Cloning And Localization Of Sex Difference Repetitive Sequences From Asparagus Officinalis Based On Genomic Subtractive Hybridization

Asparagus officinalis is an important perennial dioecious herb,its haploid genome size of 1323 Mbp,having 2n=2X=20 chromosomes.Its composition is the same type of XY sex chromosomes,asparagus sex chromosomes is still in the early stages of the evolution,it can be more conducive to study the origin and evolution of plant chromosomal mechanisms.The present study showed the plant XY sex chromosomes originated a pair of autosomes,one of which for some unknown reason accumulated retrotransposons and other repetitive sequences and gradually expanded in size,and then gradually evolved from a form of profiled XY sex chromosomes.However,the cumulative repeats if it is a common phenomenon in sex chromosomes volume expanded,the sex chromosomes is involved in the early evolution of other issues is still unclear.Therefore,this paper asparagus as materials,genomic subtractive hybridization and bioinformatics analysis of isolated genomic differences between male and female repeat,metaphase chromosomes using fluorescence in situ hybridization to obtain gender differences in the sequence by chromosome location,analysis of the possible role of repeats in the origin and evolution of sex chromosomes in the process.It provides theoretical data to reveal the origin and evolution mechanism of sex chromosomes asparagus.The main findings are as follows:1.Asparagus with male and female genomic DNA as material,the use of universal primers M13 for asparagus 6600 product subtractive hybridization clones were screened,get 98 male and female differences in sequence after three verification by dot hybridization.Sequencing successful 52 Sequence analysis showed that the length of obtained sequence distribution of 63 bp-297 bp.Among them,33 are non-chloroplast genomic sequence(named Ao1-Ao33),comprising three retrotransposons,15 unnamed sequences,6 mRNA sequence,and 9 other sequences.In addition,19 sequences belong to the chloroplast source sequence(named Ao34-Ao52),Ao34-Ao50 in nucleic acid databases are homologous sequence,similarity in the distribution of 84%-100%,Ao51 lowest similarity was 37%.Ao52 sequence homologous sequences not found in the database,the sequence similarity with asparagus chloroplast genome is 100%.To further verify whether 52 sequences are a sequence of gender differences,the use of genomic semi-quantitative technologies for 52 dot blot verify differences sequence analysis.The results show that 9 differences sequence between male and female differences in sequence,comprising five sources of non-chloroplast sequence(Respectively Ao6,Ao13,Ao15,Ao19,Ao29).4 chloroplast genomic sequences(Respectively Ao34,Ao36,Ao43,Ao51),Targeting sequences found in nuclear integrants of plastid DNA(NUPTs)mainly in the chloroplast genome of inverted repeat region(IR)(containing IRa and IRb area)(respectively Ao34,Ao36,Ao43).It shows chloroplast inverted repeat sequences more easily transferred to the nuclear genome,the sequence in the region may be related to high copy number.2.In order for the gender differences sequence obtained was chromosomal location,the five difference between the non-chloroplast origin sequence were long fragment was amplified from the genome.which,Ao6 and Ao15 two sequences and length are successful long sequence of 3048 bp and 528bp(named Ao6-L and Ao15-L).Metaphase chromosome fluorescence in situ hybridization showed that,Ao6-L sequence hybrid signal sequences in all chromosomes with the centromere and the centromere on the signal strong and clear.Ao15-L sequence is hybridization signals on all chromosomes,and the signal is concentrated in and near the centromere,the stronger the closer the distance centromere signal.It follows that the position of the centromere enrichment largest differences repeats.Asparagus contains a lot of centromeric repeats,only to hybridization signals in the centromeric and stacked nearby.3.In order to obtain sequences of chloroplast-derived chromosome location,first long sequence amplification according asparagus chloroplast genome sequences of primers were designed,the results show,Ao52 sequences were amplified length of 2859bp(named Ao52-L).Metaphase chromosome localization found Ao52-L sequence are hybridization signals on all chromosomes,and the signal from telomere to centromere showed a gradual strengthening.It is characterized by the sequence described NUPTs enrichment up to centromere position,and the distribution of rice metaphase chromosomes NUPTs consistent.Secondly,more repeat sequence is present in the centromere,and further the shift from the chloroplast to the nuclear genome sequence is composed of one of the important components of centromeric flanking repeat sequences.Asparagus chloroplast sequence is inserted in the nuclear genome,deletion,and rearrangement is an important evolution in the production of new nuclear genes.