Development Of InDel And SV Markers For Citrus Suavissima 'Wuzi Ougan'

‘Wuzi Ougan',a bud variation of Ougan(Citrus suavissima),has all of the same excellent characters as Ougan.The fruits are extremely resistant to storage,and the flavor remains the same after storing up to the next may since its mature period under the room temperature.Seedless fruits now occupy an important position in the domestic and international market,specially the parthenocarpy.It is meaningful to study the seedless mechanism in order to provide theoretical guiding for the development of seedless citrus species.In this study,genome-wide In Del and SV variations were detected in the leaf of ‘Wuzi Ougan' and its wild-type,and developed molecular marker based on PCR method.These markers were applied to the location of meiosis-related genes,and the main results were as follows(1)A total of 2,729,553 SNPs and 276,205 InDel were obtained between ‘Wuzi Ougan' and its wild-type compared with the reference genome by genome-wide sequencing analysis,with an average of 18,896 SV variants per individual.2,615,477 and 2,573,968 SNP loci were obtained in Contig with and without Osmanthus caryophyllus,and the SNP loci were 1,705,820 and 1,680,199 in total,And65.22% and 65.28%,respectively.The transversal SNP loci were 909,657 and 893,769,accounting for34.78% and 34.72% of the total.(2)According to the data of the whole genome re-sequencing,we analyzed the distribution of InDel and screened the InDel mutations.Through PCR amplification and polyacrylamide gel electrophoresis to validate the InDel,we found 99 InDel sites,84 pairs of PCR primers were designed.In the annotation of InDel,gene MYB063,NAC DOMAIN,SMC,XRI1 and AGO5 were found related to meiosis.(3)The genome re-sequencing analysis showed that 19,283 and 18,509 SVs were detected in ‘Wuzi Ougan' and its wild type,respectively.There were 1,587 and 1,853 insertion respectively,accounting for 8.23% and 10.01 %;11705 and 10861 deletion respectively,accounting for 60.7% and 58.68%;328and 346 inversion respectively,accounting for 1.7% and 1.87%;1,307 and 1,275intra-chromosomal translocation,accounting for 6.78% and 6.89%;4,356 and 4,174 inter-chromosomal translation,accounting for 22.59% and 22.55% respectively.The primers were designed on 9 chromosomes with20 bp forward and reverse primers designed based on the sequence of 300 bp upstream and 300 bpdownstream of the SV mutation site.A total of 14 pairs of SV primer can effectively distinguish between ‘Wuzi Ougan' and its wild type.