Tetraploidy enhances the cold-resistance in yellow kiwifruit (Actinidia chinensis)

Yellow kiwifruit ( Actinidia chinensis ) is highly susceptible to severe weather, such as low temperature and frost, which may affect the production in the coming year. And the cold-resistant mechanism of kiwifruit associated with gene regulation is poorly investigated. To botain cold-resistant germplam, to provide insight into the causes of differences in low temperature tolerance due to ploidy and to better understand cold-adaptive mechanisms in tetraploid kiwifruit, the diploid yellow kiwifruit ‘SWFU03’ and its tetraploid plantlets were subjected to cold-tolerant screening with L-hydroxyproline (L-Hyp) and low temperature, the selected ones were then analyzed by transcriptome data and conrmed by RT-qPCR. The results showed that the survival rate of tetraploid plants was 62.22% when treated with 8 mmol/L L-Hyp for 30 days, while all the diploid ones died. After treated with 0°C for 12 h, then at room temperature for seven days, the survival rate of tetraploid plantlets was 42.22%, while all diploidy died. Hence, cold tolerance of the tetraploid plantlets was stronger than that of the diploid genotypes. Using these two screening systems, 126 cold-resistant tetraploid tissue culture plantlets were obtained. A total of 1630 differentially expressed genes (DEGs) were identied, of which 619 were up-regulated and 1011 were down-regulated in the low temperature treatment goup. The DEGs enriched in the cold-tolerance related pathways mainly included plant hormone signal transduction, and starch and sucrose metabolism pathway. RT-qPCR analysis conrmed the expression levels of eight up-regulated genes in these pathways in the cold-resistant mutants. In conclusion, this study has identied cold-resistant yellow kiwifruit plantlets and cold-tolerance related genes. Moreover, the dataset got in this study advances our knowledge of the cold-adaptive genes in the regulatory networks and leads to understand the cold tolerance mechanisms in the tetraploid yellow kiwifruit. out using the tetraploid seedlings induced and identied in our previous research, and the cold-tolerant mutants were screened by using L-hydroxyproline (L-Hyp) and low temperature. To provide insight into the causes of differences in low temperature tolerance and better understand cold-adaptive mechanisms, selected cold-tolerant mutants were subjected to transcriptome analysis and validation by Real time quantitative polymerase chain reaction (RT-qPCR) analysis. of indexes diploid tissue for inoculated in one bottle. survival rate and weight of plantlets, plantlets and after The changes of indexes were after being cultured in the same environment for 7 days. transferred to normal medium L-Hyp cold-tolerant diploid kiwifruit

survival rate was only 20%). In MS medium with 10 mmol/L L-Hyp, all diploid plants died,but the survival rate of tetraploid plants was still 53.33%. The survival rate of tetraploid plantlets was signi cantly higher than that of diploid plantlets (Table 1).
According to the experimental results, it was concluded that the semi-lethal concentration of L-Hyp in tetraploid plants was 8 mmol/L. With the concentration increase of L-Hyp, the survival rate of diploidy decreased signi cantly, while the survival rate of tetraploidy decreased gradually. And the survival rates of tetraploid plants at high concentrations were signi cantly higher those of diploid plants. The results showed that the tolerance of tetraploid plants to L-Hyp was stronger than that of diploidy.
After 30 days of treatment, they were transferred to a normal medium, and the changes of fresh weight before and after treatment were recorded (Table   2). Under the concentration of L-Hyp of 0 mmol/L, the increased rate of tetraploidy the fresh weight was 110.79%, and the increase rate of diploidy fresh weight was 114.13%. Under the condition of no L-Hyp stress, diploidy grew faster and increased fresh weight more than tetraploidy. In MS medium with 2 mmol/L L-Hyp, the increased rate of fresh weight of tetraploid and diploid was 104.14% and 93.63%, respectively. The increased rate of fresh weight of tetraploid was higher than that of diploidy, and some diploidy died. Under the stress of 4 mmol/L L-Hyp, the increase rate of fresh weight of tetraploidy was 101.94%, while that of diploid was 66.24%. Under the stress of 8 mmol/L L-Hyp, the increase rate of diploid fresh weight was only 10.28%, and that of tetraploidy was 96.83%. Under the stress of 10 mmol/L L-Hyp, all the diploidy died, no fresh weight increased, and the rate of increase of tetraploidy fresh weight was 75.67%.
The seedling heights of tetraploidy and diploidy were measured before and 30 days after treatment ( Table 3). The results showed that the height increase rate of diploidy (152.94%) was higher than that of tetraploidy (140.12%) without L-Hyp treatment. Under the stress with 4 mmol/L L-Hyp, the height of diploid plants increased only slightly, and that of tetraploid plants increased, while under the stress of 6 mmol/L L-Hyp, the diploid plants basically stopped growing. Under the stress of 8 mmol/L L-Hyp, the height of diploid plantlets did not increase and the height of tetraploid plants increased slightly. Under the stress with 10 mmol/L L-Hyp, all the diploid plants died, so there was no seedling height data, and the growth of tetraploid plants basically stopped (Table 4). The results showed that the growth rate of tetraploid plants treated with L-Hyp at different concentrations was higher than that of diploid plants.
Effect of low-temperature treatment on yellow kiwifruit As shown in Figure 1, the freezing injury symptoms of tetraploid plantlets were less than that of diploid plantlets. The survival rates of tetraploid and diploid plantlets at 0 °C for 3 h were 88.89% and 17.78% respectively (Table 5). At 0 °C for 12 h, the survival rate of tetraploid plantlets was 42.22%, and the survival rate of diploidy was only 2.22%. After low-temperature stress, the tetraploid and diploid plantlets were transferred to normal MS medium and placed at room temperature for observation. Seven days later, the control plantlets grew slowly, the leaves wilted, and dead tip and edge of the leaves appeared. However, most of the tetraploid plantlets grew normally, the degree of chilling injury was obviously alleviated, and even no chilling injury occurred. Therefore, the cold tolerance of tetraploid plantlets was higher than that of diploid plantlets.

