雷氏菌。药物敏感试验结果表明:菌株SG24对链霉素、庆大霉素、壮观霉素、强力霉素、卡那霉素、阿米卡星、诺氟沙星、氧氟沙星和复方新诺明敏感。     

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.     

3种星虫线粒体16S rRNA、COI和Cyt b基因片段的序列比较

对裸体方格星虫(Sipunculus nudus)、可口革囊星虫(Phascolosoma esculenta)和澳洲管体星虫(Siphonosoma australe)的线粒体16S rRNA、COI和细胞色素b(Cytb)基因片段序列进行比较,并对其系统发生进行了初步探讨。采用PCR方法得到总长度分别为531～544bp(16S)、652～675bp(COI)和406～453bp(Cytb)的线粒体片段。片段碱基A+T比例较高(16S rRNA基因58.3%,COI基因56.9%,Cytb基因59.5%)。16S rRNA片段存在169个碱基变异位点(其中包括167个简约信息位点)和44个碱基插入/缺失,种内个体间变异较小;COI片段有512个碱基(333个简约信息位点)存在变异,79个碱基插入/缺失;Cytb片段存在347个碱基(318个简约信息位点)变异位点,16个碱基插入/缺失。数据分析结果支持3种星虫和环节动物的分类地位较近,与软体动物较远的分类观点。此外,裸体方格星虫与澳洲管体星虫之间亲缘关系较近(D=0.3159、0.3156、0.2361)。认为3种星虫线粒体16S rRNA、COI和Cytb基因在种间存在明显的多态性,证实了三种基因序列均普遍适用于星虫种及以上阶元的系统学分析。     

一株海水氨氧化细菌的系统发育分析

应用PCR技术扩增16S rRNA基因和amoA(ammonia monooxygenase subunit A)的基因片段,并测定其序列,对一株源于海水养殖水体的高效氨氧化细菌(ammonia-oxidizing bacteria,AOB)进行了系统发育分析。结果表明,经PCR扩增得到了1 098 bp的16S rRNA基因片段和491 bp的amoA基因片段,将其序列用NCBI-Blast软件在GenBank数据库中进行同源性检索后发现,该菌株的16S rRNA基因序列和amoA基因序列分别与亚硝化单胞菌Nitrosomonas sp.NS20的相对应基因片段相似性分别为98.4%和96.7%。在此基础上构建了系统发育树,表明用该菌株的16S rRNA基因片段和amoA基因片段构建的系统发育树均与亚硝化单胞菌属类聚在一起,结合该菌株形态和生理生化特性,鉴定该株氨氧化细菌属亚硝化单胞菌。     

蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌(Coelomactra antiquata)、中国蛤蜊(Mactrachinensis)和四角蛤蜊(Mactra veneriformis)3种双壳贝的16S rRNA基因片段和ITS2核苷酸序列,测序后用DNA star软件分析了核苷酸差异。结果显示:三种贝类16S rRNA基因片段长度相同,均为306bp(去除引物),核苷酸存在多态性,共有45个变异位点,54个核苷酸发生了变异,全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88.9%,与四角蛤蜊的同源性为88.6%,中国蛤蜊与四角蛤蜊的同源性为90.6%。三种蛤蜊ITS2序列分别为390 bp(西施舌)4、41 bp(四角蛤蜊)和466 bp(中国蛤蜊),存在长度多态性,ITS2核苷酸差异分析结果显示,西施舌与中国蛤蜊的同源性为70.9%-71.1%,西施舌与四角蛤蜊的为70.5%-71.0%,中国蛤蜊与四角蛤蜊的同源性为88.1%-88.8%。ITS2序列分析结果与16S rRNA基因片段分析结果一致,2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。     

3种柔鱼线粒体基因与核核糖体基因片段序列比较分析

为准确检测柔鱼（Ommαstrephesbartram川、茎柔鱼（ Dosidicus gigas）与阿根廷滑柔鱼（ IIIex argentinus ） 的种间遗传差异,对线粒体16SrRNA、细胞色素b（Cytb）与编码核糖体大亚基的基因（28SrDNA）片段序列进 行测定。经比对获得同源片段序列的长度分别为444、430、464坤,其中16SrRNA与28SrDNA基因片段上分别存在3处和47处碱基插入/缺失。核昔酸组成分析表明;3种柔鱼在3个基因片段上的核音酸组成差异不显著, 在线粒体2个基因片段上的A＋T含量（16SrRNA;69.90%、72.01%、74.66%; Cytb; 63.61%、68.91%、71.65% ） 均明显高于C＋C含量（16SrRNA;30.10%、27.99%、25.34%; Cytb; 36.39%、31.09%、28.35% ）,而在28SrDNA 基因片段上的A＋T含量（37.16%、36.74%、38.29% ）明显低于C＋C含量（62.84%、63.26%、61.71 %）0 3种柔鱼 在28SrDNA基因片段上检测到的核昔酸替代率最低,为6.68%,而蛋白质编码基因Cytb核昔酸替代率最高,为 20.93%,核营酸替代均发生在密码子第3位点上,而且未引起氨基酸替代。基于邻接法、最大简约法与最大似然 法重建的系统树显示,柔鱼与茎柔鱼的亲缘关系较近。根据C严b基因片段序列分析,柔鱼与茎柔鱼和阿根廷滑柔鱼的分歧时间分别为653-790万a和765 - 925万a,种间分化事件发生在中新世至上新世间。     

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopusjaponicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopusjaponicus individuals.     

