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(Solieriaceae,Rhodophyta)

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（Kap-paphycus 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。

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.