Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) plus Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids
Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) plus Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids
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DOI：
10.1007/s00299-002-0532-2
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发表时间：
2003-01-01
影响因子：
6.2
通讯作者：
Deng, XX
中科院分区：
文献类型：
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作者：
Cheng, YJ;Guo, WW;Deng, XX
对4个10年生瓦伦西亚橙子[Citrus sinensis（L.）采用酶切扩增多态性序列（CAPS）和限制性片段长度多态性（RFLPs）分析了两个品种的遗传多样性。利用5对叶绿体通用引物和3对线粒体通用引物进行扩增，未发现多态性。用15种限制性内切酶对PCR产物进行酶切分析，发现4个cpDNA-CAPS和2个mtDNA-CAPS多态性标记，表明体细胞杂种的cpDNA和mtDNA均来源于瓦伦西亚橙子（胚性悬浮亲本）。用5种限制性内切酶对体细胞杂种及其亲本的基因组DNA进行酶切，并与1个叶绿体探针（RbcL-RbcL）和9个线粒体探针（cox Ⅰ、cox Ⅱ、cox Ⅲ、cob、atpA、tyr、proI、atp 6和atp 9）进行杂交。结果表明，3个杂种植株的cpDNA与双亲共有一条强带，1个杂种植株在共有带的基础上又多了两条新带，而其mtDNA与瓦伦西亚橙子的mtDNA及非双亲带完全相同。当mtDNA带型的数据与田间表型表现的观察相结合时，发现更复杂的mtDNA带型与植物活力的增加相吻合。细胞器基因组的稳定性进行了研究，通过提取基因组DNA的一个杂交植物在每月的时间间隔为1年，然后分析它使用RFLP。在枝条枯死前，mtDNA有两个片段丢失，而cpDNAs保持稳定。流式细胞仪倍性分析表明，所有杂种均为稳定的四倍体。利用4对简单重复序列引物对4个杂种植株、双亲和1个植株的12个DNA样品进行了微卫星检测。结果表明，所有杂种的双亲带型一致，表明它们具有相同的核背景。这些结果表明，瓦伦西亚和欧橘体细胞杂种植株的线粒体DNA带型与表型异常有关，核质不亲和性可能是引起枯梢病的原因。
Organelle DNA inheritance of four 10-year-old somatic hybrid trees between Valencia orange [Citrus sinensis (L.) Osbeck] and Meiwa kumquat (Fortunella crassifolia Swingle) was analyzed by cleaved amplified polymorphic sequence (CAPS) and restriction fragment length polymorphisms (RFLPs). Five chloroplast (cp) and three mitochondrial (mt) universal primer pairs were amplified, but no polymorphisms were detected. When the polymerase chain reaction products were digested by 15 restriction enzymes, four polymorphic cpDNA-CAPS and two mtDNA-CAPS markers were found. Both the cpDNA and mtDNA in the somatic hybrids were derived from Valencia orange (the embryogenic suspension parent). Genomic DNA of the somatic hybrids and corresponding parents was digested by five restriction endonucleases and hybridized with one chloroplast probe (RbcL-RbcL) and nine mitochondrial probes (coxI, coxII, coxIII, cob, atpA, tyr, proI, atp6 and atp9). The results indicated that three hybrid plants shared one strong cpDNA band with both parents and that the remaining one plant had two additional novel bands besides the shared band, while their mtDNA was identical to that of Valencia orange plus non-parental bands. When data on the mtDNA banding patterns were combined with observations on phenotypic performance in the field, it was found that the more complex mtDNA banding pattern coincided with increased vigor of the plant. The stability of the organelle genomes was studied by extracting the genomic DNA of one hybrid plant at monthly intervals for 1 year and then analyzing it using RFLPs. Before the dieback of the shoots, two fragments of the mtDNA were lost while the cpDNAs remained stable. Ploidy analysis by flow cytometry showed that all of the hybrids were stable tetraploids. Four simple sequence repeat primer pairs were applied to detect microsatellite alleles of the four hybrid plants, both parents and the 12 DNA samples from one plant. The results showed that all hybrids had biparental bands uniformly, which indicated that they had the same nuclear background. These results suggest that the mtDNA pattern is correlated with the phenotypic abnormality of Valencia and kumquat somatic hybrid plants and that nuclear-cytoplasm incompatibility may be the cause of dieback.