Identification and expression profiling of GAox gene family in potato (Solanum tuberosum L.)

doi:10.21203/rs.3.rs-3081762/v1

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Identification and expression profiling of GAox gene family in potato (Solanum tuberosum L.)

https://doi.org/10.21203/rs.3.rs-3081762/v1

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Background

GAox is a key enzyme involved in GA biosynthesis pathway and plays an important role in regulating various processes in plant life cycle.

Results

In this study, GAox gene family (StGAox) in potato was identified, and their physical and chemical properties, distribution on chromosomes, gene structure, conserved motifs, gene duplication events and expression patterns were analyzed. The results showed that a total of 33 GAox proteins were identified and unevenly distributed on 10 chromosomes. According to their protein structure and phylogenetic protein characteristics, these 33 StGAoxes were divided into 5 distinct subclasses. Collinearity analysis revealed that there were 5 pairs of duplicated genes in the StGAox gene family, and all these genes evolved under purifying selection. RNA-seq data of doubled monoploid (DM) potato in different tissues, under abiotic stresses and hormone treatments were used to screen tissue-specifically expressed StGAox genes, and those responded to abiotic stress and hormone treatments. In addition, StGAox genes that might respond to drought stress in drought-sensitive and drought-tolerant tetraploid potato cultivars was further analyzed based on RNA-seq data.

Conclusions

These results provided a theoretical basis for further understanding the function of GAox genes in potato.

Potato (Solanum tuberosum)

Gibberellin dioxygenase

Expression profiles

Drought stress

Gibberellin (GAs) is a kind of diterpenoid plant hormone, which is involved in physiological processes at various stages of plant growth cycle, such as stem elongation, root development, leaf extension, root hair growth and other life processes [1–4]. The biosynthesis pathways of GAs in higher plants have been clarified, and their essential elements have been found[5, 6]. The synthesis of GAs is catalyzed by a series of enzymes, among which there are three key GA oxidases, namely GA20ox, GA3ox and GA2ox, each GA oxidase is independently encoded by a small gene family, and their domains are highly homologous[7]. Overexpression of GA20ox not only can promotes internode elongation, but also contributes to leaf expansion in plant[8, 9]. However, unlike this, overexpression of GA20ox in zucchini resulted in dwarfing of the plant[10]. GA2ox can passivate the action of gibberellin and make gibberellin lose biological activity in plants. GA3ox can synthesize bioactive gibberellins[11]. Previous research has demonstrated that the GAox gene family not only plays a role in controlling abiotic stress, but also plays a vital role in plant growth and development[12, 13]. For instance, in Camellia lipoensis, transgenic plants which overexpressing ClGA2ox1 or ClGA2ox3 had poor growth and curled leaves[14]. Overexpression of OsGA2ox5 increased the ability of rice seedlings to withstand salt stress, whereas the plant height was significantly reduced[15]. Overexpression of AtGA2ox7 and AtGA2ox8 in tobacco showing dwarf phenotype with decreased gibberellin content, whereas the flower and fruit phenotypes were normal[16].In grapes, overexpression of GA3ox severely inhibited pollen germination and pollen tube growth, resulting in seedless phenomenon[17]. In spinach, overexpression of SoGA2ox3 resulted in smaller plant height and delayed flowering[18].

At present, GAox gene family has been identified in multiple species, such as in Arabidopsis thaliana with 16 members[19], in rice with 21 members[20], and in cucumber with 18 members[21]. However, the GAox gene family in potato has been rarely reported. In the present study, the GAox gene family in potato was identified at the whole genome level, the chromosome distribution, gene structure, physical and chemical properties, protein conserved domains, and genetic relationships among genes were systematically and comprehensively analyzed. Using RNA-seq data, StGAox genes in doubled monoploid (DM) potato that might be involved in different organ development, as well as in response to abiotic stresses were screened. Furthermore, candidate genes in tetraploid potato cultivar that might be involved in drought stress were explored. The results provide a theoretical basis for further study of the function of GAox gene families in potato.

