Construction Of Sorghum 【Sorghum Bicolor (L.)】 Methylation Genetic Linkage Map With MSAP And SSR Markers

Sorghum [Sorghum bicolor (L.) Moench] is one of important crops in the semi-arid regions of the world that receives growing attentions in genetic research due to its adaptation to harsh environments, diverse germplasm collection, and relatively small genome size (750Mb). DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes, it had been related in gene regulation, transgene silencing and genomic imprinting, as well as in the control of parasitic DNA elements in plants. The role of DNA methylation in life process has recently become a focus of biological sciences. The techniques for measuring DNA methylation have emerged with the requirement. Methylation sensitive amplified polymorphism (MSAP) analysis is based on the use of isoschizomers for detection of DNA methylation. It is an adaptation of the amplified fragment length polymorphism (AFLP) technique, in which the isoschizomers HpaⅡand MspⅠare employed as'frequent-cutter' enzymes instead of the usual Mse I.Using Map maker/EXP (version 3.0), a methylation genetic linkage map of sorghum was constructed using an F2 population derived from a cross of B2V4×1383-2 based on methylation-sensitive amplified polymorphism (MSAP). The map included 7 linkage groups with 57 markers from EcoRⅠ/MspⅠenzyme digestions, covering 227.8 cM. The average distances between adjacent two individual markers and length of linkage groups were 3.9 cM and 32.5 cM, respectively; the other map included 7 linkage groups constructed with 64 marker from EcoRⅠ/HpaⅡenzyme digestions, covering 332.2 cM, the average distances between adjacent two individual markers and length of linkage groups were 5.1 cM and 41 cM, respectively. A comprehensive map including 10 linkage groups constructed with 102 marker from EcoRⅠ/MspⅠand EcoRⅠ/HpaⅡenzyme digestions covering 401.4 cM. The average distances between adjacent two individual markers and length of linkage groups were 3.9 cM and 40.1 cM, respectively.Using Map maker/EXP (version 3.0), a comprehensive methylation genetic linkage map of sorghum was constructed with above MSAP markers and certain reported SSR markers using an F2 population derived from a cross of B2V4×1383-2. The map including 11 linkage groups was constructed with 151 markers covering 483.6 cM. Among 122 MSAP markers,89 were produced from EcoRⅠ/MspⅠenzyme digestions, and 33 were produced from EcoRⅠ/HpaⅡenzyme digestions. The average and maximal distances between adjacent two individual markers were 3.2 cM and 22.7 cM, respectively. Twenty-nine previously mapped SSR markers in sorghum were used as anchors, most of the SSR markers in this map ordered collinearly to the published sorghum genetic map, and the distances of common markers intervals similarly. The mapping results also showed that no methylation marker was found on the chromosome SBI-08; methylation markers from both EcoRⅠ/MspⅠand EcoRⅠ/HpaⅡenzyme digestions were detected on SBI-02-a and SBI-05; methylation markers from only EcoRⅠ/HpaⅡenzyme digestion were detected on SBI-02-b; and methylation markers from only EcoRⅠ/MspⅠenzyme digestions were detected on the other 7 chromosomes. High-density methylation regions were revealed near SSR markers Xtxp 296 on SBI-02-a, Xtxp 69 on SBI-03 and Xtxp 160 on SBI-07.Based on polymorphism of methylation fragments between parents and their segregation among the F2 population, cytosine methylation patterns between hybrid and their parents were divided into two major groups:class "A" refers to loci with the CCGG sites that were detected in one of its parents and hybrid F1. These loci indicated demethylation in hybrid F1 corresponding to its parents. Class "B" refers to loci with CCGG sites that were present in one of its parents but not in hybrid F1, which indicated an increased level of methylation in the hybrid. The results indicated that extensive cytosine methylation alterations including hyper-and demethylation as well as the potential conversion of methylation types (from external cytosine to internal cytosine or vice versa) occurred in the hybrid compared with its parents.Importantly, MSAP method was first used in the construction of a genetic linkage map. MSAP can be used efficiently to detect DNA methylation polymorphisms, and it is also an efficient technique in constructing genetic linkage map. The methylation linkage groups of sorghum could represent cytosine methylation patterns and levels. By studying the methylation pattern and level changes between parents and F1 hybrids in sorghum, we can analyze some specific sites located on methylation linkage groups, to further explore the genetic mechanism of heterosis. By studying gene expression in regional hot spots of organisms, we can further explore epigenetic phenomena of organisms and the mechanism of target gene silencing in genetic transformation. By analyzing the loci linked to drought tolerance, disease resistance, insect resistance and yield traits, etc, the identification and cloning of related genes is possible. The methylation map could provide new data for genomics researches in plants.