【作者】 郭鲜蒲；

【导师】 柳俊；

【作者基本信息】 华中农业大学 ， 遗传学， 2011， 博士

【摘要】 马铃薯种间体细胞杂交不仅可以克服远缘杂交不亲和等生物学障碍,而且可以将野生资源中的有利性状转育到栽培种中,实现核与质的融合或重组,创造出有性杂交所不能实现的新种质。本研究以来自于两个融合组合的149个马铃薯种间体细胞杂种系为材料,通过流式细胞仪和SSR分子标记方法,对组织培养条件下长期继代繁殖的马铃薯体细胞杂种的遗传稳定性进行了分析,采用细胞学技术对杂种及其融合亲本花粉母细胞的减数分裂行为及花粉育性进行了观察,并对体细胞杂种与四倍体普通栽培种的回交后代进行了系统评价分析,主要研究结果如下：1.通过流式细胞仪分析,组织培养条件下连续继代培养4年(2003-2007)的108个马铃薯种间体细胞杂种中68个体细胞杂种的倍性水平发生了变化。与2003年的结果比较,54个六倍体中有14个杂种的倍性发生了变化,与其他倍性的杂种相比六倍体的倍性稳定性最高。其他倍性水平(混倍体、八倍体和非整倍体)的体细胞杂种的倍性均向整倍体化和均一化变化,说明具有双亲倍性之和的马铃薯种间体细胞杂种其倍性较稳定,非期望倍性的杂种在组织培养条件下表现高频率的染色体丢失。2.利用1对叶绿体SSR引物和4对线粒体特异引物对组织培养条件下长期继代培养的体细胞杂种的细胞质遗传组成及其稳定性进行了分析。143个体细胞杂种中,55个具有野生种亲本的叶绿体类型,56个具有栽培种亲本的叶绿体类型,其余32个体细胞杂种在不同年份的检测间存在变化。用于检测线粒体遗传组成的44个来自3#+C9701融合组合的马铃薯体细胞杂种中,除3个为野生种类型之外,41个杂种的线粒体类型均为双亲融合或重组类型。在2010年和2011年进行了两次检测的42个体细胞杂种中,除3C8-4由野生种类型变为重组型、3C10-2由重组型变为栽培种类型外,40个体细胞杂种的线粒体类型没有发生改变。结果表明,马铃薯种间体细胞杂种的细胞质基因组中,线粒体基因组较叶绿体基因组更易发生整合或重组,且具有较高的稳定性。3.采用花粉母细胞酸解压片法,观察了不同倍性的体细胞杂种及其融合双亲的花粉母细胞减数分裂行为,发现二倍体野生种融合亲本C9701的花粉母细胞减数分裂行为基本正常,异常花粉母细胞的比例为1.29%,四倍体栽培种融合亲本3#和8#的异常花粉母细胞比例分别为20.44%和12.12%。体细胞杂种花粉母细胞异常减数分裂的比例均高于其融合亲本,其中六倍体8C30-1的异常花粉母细胞的比例最低为21.10%,混倍体8C13-1的比例最高达到73.42%。倍性越高或倍性越复杂的杂种,其花粉母细胞异常减数分裂的比例越高。在体细胞杂种花粉母细胞减数分裂的各个时期都可以观察到异常的花粉母细胞,主要异常现象包括单价体、多价体等不同的染色体构型、落后染色体、染色体桥、染色体不均等分裂、染色体行为不同步、微核、三分体和多分孢子等现象。说明体细胞融合所导致的杂种倍性构成的复杂性是花粉母细胞减数分裂异常的重要原因之一,具有期望倍性的杂种其异常减数分裂频率较低。4.马铃薯体细胞杂种的花粉萌发率与花粉染色活力呈显著正相关。二倍体野生种融合亲本的花粉染色活力最高为99.5%,两个四倍体栽培种融合亲本的花粉染色活力分别为47.0%(3#)和49.9%(8#)。除4个体细胞杂种系之外,其余杂种的花粉活力均低于四倍体融合亲本。除2个体细胞杂种之外,所有体细胞杂种的小花粉粒比例均大于融合亲本。结果表明,花粉母细胞异常的染色体减数分裂行为是导致小花粉粒产生的主要原因,小花粉粒越多花粉活力越低。5.研究选用能够正常开花且花粉育性基本正常的12个体细胞杂种系,与12个四倍体栽培品种或品系配制杂交组合共35个,其中25个杂交组合成功获得2515个实生种子。12个体细胞杂种亲本中,以五倍体体细胞杂种3C28-1与栽培种杂交亲和力最高,共配制20个杂交组合(3C28-1作母本的杂交组合13个,作父本的杂交组合7个),18个组合成功获得实生种子共2258粒。结果证明,种间体细胞杂种中能够筛选出与四倍体栽培种杂交亲和的材料用于遗传育种。6.对体细胞杂种作为回交亲本获得的281个回交后代及3C28-1的17个自交后代的倍性水平进行了鉴定,结果显示回交后代中181个为四倍体,10个为五倍体,另外90个为介于4x-5x的非整倍体。3C28-1的17个自交后代的倍性水平从4x到6x均有分布,其中包括2个四倍体,1个介于4x-5x的非整倍体,8个五倍体,2个介于5x-6x的非整倍体以及4个六倍体。说明体细胞杂种形成的配子具有多种倍性,其中倍性为2x的配子能与四倍体栽培种产生的配子优先成功配对。7.对3C28-1的回交后代农艺性状观察发现,回交后代在株高方面表现出较强的杂种优势,植株高度均高于栽培种杂交亲本,存在广泛分离；回交后代的主茎数均在亲本范围之内,偏向于体细胞杂种3C28-1,说明主茎数具有较强的高亲遗传趋势；回交后代叶形分离较广,大多数都介于回交亲本之间；匍匐茎长度、单株结薯数、单株薯重和单个薯重的变异幅度较大,可能受多基因调控。8.利用11对SSR引物对3C28-1与四倍体栽培种E1的96个回交后代以及40个3C28-1的自交后代进行了遗传分析。11对引物共扩增出13条野生种融合亲本C9701的特异条带,4条栽培种融合亲本3“的特异条带,7条回交亲本E1的特异条带以及9条3#与E1的共有条带。遗传分析结果显示,亲本的33条特异条带在回交后代群体中都有出现,但每个株系所含特异标记的种类和数目不同,单个亲本特异条带在回交后代中出现的变异幅度大于双亲共有条带,证明体细胞杂种能将遗传信息稳定传递给回交后代,且在回交过程中能产生广泛分离。在3C28-1的40个自交后代中,野生种C9701的13条特异条带出现的频率为71.7%,栽培种3#的13条特异条带出现的频率为59.0%,并且在自交后代中检测到了新带的出现,进一步说明体细胞杂种配子形成的复杂性,同时证明在减数分裂过程中发生了染色体交换或位点重组事件。更多还原

