长心卡帕藻的形态结构观察与分子鉴定
Morphological characteristics and molecular identification of Kappaphycus alvarezii
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摘要: 通过形态构造观察和分子生物学相结合的方法,对栽培于我国海南的长心卡帕藻(Kappaphycus alvarezii)株系XC-03(陵水)和麒麟菜族未知种株系LD-02(三亚)和LD-06(三亚)进行了鉴定,并与细齿麒麟菜(Eucheuma denticulatum)株系TSNY-05进行比较分析,结果表明:长心卡帕藻株系XC-03与麒麟菜族未知种株系LD-02和LD-06之间在分枝形状、长短、分布以及突起数量等藻体外部形态特征方面具有明显差异;它们的表皮、皮层和髓部细胞大小、皮层细胞层数和胶质层厚度等显微结构特征亦不相同,但分子鉴定表明该3个株系为同一个种,即长心卡帕藻。它们的ITS序列相似度为99.69%~99.85%,株系XC-03与LD-02之间仅有1个碱基转换和1个碱基颠换,株系XC-03与LD-06和株系LD-02与LD-06之间均分别仅有1个碱基转换和1个碱基颠换。长心卡帕藻与细齿麒麟菜的ITS序列相似度只有41.28%,两者差异较大。用RAPD,ISSR和ITS标记分析表明长心卡帕藻3个株系的两两间遗传距离分别介于0.122 9~0.153 5,0.068 8~0.112 4和0.002~0.005。用neighbor-joining和UPGMA聚类分析皆显示长心卡帕藻株系XC-03与LD-06的亲缘关系较近,与株系LD-02相对较远,而细齿麒麟菜单独列为一支。该研究为长心卡帕藻的种质鉴定和遗传育种研究提供了依据。Abstract: Three lines (Line XC-03 of Kappaphycus alvarezii from Lingshui coast, Line LD-02 and Line LD-06 from Sanya coast) of eucheumatoids algae with different shapes cultivated in Hainan Province, China were identified by morphological characteristics and molecular phylogenetic analysis, and were compared with Line TSNY-05 of Eucheuma denticulatum from Tanzania. There were differences in the branch shapes, length and distributing and tuber numbers, the cell sizes of epidermis, cortex and medulla part, the counts of cortex and the thickness of the colloid among three lines (Line XC-03, Line LD-02 and Line LD-06). But molecular identification indicated that Line XC-03, Line LD-02 and Line LD-06 were same species, K. alvarezii. The genome DNA was extracted from four lines by CTAB method. 5 polymorphic primers were screened out of 30 primers for random amplified polymorphic DNA (RAPD) analyses and 39 primers for inter-simple sequence repeat (ISSR) analyses, respectively. 12 individuals were used for molecular analysis respectively, and 39 loci (RAPD) and 49 loci (ISSR) were obtained. The fragments for RAPD and ISSR markers were from 300 to 3 000 bp or from 400 to 3 000 bp, respectively. The PCR amplified products of the internal transcribed spacer (ITS) were sequenced by the ABI PRISM 3730 genetic analyzer. The sequence data were blasted and aligned, and phylogeny trees were constructed by neighbour-joining and UPGMA analyses using MEGA 4.0. The results showed that the similarity of the ITS sequence was 99.69%~99.85% among Line XC-03, Line LD-02 and Line LD-06. There was one base transition and one base transversion between Line XC-03 and Line LD-02, and there was only one base transition between Line XC-03 and Line LD-06 and one base transversion between Line LD-02 and Line LD-06. While the similarity of the ITS sequence was 41.28% between K. alvarezii.and E. denticulatum. And RAPD, ISSR and ITS analysis further indicated that the pairwise genetic distance between Line XC-03, Line LD-02 and Line LD-06 was from 0.122 9 to 0.153 5 (RAPD), from 0.068 8 to 0.112 4(ISSR) and from 0.002 to 0.005 (ITS), respectively, with software Popgen 32 and MEGA 4.0. With Neighbor-Joining and UPGMA analyses, it indicated that the relationship between Line XC-03 and LD-06 was closer than that between Line XC-03 and Line LD-02. However, the pairwise genetic distance was above 0.9 between E. denticulatum and K. alvarezii, and it showed that the relationship was very far between E. denticulatum.and K. alvarezii. These results provide a basis for the species identification and genetics breeding in K. alvarezii cultivation.
