【作者】 邓祖湖；

【导师】 林彦铨；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2010， 博士

【摘要】 选育抗旱、抗病、宿根性强的高产高糖甘蔗品种是提高甘蔗生产水平的最有效措施,而种质创新是选育突破性品种的关键。甘蔗近缘属植物斑茅种质的开发利用是甘蔗种质创新研究的重心。由于甘蔗与斑茅杂交后代花粉育性低和杂交后代遗传机理不清,斑茅杂交利用进展缓慢。本研究通过对系谱来源清楚的斑茅杂种F1、BC1及其亲本的染色体核型分析,F1、BC1体细胞染色体基因组原位杂交(GISH)来揭示甘蔗斑茅远缘杂交染色体的遗传；以花粉育性差异的斑茅杂种BC1及其亲本为材料,提取不同育性材料总RNA,反转录成cDNA与Affymatrix公司已经开发的甘蔗基因芯片杂交,利用生物信息学理论,从涉及育性的众多基因中寻找相关基因。主要研究内容如下：(1)核型分析结果,2个海南斑茅无性系海南92-105和海南92-77的核型公式都为2n=60=60m,核型属1A型,而其余5个材料的核型均属2B型；热带种拔地拉核型公式为2n=80=70m+10sm,商业栽培品种CP84-1198为2n=120=114m+6sm(4SAT)；2个甘蔗与斑茅杂种F,崖城96-66和崖城96-40的核型公式都为2n=70=68m+2sm；来自同一组合的甘蔗与斑茅杂种BC1崖城01-21、崖城01-134和崖城01-36的染色体条数差异较多,核型公式分别为：2n=114=114m、2n=104=100m+4sm(4SAT)和2n=132=130m+2sm。(2) GISH技术分析结果表明2个甘蔗与斑茅杂种后代F1崖城96-66和崖城96-40染色体都是由30条来自斑茅的染色体和40条来自甘蔗(Badila)的染色体组成；甘蔗与斑茅BC1崖城01-134的染色体构成为85条染色体来自甘蔗,29条来自斑茅；崖城01-36的染色体构成为96条染色体来自甘蔗,36条来自斑茅。甘蔗与斑茅杂种染色体以多于亲本的条数传递是首次发现。4个甘蔗斑茅杂种有丝分裂中期染色体和间期染色质观察都存在分布不均匀的现象,来自不同属染色体有聚于染取色体某部位的趋势,但不是截然分开。甘蔗与斑茅BC1崖城01-134和崖城01-36分别有1条和2条染色体发生易位。(3)核型分析和GISH结果都表明,甘蔗与斑茅杂种F1染色体是按n+n的方式传递的,而甘蔗与斑茅杂种BC,遗传复杂,崖城01-134是按2n+n的方式遗传的,崖城01-36是按特殊的方式传递的,都发生了染色体遗传的“不平衡”现象。(4)2个可育和2个不可育材料杂交芯片表达谱之间4次比较,可育对不育表达上调达4次的基因有14个,3次的有32个,表达下调达4次的基因有12个,3次的有34个。共同上调或下调表达次数达3次以上的基因共有92个。应用相对定量PCR技术对3个共同差异基因进行了验证实验。结果表明相对定量PCR结果与芯片检测结果趋具有较好的一致性,基因芯片检测结果可靠。(5)对92上调和下调3次以上的基因,进行的BLAST比对分析,从中发现一些与育性有重要关系的基因发现与育性相关的基因SR,JLP,Prm3和Retrotransposon。它们在植物花发育中调控花器官以及花分生组织特性,在营养生长转向生殖生长的调控中发挥作用,与花粉发育、花药开裂过程、发育时期等相关。(6)对甘蔗与斑茅BC1后代崖城01-36染色体构成成因进行了探讨,认为其形成原因可能是崖城96-40为甘蔗种质中渗入大量远缘种质斑茅染色体的杂种,使其代谢紊乱,不但雄性不育,其作母本育性也是非常差,只有极少数异常减数分裂形成的染色体基因“完美组合”的配子或具2n及超过2n染色体的雌配子才能完成受精、胚胎发育和发芽及生长的过程,还有可能是2n+n合子中不同来源染色体分裂不同步,造成部分加倍。由于育性差,这种后代一出现就被发现了。更多还原

