【作者】 石媛嫄；

【导师】 杨官品；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 海带?于1927年引入我国,是我国人工栽培历史最长和产量最高的经济海藻。经过几十年的发展,海带遗传育种、育苗及栽培取得了显著成效。与海带栽培相比,目前有关海带遗传多样性分析、经济性状遗传基础等研究还比较落后。因此迫切需要一套适用的分子标记,用来构建海带连锁图谱以促进海带遗传学研究,但是目前还没有密度适中、标记适用的连锁图谱构建。东方2号是通过海带(Laminaria japonica)的一个雌配子体克隆和长海带的一个雄配子体克隆杂交获得的,是世界上第一个大规模栽培的杂交海带,具有明显的杂种优势。东方2号是不是真正的种间杂交这个广泛关注的问题通过亲缘关系分析可以得到证明。研究内容包括:研制海带微卫星DNA标记并分析了海带(L. japonica)和长海带(L. longissima)配子体克隆的遗传多样性,组合使用微卫星DNA标记和AFLP标记构建了海带连锁图谱,另外,分别用AFLP标记、核糖体RNA间隔区序列(ITS)中的SNP及微卫星DNA标记对东方2号杂交海带进行了亲缘关系分析。主要结果如下:利用FIASCO方法构建了海带(L. japonica) (AC)n的微卫星DNA富集文库,测序得到138个含微卫星DNA片段的序列,根据所得到的序列设计了65对引物,其中有22对引物在海带配子体克隆及幼孢子体中能得到特异的扩增产物。在36份海带配子体克隆中,微卫星DNA标记检测到的等位基因数量从2-7个不等,揭示的基因多样性在0.21到0.75之间,香农氏指数在0.37到1.55之间。长海带和海带(L. japonica)的平均等位基因数(1.5/3.3),平均基因多样性(0.12/0.45)和平均香农氏信息指数(0.20/0.81)显著不同。根据总样本确定的前两个最丰富等位基因中,绝大多数的频率在长海带和海带(L. japonica)之间存在显著差异。长海带和海带(L. japonica)之间在基因多样性和香农氏信息指数反应的遗传多样性上的平均遗传分化分别达35.6%和36.9%。海带(L. japonica)雄配子体和长海带雌配子体克隆杂交获得杂交海带孢子体,从成熟孢子体分离、培养获得配子体克隆,随机取40个配子体克隆与杂交海带配子体亲本作为作图群体,利用AFLP和微卫星DNA标记分别根据杂交海带雄配子体克隆亲本特异等位基因和杂交海带雌配子体克隆亲本特异等位基因构建了两张海带连锁图谱。我们将所有标记按显性标记处理,不追究其对应的隐性等位基因。如果全都用通用性较高的微卫星DNA标记可以构建统一的一张图谱。根据雌配子体克隆亲本特异等位基因构建的图谱包含27个连锁群共100个标记(94个AFLP和6个微卫星DNA标记),总图距为678.4cM,图谱覆盖率为56.4%。根据雄配子体克隆亲本特异等位基因构建的图谱包含30个连锁群共121个标记(115个AFLP和6个微卫星DNA标记),总图距为745.7cM,图谱覆盖率为53.6%。目前我们正在构建中等密度的微卫星DNA标记连锁图谱。AFLP分析发现东方2号杂交海带和海带(L. japonica)共有的条带有70条,和长海带共有55条带,而海带(L. japonica)和长海带仅有11条带是相同的。核糖体RNA间隔区序列分析发现,东方2号在847位核苷酸存在碱基替换(T-C转换),在海带中此位点全部为T,而在长海带中为C。东方2号在两个微卫星位点同时具有父本和母本的等位基因型。以上证据表明,东方2号是海带(L. japonica)和长海带的杂交后代。然而,根据核酮糖1,5-二磷酸羧化/氧化酶基因间隔区序列并不能确定东方2号叶绿体的来源。更多还原

【Abstract】 Laminaria* has the longest history of artificial cultivation and the highest yield among macroalgae cultured in China. Laminaria japonica was introduced into China for cultivation in 1927, and L. longissima and other species later. China has achieved richly in cultivation method, seedling raising technique and breeding of Laminaria, however, the genetic studies (e.g., evaluation of genetic diversity and determination of genetic basis of economic traits of Laminaria) were far behind the cultivation of Laminaria. An appropriate molecular marker linkage map (markers are transferable and dense enough) will certainly facilitate the genetic study of Laminaria. Unfortunately, such a map was not available currently.Dongfang No.2 is the first interspecific hybrid Laminaria commercially cultivated in the world, which was raised through crossing a female gametophyte clone of L. japonica with a male one of L. longissima. The hybrid holds high heterozygous vigor (heterosis) in yield and yield related traits. Whether Dongfang No. 2 is a true interspecific hybrid remained an attention from both scientific community and farmers. The parentage of Dongfang No. 2 hybrid Laminaria should be determined.A group of microsatellite DNA markers were developed and used to determine the genetic diversity of gametophyte clones isolated from L. japonica and L. longissima and construct a linkage map of Laminaria in combination with amplified fragment length polymorphism markers (AFLP). In addition, AFLP markers, single nucleotide polymorphism (SNP) of internal