远缘物种基因组DNA导入水稻的研究
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摘要
为揭示转移远缘物种基因组DNA创新植物种质的规律，在分别导入属于第2、第3和第4资源库远缘物种基因组DNA获得变异系的基础上，对变异系和它们的受体水稻的表型性状进行了比较研究，采用AFLP及SSR等分子标记技术对转移远缘物种基因组DNA创制的变异系与其供受体的遗传多态性进行了定性、定量研究，在RAPD分析基础上对只在变异系和供体存在的特异DNA片段进行了核苷酸序列的比较研究。
将小粒野生稻(O.minuta)、稗草(Echinochloa crusgalli)及钝顶螺旋藻(Spirulina platensis)等远缘物种的基因组DNA分别导入保持系V20B、恢复系先恢207、蜀恢527及盐恢559等3系杂交水稻亲本，D_1均获得变异株；从它们的后代分别选育出新的保持系野威B、恢复系RB207及恢复系株系RS527、RS559等。在植株形态、产量、品质、抗性或(和)耐逆性等性状上，野威B、RB207、RS527、RS559与各自受体相比，有较大改良。将高粱基因组DNA导入保持系IR58025B，在第1代(D_1)获得的1变异株香粱5，从D_2起在形态上一直不出现分离现象。
AFLP分析表明：导入野生稻或稗草DNA获得的变异系，与各自受体均存在4.4％的遗传多态性，并含有供体特异分子标记。另外，AFLP分析发现突变存在“热点”现象。
SSR分析表明：变异系野威B、香粱5均与受体存在遗传多态性，并含有供体特异的分子标记带型；而香粱5的5个第2代单株的SSR扩增产物间没有遗传多态性，并与第10代的一致，从分子水平证明，在外源DNA导入过程确实存在变异株一经获得即不分离的现象。
RAPD分析及DNA序列分析表明：变异系野威B与受体V20B存在遗传多态性；变异系和供体具有而受体中没有的1对RAPD特异带DNA片段(~VOPG-11_(975)与~DOPG-11_(975))均含有975个碱基对，彼此间的相似性高达97％；在水稻基因组数据库中没有发现与它们同源的DNA序列。
变异系与供受体的表型及DNA多态性比较研究结果说明，导入远缘物种基因组DNA可创造优异水稻新种质。DNA多态性研究结果进一步证明了远缘物种DNA可向栽培稻的转移；同时发现，是远缘物种基因组和受体基因组2者的编码序列间、非编码序列间的互作，使受体发生变异：通过不同分子标记技术对不同变异系和各自受体的研究还发现，变异系与受体基因组DNA间存在5％左右的多态性。后者可能揭示了远缘物种基因组DNA引起受体变异的量的一般规律。
This study aims at learning the principle of creating new germplasm in plant breeding by using the approach of transformation of the genomic DNA of distant resources.
Useful germplasm for hybrid rice breeding has been developed from variants derived from transformation of genomic DNAs of distant species: new CMS line Yewei A and its maintainer line Yewei B, developed'from variant of V20B (rice maintainer line) through transformation of genomic DNAs of 0. minuta; variant also selected from another maintainer line, IR58205B, after introducing the genomic DNA of sorghum, and no morphological segregation has been found among different offspring plants derived from the variant from the D2 on; new restorer line RB207 developed from variant of R207 (rice restorer line) through transformation of the genomic DNA of Echinochloa crusgalli; and new restorer line RS527 , RS559 developed from variant of R527, R559 separately through transformation of genomic DNA of Spirulina platensis. Great improvements in traits of yield, grain quality and resistance to diseases can be found in variant lines compared with their receptors.
After research of polymorphism of DNA between variants and its receptors with methods of SSR, AFLP, RAPD, and DNA sequence, a few facts have been revealed for the first time:
The results of AFLP analysis show much polymorphism of DNA between variants and its receptors, and indicate special DNA segments from Echinochloa crusgalli and 0. minuta may be integrated into the genome of rice, and also indicate some loci in the receptor genome are tend to be mutable.
The results of SSR analysis show much polymorphism of repetitive DNA sequence between variants and its receptors, indicating some special simple repeat DNA segments from distant relatives integrated into the genome of rice probably, and prove variants from DNA transformation can be stable in the second generation.
RAPD and DNA sequence analysis reveals DNA polymorphism exists between Yewei B and 0. minuta, and find a pair of DNA fragments in Yewei B and 0. minuta (not in the receptor V20B), VOPG-11975 and DOPG-11975, both with 975 base pairs (bp), are homologous, while no significant homologous DNA sequence with v0PG-11975 or DOPG-11975 has been found in all data libraries of rice genome.