【Abstract】 Plant somatic hybridization that can transfer desirable genes from wild species into cultivates and recombine nuclear DNA and organelle DNA between sexually incompatible species has been adopted to create novel germplasms for plant breeding. One hundred and forty-nine potato somatic hybrids had been previously obtained from two fusion combinations in our laboratory. To provide with theoretical base for further use of the somatic hybrids, we analyzed the genetic stability, the meiotic chromosomal behavior of the pollen mother cells, pollen viability and variation in agronomic traits and the genome components of the somatic hybrids, as well as the genetic variation of the hybrids and inbreds derived from them. The main results were as follows:1. Flow cytometric analysis demonstrated that68out of108somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures (from2003to2007), which mainly occurred in octaploids, aneuploids, and mixoploids, while74%hexaploids were still stable in their genome dosage, indicating the somatic hybrids with a sum of parental chromosomes have more ploidy stability than those with unexpected chromosome number.2. One pair of cpDNA SSR primer and4pairs of mtDNA specific primers were used to detect the cytoplasmic components of the somatic hybrids and their stability during long-term culture in vitro. There were77.6%of the143hybrids which showed the chloroplast pattern of one parent and the rest exhibited changed patterns among the three tests conducted in2003,2005and2010. Most somatic hybrids (41out of44) presented a fused mtDNA pattern of the fusion parents, although some specific bands altered in8hybrids. The results suggested that, in comparison with the chloroplast genome, the mitochondria genome has a higher recombination frequency which is more stable in in vitro subcultures.3. The meiotic behavior of the somatic hybrids different ploidy levels was examined and compared with their fusion parents. The meiotic analysis revealed that the wild fusion parent C9701had normal meiosis with abnormal rate of1.29%while the tetraploid cultivated parents3#and8#had abnormal rates of20.44%and12.12%, respectively. The chromosome meiotic behavior in most potato somatic hybrids was more complicated than in their fusion parents, a hexaploid8C30-1had the lowest abnormality (21.10%) and a mixoploid8C13-1had the highest abnormal meiosis (73.42%). The meiotic abnormity was associated with the genomic complexity. Different types of abnormal meiotic behavior were observed during development of the pollen mother cells (PMCs) such as formation of univalent and multivalent, laggard chromosomes, chromosomal bridges, unbalanced separated chromosome at different phases in a PMC or anther as well as triads and polyads. The results demonstrated that ploidy complexity resulted from somatic hybridization in somatic hybrids is a main effect influencing meiosis of pollen mother cells. The hybrids with expected ploidy level had a lower frequency of meiosis abnormity.4. There was positive relationship between pollen germination rate and the pollen fertility identified by pollen staining. The results showed that the pollen viability of C9701was99.5%while of3#and8#was47.0%and49.9%, respectively. Except