【作者】 徐燕；

【导师】 陈昌生；

【作者基本信息】 集美大学 ， 水产养殖学， 2007， 硕士

【摘要】 本文利用⁶⁰Co-?射线辐射处理野生坛紫菜（Porphyra haitanensis）丝状体，从其后代中选育经济性状优良的诱变品系；利用杂交育种技术，以诱变选育品系为父本，野生选育品系为母本进行杂交实验，通过对杂交子代的培养观测，选育出杂交色素突变体和具有杂种优势的品系；采用体细胞酶解技术和单克隆技术使诱变和杂交选育获得的纯系得到快速繁殖，并对其后代的主要经济性状进行了评估和比较；同时通过对杂交亲本和子代的ISSR扩增带谱进行分析，以期能快速准确地对杂交后代进行分析鉴定，在分子水平上为坛紫菜杂交育种及杂种优势的研究提供理论依据。1 ⁶⁰Co-γ射线作为坛紫菜诱变育种的诱变剂是有效的，在300～1100Gy范围内，它可以使坛紫菜丝状体发生变异并在子代中表现。诱变选育出3个品系：①品系7号Ⅰ：叶片宽（2.99±0.61cm）、生长快（30cm以上的藻体长度平均日增长量可达6.51±1.39cm）、耐高温（29℃）、总藻胆蛋白（total phycobiliprotein）含量高（104.858±7.376mg/g干品）；②品系7号Ⅱ：生长快（长度30cm以上的藻体平均日增长量可达4.26±1.23cm）、叶片薄（藻体中部厚22.50～27.50μm）、总藻胆蛋白含量高（104.239±5.064mg/g干品）；③紫色突变体3号Ⅰ：藻体紫色；叶片薄，藻体中部厚25.00～32.50μm。2坛紫菜诱变品系和野生选育品系杂交其子代叶状体表现出较大的杂交优势，杂交子代既具有亲本优势，又具有超亲优势。杂交选育出四个经济性状优良的品系Ⅱ、Ⅲ、Ⅳ和Ⅴ，3～5cm的幼苗经过10d培育平均日增重量超标优势分别达12.35％、139.78％、107.57％和173.52％；杂种优势指数达99.83％、213.07％、184.44％和243.05％。其中品系Ⅱ耐高温能力突出，可在29℃培养液中可正常生存。3杂交产生了褐黄、翠绿和褐绿突变体，与亲本相比，3种色素突变体主要藻胆蛋白（藻红蛋白、藻蓝蛋白和别藻蓝蛋白）含量发生了较大变化，明显低于亲本，仅为母本的40.55％～73.17％。翠绿色突变体生长快速，30cm以上藻体日生长量可达（7.75±1.78）cm，并且翠绿色丝状体发育方式特殊，营养藻丝不经过丝状藻丝加粗阶段，由球形细胞直接发育成孢子囊枝和壳孢子囊。4应用ISSR（Inter-Simple Sequence Repeat）标记技术对坛紫菜杂交亲本及其5个子代品系（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ）的杂种优势进行了研究，从67个引物中筛选出了21条能扩增出清晰、稳定条带的引物，共得到366个扩增位点，其中347个位点具有多态性，多态位点百分率达94.8％。通过对扩增带谱的统计分析，发现5个坛紫菜杂种子代品系同父本和母本的遗传相似性平均值分别为0.5191和0.5551，都明显低于两亲本间的遗传相似性0.6120；并且5个杂种子代的有效等位基因数，期望杂合度和Shannon多样性指数都要明显高于两亲本的相应数值。由此表明杂交使得坛紫菜的遗传多样性水平明显提高，产生了显著的杂种优势。从数据统计结果还可发现5个杂种子代同两亲本的遗传距离不是对等的，全部都稍偏向母本，聚类分析结果也同样证明了这一点。更多还原

【Abstract】 Free-living conchocelis of wild Porphyra haitanensis were treated with ⁶⁰Co-? ray in orderto obtain mutations with excellent practical characters. Then crossed experiments between themutations（?） and selected wild strain（?） were carried out. Through cultivated and observed F₁gametophytic blades of hybrids, the pigmentation mutants and crossed strains with heterosiswere picked up. Single cell clone and enzymolysis were used to propagate fast in pure strains ofinduced mutations and hybrids. Moreover, the central practical characters of their offspring wereevaluated and compared. At same time, inter-simple sequence repeat（ISSR） was used in statisticand analyzing amplified bands of parents and hybrids, just expect to analyze and identify filialgeneration quickly and exactly, and provid the theories gist in research of hybridization breedingand heterosis analysis of P. haitanensis on the molecule level.1. Three strains have been selected. The first one was 7-Ⅰwhich has the quickest growth(6.51±1.39 cm·d^-1, after blade length overed 30 cm), wide blade （2.99±0.61 cm）, hightemperature resistant（29℃） and high phycobiliprotein content(104.858±7.376 mg·g^-1) of drysample. The second one was 7-Ⅱwhich has quick growth(4.26±1.23 cm·d^-1, after blade lengthovered 30 cm), thin blades（22.50～27.50μm in the middle part of the blade） and highphycobiliprotein content(104.239±5.064 mg·g^-1) of dry sample. The third one waspigementation mutant, 3-Ⅰ, which possessed of purple and filmy blade（25.00～27.50μm）。Itproved that ⁶⁰Co-? ray was a useful mutagen in inducing breeding of P. haitanensis. Whenirradiation rang was about 300～1100 Gy, conchocelis could be induced and represented inoffsprings.2. Filial generation （mutations（?）and selected wild strain（?）） had significant hybrid vigor,not only had parental dominance but heterobeltiosis. Four strains（Ⅱ、Ⅲ、ⅣandⅤ） with excellentpractical characters were selected by hybridization breeding. After the thalli （which length wasabout 3～5 cm） were cultivated for ten days, the over-standard heterosis of average daily gaincould reach to 12.35%, 139.78%, 107.57% and 173.52%, and the index of heterosis couldreach to 99.83%, 213.07%, 184.44% and 243.05 % respectively Thereinto, strainⅡwas stoodout in hih temperature resistant which grew normally in 29℃.3. Brown yellow, emerald and breen pigementation mutants were obtained after crossing.They were low in contents of three phycobiliprotenin （R-phycoerythrin, R-phycocyanin andallophycocyanin） , only about 40.55%～73.17% of maternal. Among them, the thallus of emeraldgrew quickly(7.75±1.78 cm·d^-1, after blades length overed 30 cm). Furthermore, the emeraldconchocelis were easy to mature and had special developmental mode, that was the spherulocytes could develop to sporangial branchlets and conchosporangia without thickconchocelis stage directly.4. ISSR method was used to study the heterosis of the P. haitanensis parents and its fivehybrid strains（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ）, 21 ISSR primers （were screened from 67 primers） gave riseto 366 discernible DNA bands of which 347 （94.8%） were polymorphic. By statistic andanalyzing these amplified bands, it can find that the average genetic similarity between the 5hybrids and female parent and male parent are 0.5191 and 0.5551, which less the geneticsimilarity between the parents （0.6120）. The effective number of alleles （Ne）, expectedheterozygosity （h） and shannon’s information index （I） among 5 hybrids were 1.5501, 0.3200and 0.4720 respectively, which also greater than the corresponding values between the parentsevidently. From these datas, it can conclude that the hybrid can enhance the genetic diversity ofP. haitanensis and produce distinct heterosis. From these datas, it also can find that the geneticdistances between the 5 hybrids and female parent and male parent are no equality, all secund tothe female parent.更多还原