Plant materials and treatments

The experiment was carried out in the field under rain-proof shed in Dingxi Academy of Agricultural Sciences, Gansu Province, China. The drought-sensitive cultivar ‘Atlantic’ (A) and drought-tolerant variety ‘Qingshu No.9’ (Q) were used as experimental materials. For specific methods, see Li et al[22].

Identification of GAox in potato

The potato's genome sequence, proteins, CDS and chromosomal locations are downloaded from the Potato Genome Sequencing Consortium (PGSC)[23]. In the process of identifying potato GAox members, two conditions were used as follows: (1) GAox conservative structural domain was downloaded from the Pfam database as template sequence[24], the Arabidopsis Information Resources (TAIR) were used to retrieve the amino acid sequences of members of the GAox family as well [25]. HMM models were created using the HMMER 3.1program with a cutoff value set to 0.01, which were subsequently used to search for GAox members in potato genome database. (2)The BLASTP algorithm (threshold E < 1e-5) was used to find GAox members in the Arabidopsis GAox amino acid sequence[26]. Based on previous research, the TAIR was used to obtain the AtGAox protein sequences[25]. In the end, candidate members were manually verified in the SMART website [27] and the NCBI-CDD[28].

Sequence analysis and structure characterization of StGAox genes in potato

The gene characteristics, such as AA (amino acids), MW (molecular weight), PI (theoretical isoelectric point), and other physical and chemical properties, were obtained from online software ExPASy Protparam Server[29]. Using the CELLO website to predict subcellular localization of potato GAox protein [30]. The locally created GAox protein database was imported into the online software MEME (maximum motif = 10, width = 6 ~ 50) to examine the conserved structural domains of GAox proteins [31]. Using the online Gene Structure Display Server (GSDS2.0) tool, the exon-intron of each GAox gene was rigorously studied, finally the StGAox gene structure was graphically presented [32].

Chromosome distribution and gene duplication analysis

According to the position information of StGAox family members on chromosomes in PGSC database, MapChart software[33] was used to plot the chromosomal position map and relative distance of StGAox. Duplication events of StGAox genes were analyzed by MCScanX [34]and visualizedby Circus v0.69[35]. Tandem duplication events of the StGAox gene were determined according to the following two conditions: (1) the relatively constant short sequences that are more than 70% of the length of the long sequences; (2) the similarity of two aligned sequences is greater than 70%. The genes involved in tandem duplication are those that are closely spaced out along the same chromosome segment, separated by less than 100 kb, and have fewer than five genes in common. The synonymous (Ks) and non-synonymous (Ka) values of the StGAox genes were computed using the KaKs Calculator 2.0[36].

Evolutionary analysis and classification of StGAox

ClustalW software was used to perform multi-sequence alignment of 33 newly identified GAox amino acid sequences in potato and 16 known GAox amino acid sequences in Arabidopsis. An unrooted phylogenetic tree was constructed by using MEGA7.0 software (1000 iterations of bootstrap testing)[37].

RNA isolation and quantitative real-time RT-PCR (qPCR)

The RNA extraction kit (DP419) of Tiangen Biochemical Technology Corporation (Beijing) was used to extract total RNA of leaves. Subsequently, RNA integrity and concentration were determined using agarose gel electrophoresis and a Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, USA). Then, the first strand of cDNA was synthesized after removing genomic DNA contamination by using the FastKing RT kit with gDNase (KR116, Tiangen, Beijing). The SuperReal PreMix Plus (SYBRGreen FP205) kit purchased from Tiangen Biochemical Technology (Beijing) Corporation was used for qRT-PCR on CFX96 Touch™ Real-Time PCR Detection System(Bio-Rad, USA), and three biological replicates were performed for each treatment.

