Development Of Genic-SSR Markers By Transcriptome Sequencing In Juglans Regia L.and Application On Black Spot Disease Resistance Identification

Walnut(Juglans regia),also known as peca,is one of the most important “woody grain” ecological species.As the cultivated area expansion of walnut and continuous improvement of intensive culture,diseases and insect pests have became a serious problem of the walnut industry,especially walnut blight,caused by Xanthomonas campestris,declines the quality and yield of walnut.As a consequence,breeding to obtain resistant cultivars is the most efficient way to control walnut blight.Compared with conventional artificial inoculation,the method of molecular marker assisted screening(MAS)was highly sensitive and specific for finding target resistant germplasm.In this paper,we developed microsatellite markers for Juglans rejia based on transcriptome sequencing Illumina Hiseq TM 2000.In addition to this,1 pair of simple sequence repeat(SSR)primer was identified with polymorphism significantly associatied with walnut blight of Juglans rejia,and was used to verify the resistance of 37 Juglans rejia resources to walnut blight.This work studied the resistance identification of Juglans rejia resources by reliable and effective resistance identification method.The detailes is as follows:1.Disease resistance identification of Juglans rejia resourcesBy artificial inoculation and field trial,the disease index ranges of 12 text varieties(strains)were 1.11～55.44,which showed differences in resistance of walnut blight among different walnut resources.Moreover,Juglans sigllata Dode,Qingxiang,86,199,64 and 71 were resistant to black walnut;65,Liaohe-1,Chuanzao-1,Shujiang-1,Xiangling,Chuanzao-2 were susceptible to the disease.These cultivars can be used on follow-up testing.The results indicated that a number of walnut strains showed highlt resistance to black walnut,such as 199,86 and Juglans sigllata Dode,could be used as original materials for disease resistance breeding.2.Genomic DNA extraction methods optimizationWalnut is hard to extract DNA,for its rich in polyphenols and polysaccharides.In order to improve the quality of DNA extraction,we decreased the amount of the sample size,improved the method of grinding and increased reaction time.The results revealed that the optimization method of genomic dna extraction really worked.By this method,stripes on the diagram of the DNA agarose electrophoresis is complete,bright and clear without obvious trailing.3.Development of microsatellite markers for Juglans rejia based on transcriptome sequencing.Based on results of transcriptome sequencing,a total of 162310 unigenes were examined,and a number of 5097 sequences containing SSRs with 3.1% detection rates.A total of 34265 SSR loci were detected in the unigenes with the total length of 925 193 76 bp.Combined with several reference factors,such as NR description,NT description and differential expression analysis,unigenes associated with disease resistance were screened.Then,microsatellite markers for Juglans rejia were developed.The primers were designed from 63 SSR loci by using software Primer 6.0,and could be amplified steadily by agarose gel electrophoresis.4.SSR marker associated with walnut blight obtainedBased on the results of disease resistance identification of Juglans rejia resources,63 pairs of SSR primers were screened using genomic DNA of the resistant cultivar Juglans sigllata Dode and susceptible cultivar Xiangling,and 15 polymorphic SSR primers were identified.What’s more,those polymorphic SSR primers were further verified by 12 copies of genomic DNA of 6 resistant and 6 susceptible lines.Ultimately,1 pair of SSR primer was identified with polymorphic significantly associated with walnut blight resistance of Juglans rejia.5.SSR marker assisted screening application on black spot disease resistance identificationRJR-18 was associated with walnut blight of Juglans rejia,and was apllied on the black spot disease resistance identification by 25 varieties(strains).The results showed that16 walnut cultivars(strains)showed resistance to black walnut as well as 9 walnut cultivars(strains)susceptible to the disease.