Development of molecular markers based on transcriptome sequencing for assisting the identification of southern soybean crinkle leaf disease

【Objective】Molecular markers based on transcriptome sequencing（RNA-Seq）for assisting the identification of southern soybean crinkle leaf disease（SSCLD）was developed to provide technical support for quickly identifying whether the soybean leaves encountered in production were SSCLD.【Method】Transcriptome sequencing technology was used to analyze differentially expressed genes between crinkle leaf and normal leaf soybean materials with similar genetic background in the crinkle leaf-induced soil environment. Homologous sequences with highly similar nucleotide sequences were removed，DEGs with large expression differences were selected for molecular marker development，the expression specificity of molecular markers was evaluated by using crinkle leaf soybean material and stress treatment test，and the expression specificity and reliability of molecular markers were verified by different types of crinkle leaf phenotype samples. 【Result】The sequencing base error rate of seven crinkle leaf plants and five normal leaf plants were 0.0226-0.0238，the numbers of bases in Q20 and Q30 were 99.00% and 99.90%，and the GC contents were 45.60% to 46.24%，indicating good quality of transcriptome sequencing data. Transcriptome data showed that 1063 genes were up-regulated and 85 genes were down-regulated in leaves from the crinkle leaf type material， compared to that from normal leaf type material. Based on the expression level and homology of the genes，eight genes were screened according to the expression level and the identification of the homology in soybean genome， and both of them were up-regulated genes. Real-time fluorescence quantitative PCR analysis of the eight candidate genes generally agreed with the transcriptome data.The expression levels of six of these genes showed varying degrees of interference by diverse abiotic stress， including dry stress， water logging stress， cold stress， shade and salt stress， and the expression level of GLYMA_18G033400 in crinkle leaves from the crinkle leaf-induced environment was higher than that from normal soil under dry stress， waterlogging stress， cold stress， shade and salt stress. Molecular marker qCL-18G033400 was designed according to the GLYMA_18 G033400 gene sequence，and used to perform real-time fluorescence quantitative PCR on 19 leaf samples of different crinkle leaf types. The results showed that ΔCt value less than 10.00 was used as the determination criterion of SSCLD，and the identification results were consistent with the field identification results.【Conclusion】Molecular marker qCL-18G033400 developed using transcriptome sequencing can assist in the identification of SSCLD.