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Key words:
- Kapphphycus alvarezii /
- morphological characteristics /
- RAPD /
- ISSR /
- ITS
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DAWES C J, TRONO JR G C, LLUISMA A O. Clonal propagation of Eucheuma denticulatum and Kappaphycus alvarezii for Philippine seaweed farms[J]. Hydrobiologia, 1993, 260-261: 379-383. 夏邦美,张峻甫. 中国海藻志 .第2卷,第5册.北京:科学出版社,1999:115-132. HAYASHI L, OLIVEIRA E C, BLEICHER-LHONNEUR G, et al. The effects of selected cultivation conditions on the carrageenan characteristics of Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in Ubatuba Bay, So Paulo, Brazil [J]. J Appl Phycol, 2007,19(5):505-511. 吴超元,李家俊,夏恩湛,等. 异枝麒麟菜的移植和人工栽培[J].海洋与湖沼, 1988, 19(5): 410-418. 曾广兴.异枝麒麟菜人工养殖技术[J].水产养殖, 2001(3): 5-7. 郑国洪.浅海浮筏式养殖长心麒麟菜技术[J].中国水产, 2006(11): 58-59, 80. 王红勇,姚雪梅,王李. 长心卡帕藻生长适宜环境条件的研究[J].北京水产, 2008(3): 29-34. ASK E I, AZANZA R V. Advances in cultivation technology of commercial eucheumatoid species: a review with suggestions for future research[J]. Aquaculture, 2002, 206: 257-277. AGUIRRE-VON-WOBESER E, FIGUREOA F L, CABELLO-PASINI A. Photosynthesis and growth of red and green morphotypes of Kappaphycus alvarezii(Rhodophyta)from the Philippines[J]. Marine Biology, 2001, 138: 679-686. HAYASHI L, JOSDE PAULA E, CHOW F. Growth rate and carrageenan analyses in four strains of Kappaphycus alvarezii (Rhodophyta, Gigartinales) farmed in the subtropical waters of So Paulo State,Brazil[J]. J Appl Phycol, 2007.19(5): 393-399. ZUCCARELLO G C, CRITCHLEY A T, SMITH J, et al. Systematics and genetic variation in commercial Kappaphycus and Eucheuma (Solieriaceae, Rhodophyta)[J]. Journal of Applied Phycology, 2006, 18: 643-651. 刘建国,庞通,王莉,等. 导致热带产卡拉胶海藻大规模死亡原因分析与藻株抗病差异性比较[J]. 海洋与湖沼, 2009, 40(2): 235-241. 周志刚, 孙育平. 海藻分子生物学技术的应用与发展[J].上海水产大学学报, 2002, 11(2): 161-166. GUILLEMIN M L, AKKI S A, GIVERNAUD T, et al. Molecular characterization and development of rapid molecular methods to identify species of Gracilariaceae from the Atlantic coast of Morocco[J]. Aquatic Botany, 2008, 89: 324-330. DUTCHER J A, KAPRAUN D F. Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species of Porphyra (Bangiales,Phodophyta)[J]. Journal of Phycology, 1994, 6: 267-273. KUNIMOTO M, KITO H, KAMINISHI Y, et al. Molecular divergence of the ssu rRNA gene and internal transcribed spacer 1 in Porphyra yezoensis (Rhodophyta)[J]. Journal of Applied Phycology, 1999, 11: 211-216. 贾建航,王萍,金德敏,等. RAPD标记在紫菜遗传多样性检测和种质鉴定中的应用[J].