Isolation And Cloning Of Single Chromosomes And R Gene Analogs In Fruit Trees

Fruit trees are important horticultural crops in the world. However, because of their special features such as large size, long juvenile period and high heterozygosity, and even self-incompatibility in some species, genetic studies in fruit species have been much more difficult and lagged behind in comparison with other crop species such as cereals and vegetables. In recent years, although molecular marker linkage maps have been constructed in a few important fruit species, they have not yet been efficiently used for mapping genes or quantitative trait loci (QTLs) controlling important agronomical characters.Chromosome microdissection and microcloning is a fast developing technology, which has proved very useful for the study of human, animal and plant genomes. The technology could also facilitate the genomics research in fruit trees. However, no application of the technology in woody fruit plants has been reported, largely due to the general small size of chromosomes and the difficulty of chromosome preparation.Amplification of homologous sequences based on the conserved domains of known genes is another useful technology for genomics research, which has been widely used for plant gene cloning, especially for the cloning of disease resistance (R) genes. An important advantage of the technology of homologous sequence amplification is that it needs little background knowledge about the species to be studied. Therefore, the technology could be particularly useful for gene cloning in fruit trees.In the present work, application of the technologies of chromosome microdissection and microcloning and homologous sequence amplification in fruit trees was studied. Using pomelo (Citrus grandis cv. guanxi) and ginkgo (Ginkgo biboba var.huana) as material, which have very small chromosomes (2n = 18) and relatively large chromosomes (2n = 24), respectively, an effective protocol for microisolation of single chromosomes was established, and single chromosomes isolated were successfully amplified by linker adaptor PCR (LA-PCR) or degenerate oligonucleotide primed PCR (DOP-PCR) and cloned into plasmid vectors. In addition, both single chromosomes of pomelo and their PCR products were successfully used as templates to amplify RGAs by degenerate oligonucleotideprimers based on conserved motifs in the NBS region of known resistance proteins. Details are presented below:1 Microisolation of single chromosomes in fruit trees1) High frequency metaphases of ginkgo were induced by pretreatment with ice water.2) Using young embryos of pomelo as materials, many well-scattered early metaphase and metaphase chromosomes were acquired.3) A highly effective system of glass needle preparation was established.4) Single chromosomes of pomelo and ginkgo were successfully micro-isolated by glass needles with the help of micromanipulation equipment. The result indicates that chromosomes in fruit trees, no matter large or small, can be isolated on the basis of high-quality chromosome slides and suitable glass needles.5) According to the standard karyotypes, the w and the 11th chromosomes of ginkgo and the 1st chromosome of pomelo were successfully identified and isolated. However, most of the chromosomes could not be identified under microscope. So, it is much more difficult to isolate specific chromosomes, especially the specific small chromosomes.2 Amplification and cloning of single-chromosome in fruit trees1) The isolated single pomelo chromosomes were amplified by LA-PCR and DOP-PCR. After two rounds of PCR amplification, single chromosome DNA pools with fragments ranging from 0.3~2 kb and 150~600bp respectively were acquired. The results showed that the DNA fragments in LA-PCR products were much larger than in DOP-PCR products. In addition, we found that it was easier to control contamination in LA-PCR than in DOP-PCR2) The isolated w chromosome and 11th chromosome of ginkgo were amplified by LA-PCR. The w and the 11th chromosome DNA pools with fragments ranging from 0.3~-'3 kb and 0.3-3.5kb respectively...