SNP molecular marker development based on sugarcane transcriptome sequencing

【Objective】To develop SNP molecular marker of sugarcane(Saccharum officinarum spp.)based on transcriptome sequencing,so as to establish an efficient and low-cost molecular marker development method.【Method】Forty sugarcane varieties were taken as experiment materials. Transcriptomes of these materials were sequenced by high throughput sequencing platform,and they were mapped to reference genomes after quality control. GATK software was used to identify and filter SNP molecular marker,and SNP annotation and statistical analysis were performed using snpEff.Phylogenetic analysis,principal component analysis and population structure analysis were conducted with the SNP markers. Finally,KASP marker was developed and its validity was verified.【Result】After quality control of transcriptome data,6.5 Gb clean reads per sample were generated on average. Through variation analysis and multiple filter screening,a final set of 220397 biallelic SNP on chromosome were identified,with an average density of 3 SNP/kb. SNP type and distribution characteristics analysis showed that the overall missense/silent(N/S)ratio was 1.05,transition/transversion(Ts/Tv)ratio was 1.89,heterozygous SNP was 38.66%-49.91% and 44.74% of them distributed in transcripts. Phylogenetic,principal component analysis and population structure analysis showed that there were great differences in sugarcane genome among individuals,nevertheless its genetic background was narrow. 11176 KASP primers were designed,and 25 of them were used to test the polymorphism and authenticity of identified SNP,23(92%)primers were able to amplify the target products,and 7(28%)were polymorphic in 40 sugarcane materials,which verified the validity of these markers for sugarcane diversity analysis.【Conclusion】Compared with conventional SSR molecular marker or SNP marker development method based on genotyping by sequencing(GBS),sugarcane SNP identified from transcriptome can generate a larger proportion of effective SNPs with good quality and quantity. Therefore,it is better to identify functional SNP sites,providing a new way for developing SNP markers of sugarcane.