冲绳日本绒螯蟹线粒体DNA 12S rRNA序列的研究

采集日本冲绳源河川和边野古川的日本绒螯蟹样品 ,参考果蝇与蚤状氵蚤线粒体 DNA12 Sr RNA基因片段序列进行了其相同片段的引物设计、PCR扩增及序列测定。结果表明冲绳两河川 4只日本绒螯蟹的碱基序列完全相同 ,为 4 58bp,其 A、T、G、C含量分别为 16 0 bp(34.94 % )、 179bp(39.0 8% )、4 9bp(10 .70 % )、70 bp(15.2 8% ) ,同果蝇与蚤状氵蚤相同基因片段的序列含量相似。     

16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ (COⅡ) gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A+T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Polyphasic examination on Merismopedia tenuissima CHAB 7021 from Ganjiang River,China revealed the polyphyly of the genus Merismopedia(Cyanobacteria)

Species in the cyanobacterial genus Merismopedia are present in freshwaters at different trophic levels, with some species even as the components of cyanobacterial blooms. However, species diversity in this genus was not fully verified by molecular investigation and polyphasic taxonomic studies. In this study, Merismopedia-like strain tenuissima CHAB 7021 was isolated from Ganjiang River in Jiangxi Province, China, and polyphasic characterization of this strain was performed by morphological observation, ultrastructural examination, chemical detection of pigments and phylogenetic analysis based on 16 S rRNA gene sequences. Morphological identification of the strain was supported by the ultrastructural features, as the tiny species Merismopedia tenuissima Lemmermann. The phylogeny based on 16 S rRNA gene sequences revealed at least three clades formed by the strains of Merismopedia. The three M. tenuissima strains including M. tenuissima CHAB 7021 was gathered in clade III with distant relationship to the clade I formed by the six Merismopedia strains including the type species M. punctata, and such a genetic distance may propose Merismopedia tenuissima to separate from Merismopedia genus. However intermixture relationship in between strains of M. punctate and M. glauca in the phylogenetic tree still complicated the taxonomic status in the genus Merismopedia. The process for taxonomic revision in the Merismopedia genus still await for examination and further information on more strains of type species M. punctata.     

The Complete Sequence of Mitochondrial COII Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit II (COII) gene of Penaeinae shrimp Fenneropenaeus chinen- sis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COI and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COII gene has a faster evolutionary rate than that of the COI gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COΙ. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COΙ so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.     

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes （Pleuronectiformes）

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P olivaceus clustered with K.bicoloratus, but P cinnamomeus did not cluster with t3. olivaceus, which is worth further studying.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Archaeal diversity and abundance within different layers of summer sea-ice and seawater from Prydz Bay,Antarctica

Fluorescence in-situ hybridization （FISH） and 16S rRNA gene clone library analyses were used to determine the abundance and diversity of archaea in Prydz Bay, Antarctica. Correlation analysis was also performed to assess links between physicochemical parameters and archaeal abundance and diversity within the sea-ice. Samples of sea-ice and seawater were collected during the 26th Chinese National Antarctic Research Expedition. The results of FISH showed that archaea were relatively abundant within the top layer of the sea-ice, and correlation analysis suggested that the concentration of 4NH＋ might be one of the main factors underlying this distribution pattern. However, using 16S rRNA gene libraries, archaea were not detected in the top and middle layers of the sea-ice. All archaeal clones obtained from the bottom layer of the sea-ice were grouped into the Marine Group I Crenarchaeota while the archaeal clones from seawater were assigned to Marine Group I Crenarchaeota, Marine Group II Euryarchaeota, and Marine Group III Euryarchaeota. Overall, the ifndings of this study showed that the diversity of archaea in the sea-ice in Prydz Bay was low.     

Structure analysis of the complete mitochondrial genome in cultivation variety ��Rongfu��

In this report, complete mitochondrial genome sequences of Laminaria cultivation variety ‘Rongfu’ were obtained. The results showed the length of circular molecule of mtDNA was 37 638 bp (64.7% A+T), encoding three rRNAs (23S, 16S and 5S), 25 tRNAs, 35 known mitochondrial proteins and 3 ORFs. Sequence alignment indicated its mtDNA genome was very similar to that of Laminaria japonica. Phylogenetic trees inferred from concatenated 30 mitochondrial genes showed that ‘Rongfu’, Laminaria japonica, Laminaria longipedalis, Laminaria diabolica, Laminaria religiosa and Laminaria ochotensis clustered together. In addition, compared with mitochondrial genome of L. japonica, ‘Rongfu’ mtDNA lacked a non-coding region of 19 nucleotides, which was located between rRNA small subunit gene 3 (rps3) and rRNA small subunit gene 9 (rps9). Seven cultivation varieties of China were divided into two groups based on this non-coding region which was absent in ‘Rongfu’, ‘Fujian’ and ‘Sanhai’ while present in ‘Ailunwan’, ‘Dongfang No.2’, ‘Dongfang No.3’ and ‘Zaohoucheng’. So this variation can be used in germplasm identification of cultivation variety.