基于BSR-Seq对甘蔗脱叶性的研究和生物信息学分析

Bioinformatic Analysis and Identification of Genes Contributing to Sugarcane Defoliation via BSR-Seq

甘蔗的叶片是制糖过程中杂质的主要来源,育种过程中倾向于选育易脱叶的品种。为了研究甘蔗脱叶性相关的主效、稳定基因,本研究采用BSR-Seq技术,从‘ROC25’ב云蔗89-7’的F1后代中选取极端难脱叶品系和极端易脱叶品系的+6叶位叶鞘离区,分别构建混池,并和亲本一起进行RNA转录组测序分析,共得到60.46Gb的有效片段,4个样品的Q30值均在93%以上,GC含量都大于51%,与参考基因组R570比对,比对率都超过41%。4个样品共检测出4085个差异基因,候选区域定位于9号染色体上的4个位置,总长1.40 Mb,注释到基因86个,亲本间注释到的非同义突变15个,挖掘出2个主效差异基因(Sh09_g020620,Sh09_g020080),Sh09_g020080在亲本的表达差异和子代中是同向的,而Sh09_g020620在2个亲本和2个子代混池之间的差异是异向的,表明可能仅Sh09_g020080对易脱叶性有所贡献。选取BSR亚群体中极端易脱叶品系40-159和极端难脱叶品系5-94的+1~+7叶进行荧光定量分析,发现Sh09_g020080基因在单一品系的表达和子代混池的+6叶的表达是同向的。取另一个‘B35-9’בCP08-1553’的杂交后代个体中的极端易脱叶品系16-226和极端难脱叶品系16-224的+1~+7叶位的叶鞘离区进行荧光定量分析,结果显示Sh09_g020080在2个群体中+4、+5、+6叶位的表达都为易脱叶的品系高于难脱叶品系。注释显示Sh09_g020080具有σ因子活性,参与红光、远红光、蓝光反应。根据理化性质和蛋白质结构,推测Sh09_g020080编码蛋白具有4个保守区域,且具有积累叶绿素的作用。该研究为甘蔗脱叶相关主效候选基因的挖掘提出了新的见解。

Sugarcane(Saccharam spp.),a C4 tropical grass mainly for sugar production,is commercially cultivated in over 100 countries.One of the main barriers to mechanical or manual harvesting of whole sugarcane stalk is the high percentage of impurities,mainly the debris of leaves and sheathes that results in increase of harvest time and cost.The most efficient solution for this issue is to develop sugarcane cultivars that are easy to defoliate in sugarcane breeding program.In order to investigate the genes responsible to sugarcane defoliation,BSR-Seq technology was employed in the current study to analyze pools of the leaves at+6 position of extremely hard-to-defoliate and easy-to-defoliate sugarcane plants derived from the cross of‘ROC25’בYunzhe 89-7’,together with the two parental lines with contrast defoliation phenotypes.A total of 60 Gb of clean reads were obtained and the Q30 values of the four samples were greater than 93%with GC content of 51%.Using R570 genome as reference,the alignment rate was greater than 41%.A total of 4085 differential expressed genes were detected in the 4 samples.The candidate regions were found to locate at 4 loci on chromosome 9,covering a total length of 1.40 Mb in which 86 genes were annotated.There were 15 non-synonymous mutations annotated between the parental lines,and two significant differentially expressed genes(Sh09g020620,Sh09g020080)were identified.The expression level of Sh09 g020080 in the easy-to-defoliate genotype was higher than that of the hard-to-defoliate genotype and consistent with the parents but that of Sh09g020620 was opposite between the offspring pools and the parental lines,suggesting that only Sh09 g020080 may contributing to the phenotype of easy defoliation.Quantitation of Sh09 g020080 gene expression in leaves from position+1 to+7 from the extremely easy-to-defoliate representative accession 40-159 and the extremely hard-to-defoliate accession 5-94 confirmed the BSR-seq results of the two pools.In analysis of another genetic population derived from the cross of‘B35-9’בCP08-1553’,Sh09 g020080 was also found to express in a higher level in the extremely easy-to-defoliate accession 16-226 than in the extremely hard-to-defoliate accession 16-224 in+4 to+6 leaves.Analysis of the deduced Sh09g020080 protein revealed that this protein has four domains conserved inσfactor-like proteins that may function in in red,far red and blue light reactions and accumulation of chlorophyll.Overall,this study shed a new light into the endeavor of exploring essential genes for defoliation in sugarcane.