Cheng Yun-Jiang, Guo Wen-Wu, Deng Xiu-Xin
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
Yi Chuan Xue Bao. 2002 Apr;29(4):364-9.
Cleaved Amplified Polymorphic Sequence (CAPS) was successfully applied to analyze the organelle composition of three eight-year-old trees of the somatic hybrid between Cleopatra mandarin (Citrus reticulata) and Flying Dragon (Poncirus trifoliata). Five chloroplast and five mitochondrial universal primer pairs were used. All chloroplast primer pairs (rbcL-rbcL, rbcL-PSA I, TrnH-Trnk, TrnD-TrnT, TrnK-TrnK) and three (nad 1 exon B-nad 1 exon C, 18S rRNA-5S rRNA, nad 4 exon 1-nad 4 exon 2) of the five mitochondrial primer pairs, were efficiently amplified, but no polymorphism was detected, when the PCR products were digested by eleven restriction endonucleases, including, Hin6 I, Bus RI, Taq I, Msp I, HinfI, AluI, Dra I, EcoR I, Hind III, BamH I and Pst I respectively, three polymorphic cpDNA-CAPS markers (rbcL-rbcL/Hin 6 I, TrnD-TrnT/BusR I, TrnD-TrnT/Taq I) and one mtDNA-CAPS marker (nad 1-nad1/Msp I) were found. The results showed that cpDNA in the somatic hybrid plants came from Flying Dragon, the mesophyll parent, and mtDNA from Cleopatra mandarin, the embryogenic suspension parent uniformly. In order to prove the reliability of CAPS results, and to get more detailed information about the mtDNA inheritance, RFLP analyses was conducted. Genomic DNA of the somatic hybrids and their corresponding parents were digested by five restriction endonucleases (Dra I, EcoR I, Hind III, BamH I and Pst I), and hybridized with five mitochondrial probes (Cob, Pro 2, Pro I, atp 6, 26S rRNA) as well as one chloroplast probe, i.e. the PCR product of Flying Dragon with the primer pair of trnd 1-trnt 1. The results were in line with those of CAPS, and no novel bands were detected, which indicated that no organelle DNA recombination or rearrangement have been detected in the hybrid plants. The research showed that novel pattern of nuclear-mitochondria-chloroplast interaction could be reached via protoplast fusion.
切割扩增多态性序列(CAPS)已成功应用于分析克里曼丁橘(Citrus reticulata)与飞龙枳(Poncirus trifoliata)体细胞杂种的三株8年生树木的细胞器组成。使用了5对叶绿体通用引物和5对线粒体通用引物。所有叶绿体引物对(rbcL-rbcL、rbcL-PSA I、TrnH-Trnk、TrnD-TrnT、TrnK-TrnK)以及5对线粒体引物对中的3对(nad 1外显子B-nad 1外显子C、18S rRNA-5S rRNA、nad 4外显子1-nad 4外显子2)均能有效扩增,但当PCR产物分别用11种限制性内切酶(包括Hin6 I、Bus RI、Taq I、Msp I、HinfI、AluI、Dra I、EcoR I、Hind III、BamH I和Pst I)消化时,未检测到多态性,发现了3个多态性cpDNA-CAPS标记(rbcL-rbcL/Hin 6 I、TrnD-TrnT/BusR I、TrnD-TrnT/Taq I)和1个mtDNA-CAPS标记(nad 1-nad1/Msp I)。结果表明,体细胞杂种植物中的cpDNA均来自叶肉亲本飞龙枳,mtDNA均来自胚性悬浮亲本克里曼丁橘。为了证明CAPS结果的可靠性,并获取有关mtDNA遗传的更详细信息,进行了RFLP分析。体细胞杂种及其相应亲本的基因组DNA用5种限制性内切酶(Dra I、EcoR I、Hind III、BamH I和Pst I)消化,并用5种线粒体探针(Cob、Pro 2、Pro I、atp 6、26S rRNA)以及1种叶绿体探针(即飞龙枳trnd 1-trnt 1引物对的PCR产物)进行杂交。结果与CAPS结果一致,未检测到新条带,这表明在杂种植物中未检测到细胞器DNA重组或重排。该研究表明,通过原生质体融合可以实现核-线粒体-叶绿体相互作用的新模式。