Plant Materials
F1 populations were generated in 2015 with the cross of ‘Ruby-3’(♀) × ‘Kuilv’ male(♂), the cross was finished in Zhengzhou Fruit Research Institute. Two parents, ‘Ruby-3’ and ‘Kuilv’ male (2n=4x=116) are A. arguta; ‘Ruby-3’ originates from Henan Province, and ‘Kuilv’ male originates from Jinlin Province. All the populations and parents were planted at the Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, China.
Low Temperature Treatment
All the populations were planted with well-watered and nonpest plants. Plant materials were the dormancy shoots of F1 populations. Dormancy shoots were treated at -30°C for 8 h (Sun, et al., 2020), and relative electrolyte leakage (REL) was calculated to screen for extreme cold resistance. Fifty populations with the highest REL and 50 populations with the lowest REL were selected to evaluate the detailed cold resistance and treated with -15℃, -20℃, -25℃, -30℃, -35℃ for 8 h. Then, the LT50 was calculated. Twenty populations with the higher REL (tolerance pool) and twenty populations with the lower REL (sensitive pool) were chosen, the shoots were treated with -30°C for 8 h, then used for BSR-Seq.
Measurement of Electrolyte Leakage
After low temperature treatment, the shoots without buds were cut into 1 mm-2 mm thick slices. Then, 0.2 g of the slices were incubated in 30 ml of double-distilled water for 2 h with shaking at 200 rpm at room temperature. The initial electrical conductivity (C1) was measured using a digital conductivity meter (DDS-307, Rex, China). The samples were heated to boiling for 30 min and then cooled down to room temperature with shaking for 30 mins. The second electrical conductivity (C2) was taken. The REL was calculated as indicated by Eq. 1:
REL(%)=(C1/C2)*100% (1)
The FT was expressed as LT50 (half lethal temperature at which REL reaches 50%) by fitting the response curve obtained by the REL with a logistic sigmoid function (Eq. 2):
y=k/(1+ae-bx) (2)
where x is the treatment temperature, y is the REL value, k indicates the extreme value when x approaches infinity, and a and b are the equation parameters. If the correlation coefficient r is close to 1, the equation is used to calculate LT50 (Sun et al., 2020).
Measurement of β-amylase Activity and Total Soluble Sugar Content
For measurement of β-amylase activity and total soluble sugar contents, the assay was repeated at least three times, with three biological replicates for each sample, and β-amylase activity and total soluble sugar content were determined using the relevant kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.
Each treatment was repeated at least three times with consistent results. Data are presented as the means of three biological replicates ±SE from one representative experiment. The data were analyzed by Duncan’s multiple range tests in the ANOVA program of SPSS (IBM SPSS 22), using P < 0.05 and P< 0.01 to indicate statistical significance.
RNA Extraction
Forty populations and ‘Kuilv’ male RNA in the dormancy shoots were extracted using an RNA Isolation Kit (HUAYUEYANG, China). The concentration and quality of the extracted RNAs were assessed using the NanoDrop spectrophotometer. Finally, ‘Kuilv’ male RNA was subjected to Pacific Bioscience (PacBio) single-molecule long-read sequencing, while 40 populations of RNA were submitted for second-generation transcriptome sequencing in a flow cell on the Illumina HiSeq platform for BSR-Seq.
Library Preparation and PacBio Sequencing
FL cDNA synthesis was completed using the SMARTerTM PCR cDNA Synthesis Kit. Then, PCR amplification, quality control and purification were performed. Size selection was performed using the BluePippin Size Selection protocol. Then, the product was separated into cDNA fractions with lengths of 1-2, 2-3 and 3-6 kb. The cDNA products were submitted for production of SMRTbell Template libraries by SMRTbell Template Prep Kit. Finally, the three libraries were sequenced using thirteen SMRT cells.
RNA-seq and Bulked Segregant Analysis
Tolerance pools and sensitive pools were generated for BSR-Seq analysis. Two extreme pools were constructed using equal amounts of tissues from the same position of plants at the same growing stage. Shoots collected from the most cold-tolerant individuals and the most cold-sensitive individuals were mixed as tolerant and sensitive pools, respectively. Transcriptome sequencing was performed on the Illumina HiSeq 2500 platform (China Novogene, Beijing). The Illumina library was prepared according to the protocol described in the next-generation transcriptome article [50].
Gene Functional Annotation and DEG Analysis
Seven databases (NR, Swiss-Prot, GO, NT, KOG, Pfam, KEGG) were selected to map the nonredundant transcript sequences and obtain the annotation information of the transcript with e-values of 1e-5 against a total of seven databases [51-56].
RNA sequencing reads were aligned to the abovementioned SMRT transcripts obtained by Trinity using [57], and expression levels were estimated using RSEM [58]. The expression levels of the unigenes were expressed as fragments per kilobase of transcript per million mapped reads (FPKM) values to eliminate the influences of gene length and sequencing quality difference on the estimated gene expression [59].
Differential gene expression analysis was carried out between the tolerance pool and sensitive pool using DEGSeq [60]. For the samples without biological repetitions, TMM was used to standardize readcount data [61], and then DEGSeq was used for differential gene expression analysis. In this study, genes with adjusted q values less than 0.005 and fold changes greater than or equal to 1 were identified as DEGs. Gene ontology (GO) term annotation was conducted for functional classification of DEGs [62], and further metabolic pathway enrichment analysis was carried out using KOBAS (2.0) against the KEGG (Kyoto Encyclopedia of Genes and Genomes) database [63, 64].
qRT-PCR Analysis
Shoot tissues of F1 populations were analyzed in three biological replicates. Samples were ground as described in the Materials. RNA samples were converted to single-stranded cDNA using the cDNA Synthesis Kit (TOYOBO). Candidate genes were selected from previous data. Primer Premier 5 design primers were used (Table S1). SYBR Green-based RT-qPCR analyses were performed in a LightCycler 480 (Roche480) on a 96-well plate. The conditions for the PCR amplifications were as follows: 95 ℃ for 5 min, followed by 45 cycles of 10 s at 95 ℃, 20 s at 60 ℃, and 20 s at 72 ℃. At the end of each experiment, a melt-curve analysis was carried out using the default parameters (5 s, 95 ℃ and 1 min, 65 ℃). β-actin in the kiwifruit was considered the control gene for normalization. All analyses were repeated three times using biological replicates. The relative expression levels were calculated using the 2-ΔCt method. RT-qPCR primers of target genes were designed using Primer Premier 5 software.