The following reaction system for qPCR was used: cDNA(50ng/µl), forward primer 0.6µM, lower primers 0.6µM, 2×SYBRGreen MasterMix 10µl, ddH₂O 6.8µl. The following qPCR settings were used: 30 s at 95°C, 40 cycles of 5 s at 95°C, 30 s at 60°C, followed by detection of the melting curve at 65–95°C. StEF-1α (AB061263)[38] was used as the internal reference gene. The 2^–ΔΔCt method was used to calculate the relative expression level of GAox genes[39]. The primers are shown in Table S1.

Expression pattern analysis of StGAox in potato

Using Illumina RNA-seq data, StGAox gene expression in DM potatoes were analyzed in different tissues (immature fruit, ripe fruit, carpels, petals, petioles, flowers, stolons, stamens, sepals, tubers, buds, roots and leaves). The expression level of StGAox gene family under abiotic stress (salt stress: 150 mM NaCl, 24 h; Mannitol-induced drought stress treatment: 260 µM mannitol, 24 h; Heat stress: 35℃, 24 h) and hormone treatment (GA₃, IAA, ABA, BAP) were also detected. Use TBtools software[40] to draw heat map.

In order to further understand the expression of StGAox gene family under drought stress. this study used RNA-seq data, the expression of StGAox gene in two varieties of potato ‘Atlantic’ (A, drought-sensitive variety) and ‘Qingshu 9’ (Q, drought-resistant variety) under drought stress was analyzed at three time points (S1:25 d, S2:50 d and S3:75 d). For the analysis of RNA-seq data, see Liu et al[23].

Evolutionary analysis and classification of StGAox proteins

The amino acid sequences of 16 GAox members in Arabidopsis and 33 GAox members in potato were used to construct a phylogenetic tree (Fig. 1). They were divided into five subfamilies, namely GA3ox, GA20ox, GA2ox-Ⅰ, GA2ox-Ⅱ and GA2ox-Ⅲ. The evolutionary relationship showed that 17 GAox genes (5 AtGAoxes and 12 StGAoxes) were classified in the GA20ox. 9 GAox genes (4 AtGAoxes and 5 StGAoxes) were classified in the GA3ox, 10 GAox genes(2 AtGAoxes and 8 StGAoxes) were classified in the GA2ox-Ⅰ, 4 GAox genes(1 AtGAox and 3 StGAoxes) were classified in the GA2ox-Ⅱ, 9 GAox genes (4 AtGAoxes and 5 StGAoxes) were classified in the GA2ox-Ⅲ.

Physicochemical properties and subcellular localization of StGAox proteins

The amino acid length and physicochemical properties of potato GAox proteins are quite different. The amino acid length ranged from 113 (PG0036075) to 378 (PG0024249), the MWs were between 12855.48 Da (PG0036075) and 43143.76 Da (PG0024249) and the predicted PI ranged from 5.01 to 8.64. The full gene length ranged from 339bp (PG0021383) to 1848bp (PG0009427), most of the GAox members were within 1.1kb, with the exception of 6 members longer than 1.1kb, which were PG0027645, PG0027963, PG0024249, PG0039968, PG0009427 and PG0016516, respectively. In addition, subcellular localization prediction showed that StGAox genes were mainly expressed in the nucleus, cytoplasm and cytoplasmic matrix, with an exception of PG0016537 being expressed in the cell membrane; The details are shown in Table S2.

Structure and conserved motifs of StGAox

Same division as in Fig. 1, the unrooted phylogenetic tree was structured using amino acid sequences of 33 GAox proteins in potato (Fig. 2-A). The exon/intron structures of GAox genes were visualized by GSDS2.0, the number of exons in StGAox genes between 2 and 3, except for PG0002068 and PG0041097 with the most number of 4 exons. Whereas the number of introns in StGAox gene family was relatively small with the number of introns less than 3 (Fig. 2-B).

