| Laminaria is one of the major cold-water cash seaweed. It produces important economic value as it can be used as food, chemical materials, health care product and biofertilizer. In addition, with huge biomass, Laminaria contributes a large part of the primary productivity of the subtidal area in warm temperate zone and occupies a key niche in coastal ecosystem. Heterogenesis of Laminaria makes it a typical model organism for algology and genetics researches.Laminaria cultivation has played an important role in our country and its farming area and yield-production both rank first in the world. Compared with Laminaria cultivation, the research in molecular biology level is relatively backwards and study on the genome is underdeveloped. As a result, it is difficult to illustrate the genetic mechanism of Laminaria without enough genetic information standing by. For above-mentioned reasons, it is necessary to carry out Laminaria genome research as soon as possible.The objective of this paper lies in two aspects: firstly, complete mitochondrial genome sequences of Laminaria japonica×Laminaria saccharina, Laminaria longissima and Laminaria hyperborean are obtained by primer walking and sequencing. These sequences together with public database of Laminaria are resolved to discuss the mitochondrion DNA (mtDNA) structure and evolutionary characteristics. What's more, phylogenetic relationships are also deeply studied. Secondly, to know the composition and distribution character of repetitive sequences in the genome of Laminaria japonica×Laminaria saccharina through analyzing parts of the genomic sequences obtained by random sequencing for the first time. The results are shown as follows:1. Analysis of structure characteristics and phylogenetic relationship based on mitochondrial genome.The complete mitochondrial genome sequence of Laminaria japonica×Laminaria saccharina, Laminaria longissima and Laminaria hyperborean is 37,638 bp, 37,628 bp, 37,976 bp respectively. Mitochondrial genome encodes 66 or 67 genes, including 38 or 39 protein-coding genes, 3 rRNA genes and 25 tRNA genes.Tightly arranged genes with small intergenic regions are widespread in Laminaria mitochondrial genome. The gene order of Laminaria mtDNA is highly conserved with some exceptions, due to the absence of ORF157 in some mtDNAs. The sequencing analysis shows a high A+T content with mean overall value of 65% and G-bias. The universal genetic codon seems to be applicable in all of these mtDNAs. ORF157 in Laminaria digitata and Laminaria hyperborean use GTG as the initiation codon while ATG is applied in the other genes. All of the three stop codons are used (TAA, TAG, TGA), with significant preference to TAA. The usage of synonymous codon markedly correlated with the nucleotide composition at third codon position.Compared to mitochondrial genome of L. japonica,"Rongfu"mtDNA lost a non-coding region of 19 nucleotides, which is located between rRNA small subunit gene 3 (rps3) and rRNA small subunit gene 9 (rps9). Seven cultivation varieties of China are divided into two groups based on this non-coding region which is 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.Phylogenetic relationships of Laminaria are conducted with the concatenation of 30 protein-encoding genes and rRNA genes respectively, using Ectocarpus siliculosus as outgroup. Laminaria digitata and Laminaria hyperborean cluster one clade, whereas the other nine Laminaria species cluster another clade. The relationships between Laminaria species are in accordance with morphological characteristics and geographic distribution.Phylogenetic relationships of 27 Laminariaceae species belonging to 4 families are conducted with the gene concatenation of nad6 and cox1, also using Ectocarpus siliculosus as outgroup. The result exhibits that one clade in Laminaria clusters, represented by Laminaria japonica, together with Macrocystis integrifolia and Hedophyllum sessile were clustered with another clade in Laminaria characterized by Laminaria digitata. 2. Analysis of distribution and frequencies of tandem repeat sequences based on nuclear genome.Genome library was constructed and then random sequencing was carried out using the new generation of high-throughput sequencing technology (454 GS FLX Titanium). These sequences are assembled into 18,051 non- redundant sequences with a total length up to 9,397,460 bp, approximately 1.33% of the Laminaria genome.The distribution and frequencies of tandem repeat sequences are studied based on the whole genome level. The cumulative length of tandem repeats is 100,320bp, accounting for 1.00% of the total length of DNA sequences. Meanwhile, a total of 1782 tandem repeats are found, including 770 microsatellites, 992 minisatellites and 20 satellites, occupying 43.21%, 55.67%, 1.13% of all tandem repeats respectively. The distribution and frequencies of microsatellite sequences in Laminaria genome are studied. It appears that there is one microsatellite sequence every 12 kb Laminaria genome sequences. The correlation between length of repeat unit and copy number is significantly negative (γ=-0.719). Furthermore, it is found that the copy numbers of 1, 3, 5 odd-number-composed repeats are significantly more than those of repeat types beside them. Trinucleotide repeats are the most frequent type, accounting for 32.21% of the total number of microsatellite. In addition, the variation of trinucleotide repeats is higher than other repeat types. Microsatellite in Laminaria genome is AT-rich because the number of microsatellite in which A+T content is more than 50% occupies 75.58% of that of all microsatellites.The distribution and frequencies of minisatellite sequences in Laminaria genome are investigated. On average, there is one minisatellite sequence every 9 kb Laminaria genome sequences, whose abundance is richer than that of microsatellite sequences. Twenty-six-nucleotide repeats are the most frequent type, accounting for 9.07% of the total number of minisatellite. It shows that the number of sequences decreases as the length of its repeat unit increases. The copy number of repeat sequences is in the low range, mainly varying from 2 to 12. Minisatellite sequences of Laminaria genome are mostly AT-rich and of those A+T content is more than 50% accounted for 74.70% of the total number of minisatellites. Genome structure and phylogenetic analysis in this paper provide direct information for the exploration of Laminaria genomics, and lays a solid foundation for Laminaria biological research and application. |
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