Results of transcriptome analysis
Results of sequence data ltering The original data of 6 tetraploid samples were shown in Table 6. To improve the accuracy of the analysis results of the transcriptome data, the sequence data were ltered before the data analysis, and the ltered data were shown in Table 6. The sequence data of sample transcriptome was of high quality.

Detection of DEGs
There were 1630 DEGs between control group and treatment group. By analyzing the DEGs between control group and treatment group, it was found that 619 DEGs were up-regulated and 1011 DEGs were down-regulated in the treatment group compared with the control group (Table 7).

Functional classi cation and enrichment analysis of DEGs
According to the results of differential gene detection, the DEGs were classi ed and enriched in the GO database. GO enrichment analysis could be divided into three main functions including molecular function, cellular component, and biological process. A total of 1025 DEGs were classi ed into 44 functional categories. There were 10 functional classi cations of molecular function, 13 functional classi cations of cellular component and 21 functional classi cations of biological process ( Figure 2).

Functional classi cation and enrichment analysis of KEGG pathway
A total of 410 differentially expressed genes were involved in 89 metabolic pathways in control group and treatment group.The top ve pathways enriching DEGs were plant hormone signal transduction (including 25 DEGs), phenylpropanoid biosynthesis24 DEGs), plant-pathogen interaction (20 DEGs), carbon metabolism (14 DEGs), and cysteine and methionine metabolism (13 DEGs). Among them, plant hormone signal transduction was the pathway with the largest number of DEGs annotated to this pathway, which belonged to environmental information processing in ve branches.
The number of up-regulated DEGs enriched by phenylpropanoid biosynthesis was 16 in the rst three pathways of the KEGG metabolic pathway, and 14 DEGs were enriched in plant-pathogen interaction. A total of 11 DEGs were enriched by carbon metabolism. The top three pathways of down-regulation were plant hormone signal transduction, enrichment of 19 DEGs, phenylpropanoid biosynthesis, enrichment of eight DEGs, and six DEGs were enriched in carotenoid biosynthesis.

