An improved suppression subtractive hybridization technique to develop species-specific repetitive sequences from Erianthus arundinaceus (Saccharum complex)

Muqing Zhang,Rukai Chen,Xueting Li,Zuhu Deng,Fan Yu,Ling Luo,Jiayun Wu,Yongji Huang

doi:10.1186/s12870-018-1471-6

Abstract

BackgroundSugarcane has recently attracted increased attention for its potential as a source of bioethanol and methane. However, a narrow genetic base has limited germplasm enhancement of sugarcane. Erianthus arundinaceus is an important wild genetic resource that has many excellent traits for improving cultivated sugarcane via wide hybridization. Species-specific repetitive sequences are useful for identifying genome components and investigating chromosome inheritance in noblization between sugarcane and E. arundinaceus. Here, suppression subtractive hybridization (SSH) targeting E. arundinaceus-specific repetitive sequences was performed. The five critical components of the SSH reaction system, including enzyme digestion of genomic DNA (gDNA), adapters, digested gDNA concentrations, primer concentrations, and LA Taq polymerase concentrations, were improved using a stepwise optimization method to establish a SSH system suitable for obtaining E. arundinaceus-specific gDNA fragments.ResultsSpecificity of up to 85.42% was confirmed for the SSH method as measured by reverse dot blot (RDB) of an E. arundinaceus subtractive library. Furthermore, various repetitive sequences were obtained from the E. arundinaceus subtractive library via fluorescence in situ hybridization (FISH), including subtelomeric and centromeric regions. EaCEN2-166F/R and EaSUB1-127F/R primers were then designed as species-specific markers to accurately validate E. arundinaceus authenticity.ConclusionsThis is the first report that E. arundinaceus-specific repetitive sequences were obtained via an improved SSH method. These results suggested that this novel SSH system could facilitate screening of species-specific repetitive sequences for species identification and provide a basis for development of similar applications for other plant species.

Highlights

Sugarcane has recently attracted increased attention for its potential as a source of bioethanol and methane
The suppression subtractive hybridization (SSH) optimization system Restriction enzyme digestion of E. arundinaceus genomic DNA (gDNA) To determine whether E. arundinaceus gDNA was completely digested, the digested gDNA was analyzed by agarose gel
Results showed that the size distribution of either HaeIII or AluI-digested gDNA was longer than 2 kb, even though the digestion time was as long as 4 h (Fig. 1, Lanes 5 and 6)

Summary

Introduction

Sugarcane has recently attracted increased attention for its potential as a source of bioethanol and methane. Erianthus arundinaceus is an important wild genetic resource that has many excellent traits for improving cultivated sugarcane via wide hybridization. Species-specific repetitive sequences are useful for identifying genome components and investigating chromosome inheritance in noblization between sugarcane and E. arundinaceus. S. spontaneum is widely recognized as the most primitive species within the genus Saccharum, whereas S. robustum has been postulated to be the progenitor of the high sugar content species, S. officinarum [9]. An increasing amount of cytological and genomic sequencing data revealed that repetitive sequences play a significant role in chromosomal rearrangements, genomic differentiation and evolution [26] Many of these repetitive DNA sequences have been identified as molecular markers in plants [28, 29]. The amount of available sequence information that would allow the identification of molecular markers is limited and accurate identification at a species level that is fundamental for research on these plants is difficult

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