Abstract:

Screening the appropriate reference genes for qRT-PCR analysis on different progenies of Pyrus betulaefolia Bunge. under salt stress,in order to accurately evaluate expression of target genes. In this study,eight reference genes including Actin2（Chr15.g01351）,EF1α-1（Chr3.g19898）,EF1α-2（Chr4.g38173）,EF2（Chr5.g06899）,GAPDH-1（Chr16.g30426）,GAPDH 2（Chr13.g23532）,TUBB（Chr5.g06472）,UBQ（Chr4.g40121）were selected as candidates based on the whole genome and transcriptome sequencing data of P. betulaefolia Bunge. Ten pairs of qRT-PCR primers including Actin2,EF1α-1,EF1α-2A,EF1α-2B,EF2,GAPDH-1,GAPDH-2,TUBB-A,TUBB-B,UBQE were designed or cited from previous publications,and the amplification efficiency of the primer pairs was determined. Two families（Yancheng and Lianyungang）of P. betulaefolia Bunge. were treated with 200 mmol · L^-1 NaCl for 0,24,48,and 72 h respectively and leaf samples were collected. Total RNAs were extracted from 24 leaf samples and cDNAs were synthesized by reverse transcription as templates. The stability of amplification products by 10 primer pairs was analyzed by delta Ct,BestKeeper,geNorm and NormFinder respectively,and then analyzed comprehensively using RefFinder software based on four evaluation results to determine the most stable reference gene and the reference gene primers. The TUBB-A and GAPDH-1 have good stability and applicability. Finally,the expression of a HKT（Chr16.g29024）that may be involved in P. betulaefolia response to salt stress was measured by using those two selected primer pairs. The result showed that:the FPKM values（Fragments Per Kilobase Million）of the eight candidate reference genes were all greater than 30 and the FPKM variation coefficients under salt treatment at different times were 0.087-0.260;the amplification efficiency of 10 qRT-PCR primer pairs was between 91.82% and 112.99%,with primer pairs TUBB-A and GAPDH-1 being closest to 100%;The stability of 10 pairs of primer amplification products are ranked as GΑPDH-1,TUBB-Α,EF1α-1,TUBB-B,EF1α-2Α,EF2,UBQE,GAPDH-2,Actin2,EF1α-2B;Two genes（GΑPDH and TUBB）and primer pairs（GΑPDH-1 and TUBB-Α）were determined as the most suitable reference genes and the most stable primer pairs in this study. It was also found that the stability of the amplified products of different primer pairs of the same gene（such as TUBB-A and TUBB-B,EF1α-2A and EF1α-2B）is also significantly different. GAPDH-1 and TUBB-Α primer pairs are suitable for qRT-PCR analysis in P. betulaefolia under salt stress.

Key words: Pyrus betulaefolia, reference gene, qRT-PCR, gene expression stability