【作者】 李小湘；

【导师】 刘勇；

【作者基本信息】 中南大学 ， 植物学， 2007， 博士

【摘要】 普通野生稻(Oryza rufipogon Griff.)是亚洲栽培稻(O.sativaL.)的祖先种,是极其重要的种质资源。广州犀牛尾的耐逆境普通野生稻、海南崖县的“野败”型普通野生稻、马来西亚的含高产基因QTLy1d1.1和y1d2.1普通野生稻均被成功利用到水稻育种上,使中国水稻单产一次又一次大幅度提高。了解普通野生稻的居群遗传动态和交配系统将为其有效原地保护、异地保存提供重要指导。本研究采用SSR标记技术比较了普通野生稻原地保护与异地保存居群之间、原地保护与未保护的自然居群之间、自然居群子代与母代之间的遗传多样性水平;检测了6个自然居群的交配系统,并分析了这些居群的子代群体和母代群体的遗传动态,取得以下结果。1.湖南江永普通野生稻原地保护和异地保存居群的SSR多样性差异用48对SSR引物对湖南江永普通野生稻异地保存和原地保护居群进行遗传多样性分析。48对SSR引物在异地保存和原地保护中分别检测出166和119个等位基因,平均每对引物检测到的等位基因数分别为3.55和2.60,多态位点百分率为99.4%和95.2%,平均等位基因观察数为1.99和1.95,平均有效等位基因数为1.428和1.508,Nei基因多样性指数为0.272和0.304;原地保护居群遗传变异大,但异地保存样本中发现新的变异单株。江永野生稻原地保护4个亚居群(G4-1、G4-2、G4-3、G4-4)的遗传分化系数为0.434,江永野生稻4个亚居群间变异量占总变异量的比值差异较大,总变异的43.4%存在于亚居群之间,说明遗传变异主要存在亚居群内;4个亚居群的基因多样性水平从高至低的顺序为G4-1＞G4-3＞G4-4＞G4-2。2.普通野生稻保护和未保护居群遗传多样性的比较为了评价普通野生稻自然居群遗传多样性,用24对SSR引物对5个保护居群(江西东乡庵家山JXD、湖南茶陵HNC、湖南江永HNJ、广东高州大岭GDD、海南儋州HND)和3个未保护居群(广西武宣GXW、广西来宾GXL、广东高州朋山GDP),共计356个单株进行了遗传多样性分析。24个位点均表现为多态,其中18个位点表现杂合子不足,RM339位点观察杂合度最高,RM336位点预期杂合度最高。SSR分析结果表明,8个居群的遗传多样性都较高,其中5个保护居群的Ae变幅为1.780(JXD)～2.504(HNJ),He变幅为0.397(JXD)～0.555(HNJ);3个未保护居群Ae变幅为2.153(GDP)～3.226(GXL),He变幅为0.492(GDP)～0.640(GXL)。5个保护居群与3个未保护居群之间的遗传距离较大(0.6585)。这说明虽然有些居群得到了保护,但未保护居群的保护、收集价值仍然很大。居群间遗传分化明显(Fst=0.399),居群间遗传距离最小的HNJ居群与GXL居群的相似系数也只有0.43。F—统计显示所用居群都偏离了Hardy-Weinbery平衡(Fis=0.147),其中居群GDD、GDP、HND居群Fis＜0,表现为杂合子过剩;居群JXD的Fis为0.109,表现轻微的杂合子缺乏;HNC、HNJ、GXW、GXL的Fis＞0,Fis值变幅为0.315(GXW)～0.473(HNJ),说明这4个居群中杂合子不足,可能与这些居群自交比例高有关。3.普通野生稻自然居群的交配系统与遗传动态为了了解普通野生稻自然居群的交配系统和居群遗传动态,以取自6个自然居群(江西东乡居群JXD、湖南茶陵居群HNC、湖南江永居群HNJ、广西武宣居群GXW、广东高州大岭居群GDD、广东高州朋山居群GDP)的267个母株中150个家系(母株)的2353个子代个体为研究对象,用14对SSR标记检测了普通野生稻居群子代与母代的遗传多样性;分析了各个居群的单点异交率、多点异交率、近交系数。3.1子代居群与母代居群的遗传多样性比较研究结果表明,统计子代遗传多样性时可以按家系分组随机抽取的3次样本,分别统计每次抽样样本分析数据,然后计算平均值。6个子代居群之间遗传多样性存在差异,抽样平均Ae的变幅为1.7193～2.7781,抽样平均Nei sHe变幅为0.3218～0.5891,显著性检验结果因遗传多样性参数不同而不同。6个居群间子代分化明显,6个居群间子代Fst的3次重复平均为0.4181,说明41.81%的遗传多样性存在子代居群间;6个子代居群总的固定指数(FI)三次重复变幅为0.4537～0.4767,平均为0.4635,推测总的子代群体明显偏离Hardy-Weinbery平衡,各居群子代的FI都大于0,都表现纯合子过剩。6个子代居群间存在一定基因流,3次重复抽样Nm变幅为0.3443～0.3518,平均为0.3479。比较6个居群的子代与母代遗传多样性,表明子代居群的观察杂合度Ho都低于母代居群,子代居群固定指数FI都高于母代居群,说明子代居群的杂合体少于母代居群。各个居群的子代与母代的Neis He比值变幅为0.9431(HNJ)～1.1412(GDD)。6个子代居群之间Nm和6个母代居群之间Nm分别为0.3479、0.3424,差别很小;6个母代居群间Fst(0.4196)与6个子代居群间Fst(0.4181)相差也很小;各居群的母代与子代相似系数都在0.98以上,说明抽样子代居群达到了代表取样母代居群遗传多样性98%以上的要求。3.2普通野生稻自然居群交配系统参数分析普通野生稻6个取样居群交配系统为混合交配类型。居群之间的异交率相差较大,多点异交率(tm)变幅15.1%(HNJ)～41.8%(GXW),单点异交率(ts)变幅10.1%(HNJ)～28.5%(JXD)。6个取样居群的多位点异交率都高于单位点异交率,说明居群内存在一定的自交。6个取样居群的rp(m)都比较大,变幅为0.607(HNJ)～0.718(GDP),说明各居群内子代的双亲有可能是姊妹关系的个体都比较多。GDD和GDP居群的亲本近交系数F＜0,说明居群中有过剩的杂合子;JXD、GXW、HNJ和HNC居群的F＞0,说明这4个居群中有过剩的纯合体。6个取样居群的rp(s)-rp(m)差值都大,变幅为0.807(HNJ)～0.918(GDP),说明取样居群存在亚结构。3.3本研究结果对普通野生稻异地保存和原地保护具有指导意义本研究为湖南江永普通野生稻原地保护和异地保存提出了建议。本研究结果支持普通野生稻的子代种子收集保存不容忽视。提出以单穗为单元进行异地繁种套袋、收集、保存。探讨了6个取样居群的近交衰退存在的可能性和监测原地保护居群遗传多样性动态的必要性。提出进一步改善普通野生稻原地保护措施。更多还原

【Abstract】 Oryza rufipogon Griff.known as the ancestor of Asian cultivated rice(O.sativa L.),is the most important germplasm for rice improvement.It has made great contributions to improvements in rice yields.For example,from O.rufipogon,the gene tolerant to abiotic stress in Xiniuwei of Guangdong Province,the male sterility gene in Sanya county of Hainan Province,QTLyldl.1 and yld2.1 genes in Malaysian are utilized successfully in increasing rice yields.Unfortunately,the wild rice is now under seriously endangered status.To better understand genetic dynamics and mating systems can be particularly informative to obtain a conservation perspective of the species for further in situ conservation and germplasm management.By using SSR methods,the present study was conducted to compare the genetic variability between in-situ and ex-situ conserved O.rufipogon populations,between in-situ conserved and not conserved O.rufipogon populations,between maternal and progeny;to estimate mating system of natural O.rufipogon populations;and to analysis the genetic dynamic between maternal and progeny populations of O.rufipogon.The main results were as the