Construction Of Sugarcane Genetic Population And Analysis Of The Tillering Trait By BSR-Seq

Sexual hybridization is the main channel for breeding new sugarcane varieties.Sugarcane is an aneuploid and allopolyploid crop with high chromosome numbers and its F₁ generation arrived from the crossing of two cultivars segregate,in sharp contrast with a diploid plant,in which the F₁ generation is with a unique phenotype and segration occurs in F₂ generation.The main purpose of sugarcane breeding by crossing is to integrate the superior traits of both parents into a hybrid progeny for deveploment of new cultivars.However,selfing due to incomplete emasculation of the female parent oftern results in a mix of true hybrid and self-bred seeds.Therefore,it is necessary to identify the authenticity of hybrid progeny.In this study,a total of 17,923 F₁ progenies was obtained by sexual hybridization with ROC25 as female and Yunzhe 89-7 as male parents.Yunzhe 89-7-specific SSR primer pair SMC31CUQ-F/SMC31CUQ-R was screened out from 54 sugarcane SSR markers.After analyzing the polymorphic sequences,a specific primer pair SMC31CUQ-F/SMC31CUQ-1-R was redesigned to allow the successfulTransformation of the PCR polymorphic bands by SMC31CUQ into a unique band for the male parent and a portion of the F₁ progenies,greatly improving the detection efficiency.By using this primer pair to screen the whole F₁ population,9,310 progeny were found to have the male parent-specific SSR marker,accounting for 51.94%of the population.The avaiability of a large number of true hybrids lays a solid foundation for the construction of subpopulation with specific agronomic traits of interest.Tillering is an important agronomic trait in sugarcane and an important criterion for sugarcane breeding.Through the screening of the F₁ real hybrid seedlings from the cross of ROC25×Yunzhe 89-7,40 non-tillering plants with a single millable cane per clump and 80 multi-tillering plants with ten or more millable canes per clump were obtained to form a non-tillering/multi-tillering subpopulation.Then the mixed pool samples of these two groups and their parents were analyzed by using Bulked Segregant RNA-Seq（BSR-Seq）.Data sets of 67.52 Gb base paires with Q30 93.66%were obtained,and De novo assembly yielded 177,633 unigenes.A total of 127,308 SNPs was identified and2,508 genes were differentially expressed,including 781 upregulated and 1,727downregulated genes between the two pools.ED association analysis resulted in 215 genes that may be related to tillering,of which 131 genes were annotated（95 by GO,34 by KEGG,22 by COG,and a total of 131 by NR,NT,Swiss Prot and other databases）.Ten out of 11 differentially expressed genes were in good accordance in gene expression trend between the RNA-seq and the qRT-PCR.Among the differentially expressed genes was the gene c120003.graph＿c0 which encodes a cytochrome P450.The expression of this gene was 2.85-fold higher in the non-tilering group than in thestrong-tilering group.Since cytochrome P450 has been shown to be involved in the regulation of rice tillering through monolactone synthesis,it is speculated that the c120003.graph＿c0 gene may function the same way in sugarcane and thus be worthy to investigate further.Twelve genes related to the synthesis or signal transduction of plant hormone monolactone were obtained from the transcriptome database established in this study.There is a certain degree of difference in the expression levels of these genes between non-tillering and multi-tillering pools.Therefore,the differences in the tiller phenotypes between the tillering and multi-tillering plants in this study may also be related to the trigolactone pathway.