Development And Application Of Retrotransposons-based Molecular Markers In Avena L.

The genus Avena L.in the tribe Aveneae,Poaceae,consists of 30 species,including four types of genome(A,B,C and D),three types of varieties(diploid,tetraploid and hexaploid),and five genome constitution(AA,CC,AABB,AACC and AACCDD).Retrotransposons,a class of mobile elements in eukaryotes genome,are widely spread in plant genome.They are an important driving force of plant evolution showing high copy and high heterogeneity.The molecular markers developed from retrotransposons,which have many advantages,such as high sensitivity,wide coverage and high polymorphism,have been applied in different studies in many species,however,limited research has been done in Avena L.In this study,we identified the reverse transcriptase of retrotransposons and developed molecular markers based on retrotransposon.The molecular markers were used to study the genetic diversity of hexaploid oat and the genome evolutionary of diploid,tetraploid and hexaploid oats,which provided a theoretical basis for genetic improvement and genome evolution of oat.The main results are as follows:1.The degenerate oligonucleotide primers were designed based on the conserved domains of the Ty1-copia retrotransposon reverse transcriptase.The 23 Ty1-copia retrotransposons sequences were amplified from the naked oat ‘Pinyan 1' by PCR.The characterization,phylogenetic relationship and transcriptional activities of these retrotransposons were carried out.The results showed that the retrotransposons sequences had high heterogeneity,with 45% to 98% identity between sequences.The DNA sequences displayed mutations,including insertions,frameshifts,and stop codons,but the frequencies were not high.Phylogenetic analysis showed that the Ty1-copia retrotransposons of oats were mainly transmitted vertically during evolution.Five Ty1-copia retrotransposons with transcriptional activity were found by searching the oat gene expression database.2.Using the i PBS primers,the LTR sequences of five retrotransposons were isolated from diploid oats by PCR.Two types of IRAP and REMAP markers were developed based on one LTR sequence and one LTR of the OARE-1 retrotransposon reported in the hexaploid oats.Finally,we established stable technical system for these two molecular markers in oats through optimization of experimental conditions.3.The genetic diversity of 92 samples of hexaploid oats was analyzed by IRAP and REMAP markers.It was found that the diversity of husked oats was higher than that of naked oats;the cultivated species was higher than that of wild oats;among the seven groups divided by geographical location,Africa was the highest and West Asia the lowest.In terms of groups genetic relationship,the European and North American groups are the closest,while the African groups are the farthest from the other six groups.Cluster analysis of 92 materials showed that the geographical origin was not obvious,but the varieties from the same breeding unit and the related materials were in one branch.In addition,the division between wild species and cultivated species was not obvious.4.When analyzing the genomic genetic relationship of tetraploid oats using IRAP and REMAP marker,it was found that A.agadiriana was far from A.barbata,A.abyssinica and A.vaviloviana oats with AB genome,but close to A.murphyana and A.maroccana with AC genome.This result indicated that it was not appropriate to use the AB genome to represent A.agadiriana.This result also confirmed that the northern part of Africa was the origin of allogeneic tetraploid and hexaploid.5.When analyzing the genetic relationship of the diploid genome,the results from the two molecular markers were inconsistent in the classification of the As genome.The IRAP marker divided the As genome into a branch with A.longiglumis of the Al genome,but REMAP marker divided the As genome into species.Our result suggestde the reliability of the previous research about the division of As genome.This study provided a new marker system for the analysis of genetic diversity and genetic evolution of oat genome.The results provided a reference information for the collection of oat resources,the selection of breeding materials and the evolution of oat species.