Screening and analysis of cold tolerance related genes
In this study, the largest number of DEGs was annotated to plant hormone signal transduction pathway. Many cold-tolerance related studies have found that most of the DEGs were enriched in this pathway. Signal transduction pathway plays a key role in the response of plants to low temperature (Janská et al., 2010). Therefore, the DEGs enriched in this pathway might be related to cold tolerance, so the DEGs in this pathway were analyzed in detail. The pathway map (KO04075) was shown in Figure 3.
Abscisic acid affects plant cold tolerance and gene expression regulation and can improve plant cold tolerance. Abscisic acid signal transduction was due to the interaction between abscisic acid receptors in the process (Hubbard et al., 2010). Abscisic acid receptor PYL was one of the receptors, and CEY00_Acc03316 was annotated as abscisic acid receptor PYL, so it could be inferred that CEY00_Acc03316 may be a cold tolerance gene. The overexpression of bZIP transcription factor family proteins under stress was reported to improve the cold tolerance of plants (Chang et al., 2017;Ma et al., 2018). Therefore, CEY00_Acc13130 gene may be a cold tolerance gene. TIFY protein was induced and expressed at low temperature, and its gene family might be involved in low-temperature response and adaptation (Ye et al., 2009;Wang et al., 2017). Therefore, it could be inferred that CEY00_Acc33627 gene may be a cold tolerance gene.
Trehalose-6-phosphate synthase can protect biological cell membrane and protein from damage, and is widely used in transgenic experiments with plants for the improvement of cold tolerance of plants (Kim et al., 2017). CEY00_Acc26744 and CEY00_Acc16756 were annotated as alpha-trehalosephosphate synthase genes and inferred as cold-resistant genes. Maltose, which is produced by starch decomposition catalyzed by beta-amylase, may help protect the electron transport chain and protein in the stress environment. Cold tolerance of plants could be regulated by soluble sugar (Tarkowski and Van den Ende, 2015). Beta-amylase might reduce cold stress injury by increasing the content of sugars and enhance the cold tolerance of plants (Lv et al., 2017). Hence, unigenes of CEY00_Acc28966 (beta-amylase), CEY00_Acc16695 (beta-amylase) and CEY00_Acc08918 (beta-amylase) were inferred as cold-resistant genes.

RT-qPCR validation
The results RT-qPCR analysis of selected DEGs showed that the expression levels of all the genes selected were consistent with the transcriptome results ( Figure 5). The ndings suggested that there were statistically signi cant differences between the expression levels of these genes in low-temperature treated and control plantlets.

Discussions
The methods of inducing low-temperature stress in plants are mainly in the low-temperature environments, such as low-temperature natural environment, temperature control box or low-temperature refrigerator for direct screening. For example, Yuan et al. (1996) induced the natural low-temperature in Robinia pseudoacacia for 3 years, which improved the root cold tolerance of R. pseudoacacia. Chemical reagents can also be used as a selection pressure for stress and screening, in which L-Hyp is often used as a selection pressure in the study of plant cold tolerance. L-Hyp is a proline analog, which has an inhibitory effect on plants. It is often used as a selective pressure to screen all kinds of stress tolerance of plants, such as salt tolerance, cold tolerance, drought tolerance and so on (Chen et al., 1995;Gong et al., 2012). L-Hyp is often used in low-temperature stress experiments, but also can be used as a cold-resistant mutagen. Peng et al (2014) induced ornamental pineapple with EMS and screened for tolerance to HYP to induce coldresistant mutants. In this study, L-Hyp selective pressure and low-temperature stress were used to screen the cold-resistant kiwifruit plantlets, and these two methods were found to be effective. Janská et al. (2011) found that plant signal transduction pathway plays a key role in the response of plants to low temperature. In this study, it was found that the DEGs enriched in plant hormone signal transduction pathway were related to cold tolerance, which was consistent with the results of Janská et al. (2011). The starch metabolic pathway was reported to be closely related to the cold tolerance response of plants . In this study, the genes of the plant hormone signal transduction, and starch and sucrose metabolism pathway were found to be up-regulated in the treatment group, including CEY00_Acc03316 (abscisic acid receptor PYL), CEY00_Acc13130 (bZIP transcription factor), CEY00_Acc33627 (TIFY protein).
CEY00_Acc26744 (alpha-trehalose-phosphate synthase), CEY00_Acc16756 (trehalose phosphatase), CEY00_Acc28966 (beta-amylase), CEY00_Acc16695 (beta-amylase), and CEY00_Acc08918 (beta-amylase). ABA has a certain effect on improving the cold tolerance in plants . Therefore, the differentially expressed gene CEY00_Acc03316 (abscisic acid receptor PYL) might play an important role in the improvement of plant cold tolerance. BZIP transcription factors were found to play an important role in regulating plant cold tolerance and other stresses (Banerjee and Roychoudhury, 2017). Hence, CEY00_Acc13130 (bZIP transcription factor) might be a cold-tolerance related gene. Trehalose-6-phosphate synthase, a key enzyme in trehalose synthesis, was found to play an important role in the low-temperature response in different plant species (Miranda et al., 2007;Ge et al., 2008). Therefore, alpha-trehalose-phosphate synthase (CEY00_Acc26744) identi ed in the study, might be a cold-tolerance related gene. Betaamylase affected the cold-tolerance of plants mainly by regulating the content of starch (Peng et al., 2014). Beta-amylase-mediated starch degradation was found to play an important role in cold tolerance of plants (Lv et al., 2017). So beta-amylase (CEY00_Acc28966, CEY00_Acc16695 and CEY00_Acc08918) might be important cold tolerance genes in the kiwifruit.
At present, the researches on kiwifruit transcriptome analysis are mainly focused on the changes of fruit pulp color, fruit pigment, fruit development, bacterial canker and so on (Asiche et al., 2018;Song et al., 2019). There were few studies on the stress tolerance of kiwifruit transcriptome analysis, especially on cold tolerance, and there was no study on cold-resistant genes of kiwifruit, which needs to be studied in the future. The genes related to cold tolerance identi ed in the study need further functional veri cation.