follows.1.Comparisons of genetic variability between the in-situ and ex-situ conserved O.rufipogon populations in Jiangyong county of Hunan Province.Comparisons of genetic diversity levels of Jiangyong O.rufipogon were performed between the ex-situ and in-situ conservation populations by using 48 polymorphic SSR markers.A total of 166 and 119 alleles were scored in the ex-situ(G3) and in-situ(G4) populations,with the average of 3.55 and 2.60 per marker,respectively.The percentage of polymorphic loci(P),the mean number of observed alleles(A),the mean effective number of alleles(Ae),and gene diversity(He) in population G3 and G4,were 99.4%vs.95.2%,1.99 vs.1.95,1.428 vs.0.508,0.272 vs.0.304,respectively.It was indicated that relatively richer genetic variability existed in the population G4,although some unique variants existed in the population G3.For the in-situ population G4,43.4%of total variation existed between subpopulations(Gst=0.434),suggesting genetic variations were mainly held within subpopulations,and the genetic diversity levels between the four subpopulations showed the order from higher to lower were G4-1＞G4-3＞G4-4＞G4-2.2.Comparative studies on the genetic variations between Conserved and non-conserved Oryza rufipogon populationsEstimations of the levels of genetic diversity of the in-situ conserved populations of Oryza rufipogon in comparisons with the populations without conservations were conducted by using 24 SSR loci.The samples included 356 individuals from 5 conserved populations located in Dongxiang county,Jiangxi province(JXD),Chaling county and Jiangyong county,Hunan province(HNC and HNJ),Gaozhou city, Guangdong province(GDD),and Danzhou city,Hainan Province (HN),and 3 non-conserved populations in Wuxuan county and Laibin city,Guanxi(GXW and GXL) Automonous Region and Gaozhou city, Guandong Province(GDP).All the 24 loci were polymorphic in this study,among which,18 loci exhibited heterozygosity deficit.The highest observed heterozygosity (Ho) was generated by RM339,while the highest expected heterozygosity(He) appeared at RM336.In General,all these wild populations showed a high level of genetic diversity.Among the 5 conserved populations,the mean effective number of alleles(Ae) ranged from 1.780(JXD) to 2.504(HNJ),He ranged from 0.397(JXD) to 0.555(HNJ);For the 3 populations without conservations,Ae varied from 2.153(GDP) to 3.226(GXL),He from 0.492(GDP) to 0.640(GXL), respectively.Nei’s genetic distance was 0.6585 between 5 Conserved populations and 3 non-conserved populations.It demonstrated that the populations without conservations had great value of Conservation and collection in spite of some populations conserved.The evaluation of genetic differentiation showed that 39.9%SSR variation was distributed among populations(Fst=0.399).F-statistics showed that these populations presented a slight deviation from the Hardy-Weinberg equilibrium(Fis = 0.147).The population GDD,GDP、HND showed slight heterozygosity excesses with Fis ranged from -0.03(GDP) to-0.186(HND).The population JXD showed slight heterozygosity deficits with Fis= 0.109,and the population HNC,HNJ, GXW and GXL showed significant heterozygote deficits in tests of Hardy-Weinberg equilibrium and significantly positive Fis values ranged from 0.315(GXW) to 0.473(HNJ),such pattern might be related to the relatively higher inbreeding in these wild populations.3.SSR analysis for mating system and genetic dynamics in O.rufipogon populations.To understand the mating system of O.rufipogon and the population dynamics,2353 progenies from 150 open-pollinated families sampled from 267 maternal parents of 6 populations from Jiangxi Province (JXD),Guangdong Province(GDD and GDP),Guangxi Province (GXW),and Hunan Province(HNJ and HNC) were analyzed by the SSR assays using 14 SSR loci.Genetic diversity of maternal and progeny populations were compared and the multilocus population outcrossing rate(tm),the single-locus population