植物学报, 2000, 42(4): 403-407. PATWAY M U, VAN DER MEER J P. Application of RAPD markers in an examination of heterosis in Gelidium vagum (Rhodophyta)[J]. Journal of Applied Phycology, 1994, 30: 91-97. BOUZA N, CAUJAPE-CASTELLS J, GONZALEZ-PEREZ M A,et al. Genetic structure of natural populations in the red algae Gelidium canariense (Gelidiales, Rhodophyta) investigated by random amplified polymorphic DNA (RAPD) markers[J]. Journal of Phycology, 2006, 42: 304-311. SIMONAS V, ANTTA M, TAPANI Y M. Analysis of genetic diversity of Furcellaria lumbricalis(Gigartinales, Rhodophyta) in the Baltic Sea by RAPD-PCR technique[J]. Phycologia, 2000, 39: 109-117. HU Z M, ZENG X Q, CRITCHLEY A T, et al. Phylogeography of the northern Atlantic species Chondrus crispus(Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences[J]. Hydrobiologia, 2007, 575: 315-327. 李文红,胡自民,覃志彪,等. 细基江蓠及其繁枝变种的RAPD和ITS分析[J].海洋学报, 2004, 26(6): 89-95. 李文红,姚建亭,王继承,等. 龙须菜(Gracilaria lemaneiformis)选育品系及其野生型的ISSR指纹分析[J]. 海洋与湖沼, 2005, 36(3): 241-247. SIM M C, LIM P E, GAN S Y, et al. Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of Gracilaria changii(Rhodophyta)[J]. Journal of Applied Phycology, 2007, 19: 763-769. LLUISMA A O, RAGAN M A. Relationships among Eucheuma denticulatum, Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales, Rhodophyta) based on nuclear ssu-rRNA gene sequences[J]. Journal of Applied Phycology, 1995, 7: 471-477. FREDERICQ S, FRESHWATER D W, HOMMERSAND M H. Observations on the phylogenetic systematics and biogeography of the Solieriaceae (Gigartinales, Rhodophyta) inferred from rbcL sequences and morphological evidence[J].Hydrobiologia, 1999, 398-399:25-38. CONKLIN K Y, KURIHARA A, SHERWOOD A R. A molecular method for identification of the morphologically plastic invasive algal genera Eucheuma and Kappaphycus (Rhodophyta, Gigartinales) in Hawaii[J]. J Appl Phycol,2009,21:691-699.1007/s10811-009-9404-2. 赵素芬,何培民. 长心卡帕藻ISSR-PCR反应体系的正交优化研究[J].中国生物工程杂志, 2008, 28(6,Supp)163-167. 孙雪,张学成,茅云翔,等. 几种江蓠属海藻的ISSR标记分析[J].高技术通讯, 2003(9): 89-93. WANG X L, LIU C L, CONG Y Z, et al.Assessment of genetic diversities of selected Laminaria (Laminariales, Phaeophyta) gametophyte with ISSR analysis[J]. Journal of Integrative Plant Biology, 2005, 47(6): 753-758. ZHAO F J, LIU F L, LIU J D, et al.Genetic structure analysis of natural Sargassum muticum(Fucales, Phaeophyta) populations using RAPD and ISSR markers[J]. J Appl Phycol, 2007, 19: 409-416. 赵素芬,张婷,应成琦,等. 长心卡帕藻RAPD-PCR 反应体系的正交优化研究[J].生物技术通报, 2008(4): 161-165. AZANZA-CORRALES R. The farmed Eucheuma species in Danajon Reef, Philippines: vegetative and reproductive structures[J]. Journal of Applied Phycology, 1990, 2: 57-62. 刘晨临,黄晓航,刘建国.4株形态不同的长心卡帕藻(Kappaphycus alvarezii)的初步ISSR分析[J].海洋科学, 2009, 33(4): 50-53. JOS DE PAULA E, PEREIRA R T L, OHNO M. Strain selection in Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) using tetraspore progeny[J]. Journal of Applied Phycology, 1999, 11: 111-121. 王建波,张文驹,陈家宽. 核rDNA的ITS序列在被子植物系统与进化研究中的应用[J].植物分类学报, 1999, 37(4): 407-416. 谢恩义,孙彬,马家海. 三种礁膜属藻类的分子遗传多样性和亲缘关系的RAPD分析[J].上海水产大学学报, 2005,14(1): 6-11.
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