Conclusions
The tetraploid plantlets were treated and screened by L-Hyp selective pressure and low-temperature stress, and the cold-tolerance of kiwifruit was determined by using growth indexes. The highest tolerance of tetraploidy to low-temperature treatment was 0 °C for 12 h, and the survival rate was 42.22%. A total of 126 cold-resistant plantlets were obtained.
Based on the transcriptome analysis of tetraploid cold-resistant plants after low-temperature treatment, two pathways related to cold tolerance were obtained, which were plant hormone signal transduction and starch and sucrose metabolism pathway. In these two pathways, a total of 8 DEGs of coldtolerance in kiwifruit were screened. And these genes were con rmed to be up-regulated by RT-qPCR when the tetraploid plantlets were treated with cold temperature.

Materials
The experimental materials were diploid plants of yellow kiwifruit variety 'SWFU03' from the Plant Nursery, Southwest Forestry University and its tetraploid plants which were induced and identi ed by . In-vitro propagated tetraploid and diploid plants come from three mother tetraploid plants and three diploid plants. Medium, grow chamber conditions, and photoperiod of the plantlets were described previously by .

Screening of cold-resistant mutants in tetraploidy
Screening for cold-tolerant mutants with L-Hyp The tissue culture plantlets of puri ed kiwifruit tetraploidy were propagated. The tetraploid tissue culture plantlets of A. chinensis, whose ploidy had been identi ed as tetraploidy, were inserted into the basic medium supplemented with L-Hyp at different concentrations (0 mmol/L, 2 mmol/L, 4 mmol/L, 6 mmol/L, 8 mmol/L and 10 mmol/L). The survival rate, fresh weight of plantlets, the total number of leaves and the height of plantlets were measured before and after treatment. The culture temperature was (25 ±3) °C, and the changes of these indexes were observed after 30 days.
At the same time, the diploid tissue culture plantlets of A. chinensis were inserted into the basic medium supplemented with L-Hyp at different concentrations (0 mmol/L, 2 mmol/L, 4 mmol/L, 6 mmol/L, 8 mmol/L and 10 mmol/L). There were 5 bottles for each concentration, and one tissue culture seedling was inoculated in one bottle. The survival rate and fresh weight of plantlets, the total number of leaves and the height of plantlets were measured before and after treatment. The changes of indexes were observed after being cultured in the same environment for 7 days. Then the plantlets were transferred to the normal medium without L-Hyp for 30 days, repeated three times, to screen the cold-tolerant diploid kiwifruit plantlets.
Directional screening of cold-resistant mutants by low temperature treatment Yellow kiwifruit diploid plantlets and tetraploid plantlets identi ed were used as experimental materials. The plantlets were treated with low temperature at 0 °C for 3 h, 5 h, 8 h, 12 h and 24 h, respectively (n = 15, repeated 3 times). The aseptic diploid plantlets were treated with the same time as the tetraploidy. The wilting degree, survival rate and mortality of aseptic tetraploid and diploid plantlets were measured.