The conservative motifs of StGAox proteins were analyzed using the MEME program (v. 5.0.4) (Fig. 2-C,Table S3). In the same subfamily, the conserved motifs of StGAox have similar structures, where, Motifs 1, 2 and motif 4 are conservative being shared by all members. DIOX_N (PF14226) domain is common to the GA2ox-Ⅲ and GA3ox subfamily and consists of motifs 5 and 10. 20G-FeII_Oxy (PF03171) domain, contained in the GA20ox and GA3ox subfamilies, may consist of motifs 3 and 6–9. Notably, some motifs exist only in certain subfamily, such as motifs 5, 6 are unique to the GA3ox and GA20ox subfamilies, and Motif 10 is unique to the GA2ox-II and GA2ox-Ⅲ subfamilies. In general, the StGAox gene is relatively conserved in evolution.

Chromosomal location and gene duplication of StGAox genes

The chromosome localization analyses showed that 33 StGAox genes were unevenly distributed on 10 chromosomes (Fig. 3). 7 StGAox genes were on Chr01 and Chr10 showing the largest number of distribution, followed by those on Chr02 and Chr09 with 4 StGAox genes, only one StGAox gene was on Chr04 and Chr08, respectively. Chr11 and Chr12 had no StGAox gene distribution.

We analyzed the duplication events of the StGAox genes and found that 5 pairs of StGAox genes (9/33, 27.27%) were segmental duplication genes, of which, Chr03 and chr 06 encompassed 2 pairs of fragment duplication genes (PG0024249 and PG0027963, PG0005698 and PG0016516), the remaining fragment duplication genes were located on Chr01, Chr02, Chr08 and Chr10, respectively. Each pair of duplicated genes belonged to the same subfamily (Fig. 3). The results suggest that fragment duplication is essential in the evolution of the StGAox genes.

Expression profiles of StGAox genes in different tissues of DM potato

Using RNA seq data from 13 tissues (immature fruit, mature fruit, carpels, petals, petioles, flowers, stolons, stamens, sepals, tubers, shoots, roots and leaves) of DM potato downloaded from PGSC, the expression patterns of St GAox in different tissues were analyzed. it is clear that 8 StGAox genes (PG0009427, PG2003479, PG0032156, PG0027963, PG0016516, PG0027206, PG0027645, PG0027632) were expressed in all tissues, 9 StGAox genes (PG0036075, PG0042158, PG0041097, PG0038099, PG0039968, PG0036003, PG0016537, PG0040776, PG0014071) were not expressed in all tissues. In addition, a number of StGAox genes showed tissue-specific expression patterns, such as PG0009021 was only highly expressed in stolons, PG0027632 was highly expressed in carpels and sepals, 4 genes (PG0000011, PG0005698, PG0011254, PG0002068) were specifically expressed in mature flowers, PG0021095 was specifically expressed in tubers, 2 genes (PG2003479, PG0024249) were expressed specifically in carpels and shoots (Fig. 5, Table S6).

Expression profiles of StGAox genes under abiotic stress and exogenous hormone treatment

In this study, the expression pattern of StGAox in potato leaves under abiotic stress was analyzed. Compared with the control, there were 11, 10, and 9 differentially expressed genes under salt, mannitol, and heat treatments, respectively(Fig. 6, Table S7). Of which, 6 genes (PG0009427, PG0024249, PG0016516, PG0005698, PG0009021, PG0027632) were up-regulated, 2 genes (PG0027645 and PG0027963) were differentially expressed only in response to heat stress. In addition, 10 genes (PG0036075, PG0014071, PG0040776, PG0042158, PG0041097, PG0038099, PG0016537, PG0036003, PG0039968, PG0019037) were not expressed under three stress treatments (FPKM = 0).