outcrossing rate(ts) and the single-locus inbreeding coefficient of maternal parents(F) were estimated for each progeny population.3.1 SSR analysis for genetic dynamics between maternal and progeny in O.rufipogon populations.Results indicated that genetic diversity of progeny populations may be estimated using 3 times random sampling from total progeny materials after grouping by families,then compute an average of statistical results of 3 times random sampling.The levels of genetic diversity varied among the sampled progeny populations,with Ae ranged from 1.7193 to 2.7781,NeisHe ranged from 0.3218 to 0.5891.The LSR-test results of genetic diversity varied among different Parameter values showing genetic diversity.Mean Fst=0.4181 indicated that there was a significant genetic population differentiation and about 41%of the total genetic variation existed among the progeny populations.The average of the overall FI for 3 times random sampling from total progeny populations was 0.4635,with FI ranged from 0.4537 to 0.4767 and each progeny population had a positive FI value, suggesting significant heterozygosity deficits and a significant deviation from Hardy-Weinberg equilibrium.There was gene flow(Nm) between progeny populations.The average of Nm for 3 times random sampling from total progeny populations was 0.3479,with Nm ranged from 0.3443 to 0.3518.The Ho of sampled progeny population was lower than that of sampled maternal population,while the FI of sampled progeny population was higher than that of sampled maternal population,which suggested heterozygosities in sampled progeny population was lower than that in sampled maternal population for every one.The ratio of Neis genetic diversity between progeny and maternal population pairs ranged from 0.9431 in population HNJ to 1.1412 in population GDD.There was not significant difference of gene flow(Nm) between progeny populations compared with that between maternal populations(0.3479 vs. 0.3424),and Fst values showed a similar pattern(0.4196 vs.0.4181). Within each sampled progeny and maternal population pairs,the similarity coefficient was more than 0.98.These results:indicated the progeny population represented more than 98%variability of its maternal population.3.2 Analysis for mating system parameters for 6 natural populations of Orzya rufipogon.O.rufipogon possess a mixed type of mating system,although outcrossing rate was variable among populations.The multilocus outcrossing rate(tm) ranged from 15.1%(HNJ) to 41.8%(GXW),and the single-locus outcrossing rate(ts) ranged from 10.1%(HNJ) to 28.5% (JXD).There was Inbreeding because there was significant difference between tm and ts.The correlation of paternity(rp) for the multilocus ranged from 0.607(HNJ) to 0.718(GDP) Which indicated a higher fraction of Siblings shared the same father.The population GDD,GDP exhibited heterozygosity excess,while population JXD、GXW、HNJ and HNC had significant heterozygosity deficit.The correlation of paternity for the multil~cus significantly differed from the correlation of paternity for the single-locus,with rp(s)-rp(m) ranged from 0.807 in population HNJ to 0.918 in population GDP,which suggested there are substructure for every sampled population.3.3 Implications of the results to ex-situ conservation and in.situ conservation.The paper put forward suggestions for in-situ conservation and ex-situ conservation of O.rufipogon in Jianyong,Hunan.The results supported that collection and conservation seeds of progeny population of O.rufipogon can not be neglected.The present study recommended the strategy for conservation of O.rufipogon that a single panicle can be as a unit to ex-situ seed-producing,collecting and conserving.Meanwhile the possibilities of inbreeding depreesion and the necessity of monitoring genetic dynamics of in-situ conservation population were assessed.Some improving measures for further in-situ and ex-situ conservation of O. rufipogon also discussed.更多还原