transcribed spacers (ITS) region and two microsatellite DNA markers were used to determine the parentage of Dongfang No.2 hybrid Laminaria.The following are the major findings:A (AC)n microsatellite DNA containing fragment enriched library was constructed for L. japonica using FIASCO method. One hundred and thirty eight microsatellite DNA containing fragments were identified and sequenced, from them, 65 pairs of primers were designed. Of these pairs of primers (hereafter, microsatellite DNA markers), 22 amplified products from the genomic DNA of the gametophyte clones and young sporophytes of Laminaria.In 36 Laminaria gametophyte clones, the number of alleles each locus detected by these microsatellite DNA markers ranged from 2 to 7, and the gene diversity and Shannon’s information index from 0.21 to 0.75 and from 0.37 to 1.55, respectively. The average number of alleles (1.5 vs. 3.3), average gene diversity (0.12 vs 0.45) and average Shannon’s information index (0.20 vs 0.81) of L. japonica and L. longissima were significantly different. The frequencies of the majority of the two most abundant alleles of two species were also different significantly. The genetic differentiation between two species reached 35.6% (gene diversity) and 36.9% (Shannon’s information index) in average.Two linkage maps of microsatellite DNA and AFLP markers were constructed using 40 gametophyte clones randomly isolated from a sporophyte of the hybrid of a male gametophyte clone of L. japonica and a female one of L. longissima. One was done using the marker alleles specific for the male gametophyte clone parent (coupling each other) of the hybrid, and the other using those specific for the female gametophyte clone parent. Actually, a single linkage map can be constructed with microsatellite DNA markers. Two maps were constructed; the recessive AFLP alleles corresponding to their dominants were not traceable.The map constructed with the female gametophyte clone specific marker alleles consisted of 100 markers(94 AFLP and 6 microsatellite DNA)arranged into 27 linkage groups. The map length was 678.4cM, covering 56.4% of the estimated genome size. The map constructed with the male gametophyte clone specific marker alleles consisted of 121 markers(115 AFLP and 6 microsatellite DNA) assigned into 30 linkage groups, which was 745.7cM in length,covering 53.6% of the estimated genome size. We are currently trying to develop a microsatellite DNA marker linkage map at the appropriate density.Dongfang No. 2 hybrid Laminaria shared 70 and 55 bands with L. japonica and L. longissima, respectively, obviously more than 11 bands shared by L. japonica and L. longissima. Dongfang No. 2 held both“T”and“C”at position 847 of the ITS region, while“T”at this position was specific for L. japonica and“C”for L. longissima, respectively. In addition, Dongfang No. 2 also held the alleles of two parents together at two microsatellite DNA marker loci. These observations clearly proved that Dongfang No. 2 is a true hybrid of L. japonica and L. longissima. Unfortunately, the origin of its chloroplast was not determined based on the variation of RuBisCo spacer.更多还原