Tetraploidy transcriptome analysis of yellow kiwifruit
Test materials and methods Three bottles of tetraploid plantlets were treated at 0 °C for 5 h (3 plantlets in each bottle) and 3 bottles of tetraploid plants treated at 25 °C for 5 h (3 plantlets in each bottle). The bottles were placed on a clean table. In the normal temperature control groups (C1, C2 and C3) and low-temperature treatment groups (T1, T2 and T3), three plantlets (1 plant per bottle) were packed and labeled with tin foil. After sampling, the samples were quickly frozen in cryogenic liquid nitrogen and then placed in dry ice. Six samples were sent to Anhui Microanalysis Gene Technology Co., Ltd for transcriptome analysis. RNA-Seq was performed using Illumina HiSeqTM 2500, and RNA-Seq data were presented at the Genome Sequence Archive of the Beijing Institute of Genomics (BIG) Data Center (accession number CRA001986).
The data produced by the RNA-Seq sequence were compared to the reference genome of A. chinensis. The differences between samples were based on the results of quantitative expression, and Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differential genes carried out in the study.

Sequence data ltering
After the library was constructed, the Illumina HiSeqTM 2500 platform was used for sequencing, and the sequence strategy was PE150. The original data (raw reads) obtained by sequencing were ltered, and the low-quality reads, connectors, and contamination, which accounted for more than 40% of the total read, were ltered out of the alkali base with a mass value of less than 15.
Comparison of reads obtained by sequencing with the reference genome After the clean reads were obtained, the clean reads were compared to the whole genome sequence of A. chinensis by HISAT2, and the reads mapping information was obtained. The utilization rate of sequence data and the genetic relationship with the whole genome of A. chinensis were obtained by comparison.

Detection of differentially expressed genes (DEGs)
The screening threshold was FDR (false discovery rate) < 0.05, log2FC (fold change for a gene) > 1 or log2FC <-1. Based on the results of expression analysis, read count information and DEseq2 (or edgeR) software were used to analyze DEGs (Anders and Huber, 2010).

Enrichment analysis of DEGs in GO category and KEGG Pathway
The DEGs were annotated on the Gene Ontology database (GO, http://www.geneontology.org/), and the GO term with FDR ≤ 0.05 was selected as the signi cantly enriched GO entry (Anders and Huber, 2010). The DEGs were annotated on the KEGG Pathway database .

Screening of differential genes
Differential gene data were annotated to GO and KEGG were analyzed, and the cold tolerance related genes in kiwifruit were screened according to the previous studies on cold tolerance genes and related pathways (Hubbard et al., 2010;Kim et al., 2017;Li et al., 2018;Wang et al., 2017;Ma et al., 2018).

RT-qPCR analysis
The transcriptional expression levels of 3 genes in the pathway of plant hormone signal transduction and 7 genes in the pathway of starch and sucrose metabolism pathway were selected for checking by RT-qPCR in low-temperature and room-temperature treated plantlets. The total RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA), and then reversely transcribed into cDNA by random primers. The RT-qPCR analysis was conducted according to a previous report . Gene-speci c primers were designed using Primer Premier 5.0 software, and the primers used for RT-qPCR analyses were listed in Table1. The2 (-△△Ct) method  was used to analyze the data.
Abbreviations BIG: Beijing institute of genomics; DEGs: differentially expressed genes; FC: fold change for a gene; FDR: false discovery rate; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes; L-Hyp: L-hydroxyproline; RT-qPCR: real time quantitative polymerase chain reaction.

Declarations
Ethics approval and consent to participate The experiment materials does not include human being or animal. Hence, ethics approval and consent to participate is not applicable.

Consent for Publication
Not applicable.

Availability of data and material
All data generated or analysed during this study are included in this published article. RNA-Seq data were presented at the Genome Sequence Archive of the Beijing Institute of Genomics (BIG) Data Center (accession number CRA001986).  Changes of tetraploid and diploid plantlets under low-temperature treatment. The plantlets were placed at room temperature for 14 days after lowtemperature treatment at 0 °C for 12 h.

Figure 2
Bar graph of differentially expressed gene GO classi cation. Red bars represent the biological process, blue bars indicate the cellular component, and green bars represent the molecular function.

Figure 3
Plant hormone signal transduction pathway inA. chinensis tetraploid plants treated with low-temperature. Red box, up-regulated genes; green box, downregulated genes; blue bx, both.

Figure 4
Starch and sucrose metabolic pathwayin A. chinensis tetraploid plants treated with low-temperature. Red box, up-regulated genes; green box, downregulated genes; blue box, both. Figure 5