【Abstract】 It’s the most efficient measure to get a higher sugarcane production level by breeding drought-resistant, disease-resistant and strong ratooning varieties. Germplasm innovation is the key for breeding breakthrough variety. Development and utilization of Erianthus arundinaceum, a closely related genus of Saccharum is the the most important thing for the Germplasm innovation of sugarcane. The hybriding utilization of Erianthus arundinaceum make slow progress because of the infertility and unclearness of the genrtic of the chromosome. Chromosome karyotype analysis was carried out with the F1and BC1of the progenies of sugancane and Erianthus arundinaceus and their parents, GISH analysis was carried out with the F1and BC1, inorder to discover the genetic of Chromosome. Microarray of sugarcane was used to hybridize with the cDNA from the different fertility clones or parents of BC1crossed from E.arundinaceus and sugarcane. The fertility-related genes were screened from the large number genes based on the bioinformatics analysis and microarray. The results were showed as follows:(1) Chromosome karyotype analysis show that the somatic chromosome number of E. arundinaceus Hainan92-105and Hainan92-77was both2n=60=60sm, belonging to type1A. The other five tested clones belonged to type2B. The somatic chromosome number of S. officinarum Badila was2n=80=70m+10sm, commercial variety CP84-1198was2n=120=114m+6sm(4SAT), The F,s YC96-66and YC96-40that originated from Badila with E. Arundinaceus were both2n=70=68m+2sm; Clones derived from the same cross of YC96-40(female) and CP84-1198(male) had different chromosome number, YC01-21had2n=104=100m+4sm chromosomes, YC01-134had2n=114=114m, YC01-36had2n=132=130m+2sm chromosomes.(2) The reslut of GISH show that The chromosomes of F,s YC96-66and YC96-40that originated from Badila with E. Arundinaceus were both consist of30chromosomes from E. Arundinaceus and40from S. officinarum. The chromosomes of BC1YC01-134was consist of29chromosomes from E. Arundinaceus and85chromosomes from Saccharum.spp. YC01-36was consist of36chromosomes from E. Arundinaceus and96chromosomes from Saccharum.spp. It’s the first time to find that chromosome transmit by the way of more than the somatic chromosome number. The phenomenon of distribut of chromosome and chromatin was both find in the four progenies of Saccharum.spp and E. Arundinaceus. The chromosomes of E. Arundinaceus always gather togather, but not so serious. There are one and two translocation chromosome in YC01-134and YC01-36respectively.(3)The results of karyotype analysis and GISH indicated that the hybrids originated from Badila (S. officinarum) and E. arundinaceus followed the chromosome transmission pattern of n+n. and the chromosome transmission of BC1were more complex, YC01-134with the genetic pattern of2n+n and YC01-36with another special kind of genetic pattern. The phenomenon of "Disequilibrium hybridization" was found in BC1.(4)4kinds compare of microarray expression profile between2fertility clones and2sterility clones show that14genes expressed4times and32genes expressed3times as Up-regulated genes;12gene expressed4times and34genes expressed3times as Down-regulated genes. There are92genes expressed Up-regulated or Down-regulated more than3tims. Three differentially expressed ESTs with ratio more than2were selected randomly for analysis of quantitative real-time RT-PCR.The similar results from microarray and Q-PCR indicated the reliability and authenticity in the microarray analysis.(5) The92Up-regulated and down-regulated ESTs gene sequences were identified and blasted at NCBI according to gene ID. A number of fertility genes including SR (serine-arginine repressor protein), JLP (jasmonate-induced protein), Prm3and the Retrotransposon were screened from92genes. These genes control the flower organ building and the property of flower meristem, play their part in the fertility alteration。They play an important role in the pollen development, anther dehisence and development stages control.(6) Discussion was done to explain the chromosome composition of YC01-36, the reason may be attribute to that YC96-40is a hybrid with many E. arundinaceus chromosomes in S. officinarum cell, this cause the disturbance of metabolism, male sterile, poor fertility as femail also. Only very few female gamete with perfact chromosome combination or have more than2n chromosomes number caused by abnormal meiosis can put through the process of fertilization, embryonic development, germinate and growh, another reason may be causes by the the out-sync in the zygot with2n+n, so the chromosomes double partly. And these progeniese were easy to be find for few population in one cross.更多还原