All results indicate that transformation of genomic DNAs of distant species is an effective method for creating new rice germplasm. The results of SSR, AFLP, RAPD and DNA sequences analysis of variant lines, receptors and donors of exogenous DNA indicate that special DNA fragments of distant relatives can be integrated into the rice genome; and demonstrate it is the interaction of repetitive DNA sequences and genes between receptor and donor together that decides the mutation in traits of receptor in genomic DNA introduction; and hint about 5% polymorphism of DNA between variant lines and its receptors may be a general rule in genomic DNA transformation.
将小粒野生稻(O.minuta)、稗草(Echinochloa crusgalli)及钝顶螺旋藻(Spirulina platensis)等远缘物种的基因组DNA分别导入保持系V20B、恢复系先恢207、蜀恢527及盐恢559等3系杂交水稻亲本，D_1均获得变异株；从它们的后代分别选育出新的保持系野威B、恢复系RB207及恢复系株系RS527、RS559等。在植株形态、产量、品质、抗性或(和)耐逆性等性状上，野威B、RB207、RS527、RS559与各自受体相比，有较大改良。将高粱基因组DNA导入保持系IR58025B，在第1代(D_1)获得的1变异株香粱5，从D_2起在形态上一直不出现分离现象。
AFLP分析表明：导入野生稻或稗草DNA获得的变异系，与各自受体均存在4.4％的遗传多态性，并含有供体特异分子标记。另外，AFLP分析发现突变存在“热点”现象。
SSR分析表明：变异系野威B、香粱5均与受体存在遗传多态性，并含有供体特异的分子标记带型；而香粱5的5个第2代单株的SSR扩增产物间没有遗传多态性，并与第10代的一致，从分子水平证明，在外源DNA导入过程确实存在变异株一经获得即不分离的现象。
RAPD分析及DNA序列分析表明：变异系野威B与受体V20B存在遗传多态性；变异系和供体具有而受体中没有的1对RAPD特异带DNA片段(~VOPG-11_(975)与~DOPG-11_(975))均含有975个碱基对，彼此间的相似性高达97％；在水稻基因组数据库中没有发现与它们同源的DNA序列。
变异系与供受体的表型及DNA多态性比较研究结果说明，导入远缘物种基因组DNA可创造优异水稻新种质。DNA多态性研究结果进一步证明了远缘物种DNA可向栽培稻的转移；同时发现，是远缘物种基因组和受体基因组2者的编码序列间、非编码序列间的互作，使受体发生变异：通过不同分子标记技术对不同变异系和各自受体的研究还发现，变异系与受体基因组DNA间存在5％左右的多态性。后者可能揭示了远缘物种基因组DNA引起受体变异的量的一般规律。
This study aims at learning the principle of creating new germplasm in plant breeding by using the approach of transformation of the genomic DNA of distant resources.
Useful germplasm for hybrid rice breeding has been developed from variants derived from transformation of genomic DNAs of distant species: new CMS line Yewei A and its maintainer line Yewei B, developed'from variant of V20B (rice maintainer line) through transformation of genomic DNAs of 0. minuta; variant also selected from another maintainer line, IR58205B, after introducing the genomic DNA of sorghum, and no morphological segregation has been found among different offspring plants derived from the variant from the D2 on; new restorer line RB207 developed from variant of R207 (rice restorer line) through transformation of the genomic DNA of Echinochloa crusgalli; and new restorer line RS527 , RS559 developed from variant of R527, R559 separately through transformation of genomic DNA of Spirulina platensis. Great improvements in traits of yield, grain quality and resistance to diseases can be found in variant lines compared with their receptors.
After research of polymorphism of DNA between variants and its receptors with methods of SSR, AFLP, RAPD, and DNA sequence, a few facts have been revealed for the first time:
The results of AFLP analysis show much polymorphism of DNA between variants and its receptors, and indicate special DNA segments from Echinochloa crusgalli and 0. minuta may be integrated into the genome of rice, and also indicate some loci in the receptor genome are tend to be mutable.
The results of SSR analysis show much polymorphism of repetitive DNA sequence between variants and its receptors, indicating some special simple repeat DNA segments from distant relatives integrated into the genome of rice probably, and prove variants from DNA transformation can be stable in the second generation.
RAPD and DNA sequence analysis reveals DNA polymorphism exists between Yewei B and 0. minuta, and find a pair of DNA fragments in Yewei B and 0. minuta (not in the receptor V20B), VOPG-11975 and DOPG-11975, both with 975 base pairs (bp), are homologous, while no significant homologous DNA sequence with v0PG-11975 or DOPG-11975 has been found in all data libraries of rice genome.
All results indicate that transformation of genomic DNAs of distant species is an effective method for creating new rice germplasm. The results of SSR, AFLP, RAPD and DNA sequences analysis of variant lines, receptors and donors of exogenous DNA indicate that special DNA fragments of distant relatives can be integrated into the rice genome; and demonstrate it is the interaction of repetitive DNA sequences and genes between receptor and donor together that decides the mutation in traits of receptor in genomic DNA introduction; and hint about 5% polymorphism of DNA between variant lines and its receptors may be a general rule in genomic DNA transformation.
引文
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