for4somatic hybrids, the pollen viability of the most hybrids was lower than their cultivated fusion parents, and accordingly, the number of small pollens of the hybrids was higher than that of the parents. The statistic analysis revealed that small and sterile pollens were the direct result of the abnormal meiosis.5. Twelve somatic hybrids with normal flowers containing viable pollens were chosen to backcross with12tetraploid cultivated potatoes. There were25crosses yielded2515seeds. Among the12somatic hybrid parents,3C28-1had the highest crossing compatibility with the tetraploid parents (13as maternal parent and7as paternal parent) and18crosses yielded2258seeds. The results demonstrated that the inter-specific somatic hybrids could be compatible with cultivated potatoes and can be used in the breeding program.6. The flow cytometric analysis showed that, of the281backcrossing pedigrees of the somatic chybrids crossed with tetraploid cultivars,181were tetraploids,10were pentaploids and90were aneuploids (4x-5x). Among the17inbreeds of3C28-1there were2tetralploids,1aneuploid (4x-5x),8pentaploids,2aneuploids (5x-6x) and4hexaploids. The results implied that diverse gametes are formed in the somatic hybrids and most of them are compatible with the gametes of tetraploid parents.7. The backcrossing pedigrees of3C28-1with the tetraploid parents possessed plant height taller than either of the parent, indicating a strong heterosis of the hybrids. The stem number of the backcrossing hybrids was less than the cultivated parent but close to the somatic hybrid parent, showing an inheritance of stem number towarding to the high parent. The leaf index of the hybrids were the average of the parents indicating a genetic trend of mid-parents, whereas stolen length, number of tubers produced per plant, tuber weight per plant and mean tuber weight varied remarkably indicating a possible multigene-control of the traits.8. Eleven SSR primers were chosen to analyze the genetic components of96backcrossing pedigrees between3C28-1and the tetraploid cultivare E1and the40inbreeds of3C28-1. The11primers amplified33unique alleles, of which C9701had13specific bands,3had4unique bands, E1had7specific bands, and3and El had9common bands. The results showed that all the33specific alleles detected in the three parental materials appeared in the backcrossing pedigrees with some variations among individuals, proving that it is inheritable of the genetic components of the somatic hybrids and a diverse segregation could occur during the sexual crossing. In40inbreeds of3C28-1, the unique alleles of the fusion parents were71.7%and59.0%for C9701and3#respectively, while some novel bands were also detected in the inbreeds. The findings reinforced the gamete complexity of the somatic hybrids and proved the events of chromosomal exchange or site-specific recombination during the meiosis.更多还原