Characterization Of LTR Retrotransposons And Development Of IRAP Marker In Radish (Raphanus Sativus L.)

Radish(Raphanus sativus L.) belongs to the Brassicaceae family and is an important worldwide vegetable crop with high nutrition and medical value. It originates from China and has rich germplasm resources. Retrotransposons(RTNs) are the most abundant mobile genetic elements in plant genome that accomplish transposition via an RNA intermediate and can be divided into LTRs (long terminal repeat retrotransposons) and non-LTRs, in which LTRs are more studied. The LTR retrotransposons segregate phylogenetically into the Ty1-copia family and the Ty3-gypsy family according to the order of their catalytic enzymes. Many works have been reported in a variety of plants and it is accepted that retrotransposons have played an important role in the evolution of plant genome because of their replicative mode of transposition. So far, few works have been reported about the retrotransposons in radish. Retrotransposons are widely distributed in the plant genome with high copy numbers and heterogeneity, which make them very suitable for the development of molecular markers. Recently, various marker systems based on RTNs are widespread exploitation which have become powerful and useful tools for plant germplasm genetic diversity analysis in some field crops. RTNs-based markers have some manifest advantages such as the wide coverage, large informative and high polymorphism when compared to conventional DNA molecular markers. Isolation and identification of LTR retrotransposons in radish will provide the theoretical basis for the study of genome evolution and the development of RTNs-based markers can provide a new technique for the genetic breeding and improvement in radish.In this study, the domains of reverse transcriptase (RT) of Ty1-copia and Ty3-gypsy retrotransposons are amplified with degenerate primers using PCR technology in radish genome. We analyze the heterogeneity and phylogenetic within and between the sequences in radish and other plants. According to the conserved elements of 32 sequences of reverse transcriptase of Ty1-copia groups,33 primers are designed and further employed for genetic diversity analysis of 30 radish genotypes. The main factors of IRAP markers are preliminary studied for the developing of the system, a manual cultivar identification diagram (MCID) is made to discriminate the 30 radish genotypes with the polymorphic IRAP PCR bands generated from the primers. The main findings are as follows:1. Degenerate primers were used specific for the reverse transcriptase domains of Tyl-copia groups of retrotransposons with two radish genotypes (’NAU-YH’ and ’PI381011’).32 independent colonies for Tyl-copia were selected after cloning the radish genomic DNA bands using PCR technology. These sequences were AT-rich and highly heterogeneous in both the length and composition of the base. The length of Tyl-copia RT ranged from 258-267bp and homology between the sequences ranged from 17.4% to 97.7%. When translated into amino acids and analyzed for the presence of stop codons and frameshifts in their coding regions,13(40.63%) Tyl-copia clones contained stop codons or frameshift mutations. Phylogenetic trees were constructed between the sequences of LTRs from radish and other species from the GeneBank database showed that Tyl-copia retrotransposons in radish had a high homology with other species. Tyl-copia retrotransposons from radish appeared to be the same origin with Brassica rapa, Brassica juncea, Cucumis sativus and Solanum lycopersicum.1. Degenerate primers were used for the reverse transcriptase domains of Ty3-gypsy groups of retrotransposons with two radish genotypes (’NAU-YH’ and ’PI381011’) and 20 independent colonies for Ty3-gypsy were selected. These sequences were AT-rich and highly heterogeneous in both the length and composition of the base. The length of Ty3-gypsy RT ranged from 360 to 432 bp and homology between the sequences ranged from 11.6% to 99.2%. When translated into amino acids and analyzed for the presence of stop codons and frameshifts in their coding regions,3(15%) Ty3-gypsy clones contained stop codons or frameshift mutations. The 20 amino acid sequences could be divided into five families based on the genetic distance. Phylogenetic trees were constructed between the sequences from radish and other species from the GeneBank database showed that the Ty3-gypsy family in radish seemed to have a strong relation with Arabidopsis thaliana, Ginkgo biloba and Lolium multiflorum.3. Based on the domains of Tyl-copia-like retrotransposon reverse transcriptase of radish,45 IRAP primers were designed and validated in two radish genotypes.33 primers were selected and further employed for genetic diversity analysis of 30 radish genotypes. Several important reaction factors of IRAP were studied in order to establish and optimize IRAP system in radish. The IRAP-PCR was performed in a 20 μl reaction mixture containing 20 ng DNA,1×PCR buffer,0.4 μmol·L-1 primer,0.25 mmol·L--1 dNTPs,2.0 mmol·L-1Mg2+ and 1 U Tag DNA polymerase. With the development of IRAP markers in 30 radish genotypes, a total of 212 polymorphic bands were produced, with an average of 6.42. Dendrograms constructed by UPGMA method showed that these genotypes could be clustered into five groups, in which 20 radish accessions were clustered into one class. A manual cultivar identification diagram (MCID) was generated and used to discriminate these 30 radish genotypes using seven polymorphic IRAP primers. The results in this study indicated that IRAP was an efficient marker technology and could be utilized as a genetic tool for providing valuable information in radish genome analysis.