The expression patterns of StGAox genes under different hormone treatments were also examined (Fig. 6, Table S7). The results showed that the expression profile of StGAox gene was complex under GA₃, ABA, BAP, and IAA treatments. Under BAP treatment, no genes were up-regulated, the expression levels of 7 StGAox genes (PG0009427, PG2003479, PG0024249, PG0016516, PG0005698, PG0011254, PG0021383) were down-regulated, among them, the expression level of PG0021383 was down-regulated by 5 times compared to the control. Under ABA treatment, 3 genes (PG0009021, PG0021292, PG0016516) were up-regulated, among other, PG0021292 has the highest expression level, which was 4.2 times than the control. 5 genes (PG0021383, PG0032156, PG0024249, PG0027632, PG0005698) were down-regulated, among other, PG0005698 was down-regulated three times compared to the control. Under IAA treatment, 3 genes (PG2003479, PG0009427, PG0016516) were up-regulated, the expression levels were 1.23, 1.21, and 1.53 times higher than those of the control, respectively. Under GA₃ treatment, 5 genes (PG2003479, PG0032156, PG0009021, PG0021292, PG0027632) were up-regulated, among other, the most up-regulated was PG0021292, which was 2.7 times higher than the control. 5 genes (PG0009427, PG0024249, PG0005698, PG0021383, PG0032156) were down-regulated, the expression level of PG0005698 was down-regulated by 3 times compared to the control.

This study conducted that drought stress on A and Q in order to further examine if the StGAox genes responded to drought stress. The results indicated that 9 StGAox genes were not expressed in A and Q (FPKM = 0), 12 StGAox genes were expressed less than 1 in A and Q. 11 StGAox genes were up- or down-regulated (FPKM ≥ 1 and |log2FC|≥1) in Q under drought stress, of which, 4 genes were differentially expressed at three periods, 7 genes were differentially expressed at 25d (S1), 8 genes were differentially expressed at 50d (S2) and 9 genes were differentially expressed at 75 d (S3). PG0021292, PG0024249 and PG0002068 were not significantly expressed in A and Q at S1 stage, but highly expressed in Q at S2 and S3 stages. PG0032156 and PG2003479 were down-regulated in Q at S1 and S2 stages, but up-regulated in S3 by 90.2% and 80.6%, respectively. By analysing the data, it found the differential expression profiles of 5 genes (PG0024249, PG0002068, PG0021292, PG0032156 and PG2003479) genes in Q increased at first and then decreased.

Combined with the analysis of RNA-seq data under mannitol stress and drought stress, it was found that 10 StGAox genes were differentially expressed in DM under mannitol stress, of which 5 genes (PG2003479, PG0002068, PG0024249, PG0032156, PG0021292) were differentially expressed in A and Q under drought stress (Q compared with A), 3 genes (PG2003479, PG0032156, PG0024249) showed similar expression patterns under mannitol stress and drought stress. In addition, PG0011254 displayed an opposing expression pattern under the stresses of mannitol and drought, with expression up-regulated under the former treatment and down-regulated under the latter treatment (Q compared with A).

In order to confirm the accuracy of the RNA-seq data, we chose 5 StGAox genes with considerably increased expression in Q under drought stress for quantitative real-time PCR (qPCR). The results showed that although there were some differences between qPCR expression patterns and RNA-seq data, the overall trend was basically consistent(Fig. 7). The linear relationship between the RNA-Seq data and qPCR is y = 0.853x-0.06325, R² = 0.853. The resultshowed a high correlation between RNA-seq and qPCR, which further indicated that RNA-seq data were true and reliable.

In Arabidopsis, rice, cucumber and other plants, some GAox genes play important roles in abiotic stress response[16, 20, 21, 42]. The unrooted evolutionary trees were constructed to fully understand the phylogenetic relationship between potato and Arabidopsis GAox family[43]. However, the response of GAox genes to hormonal and abiotic stresses has not been reported in potato. Therefore, we identified the potato GAox gene family and analyzed the expression of these genes in different tissue and under hormonal and abiotic stresses.

In our study, we observed that these GAox proteins can be classified as 5 different subfamily, GA2ox-I, GA2ox-II, GA2ox-III, GA20ox and GA3ox, and most of GAox family proteins were situated in the nucleus and cytoplasm, as Huang et al.[44] reported that OsGA2ox6 protein was detected in the nucleus and cytoplasm. exons of GAox ranged from 2 to 4 in potato, and StGAoxes of the same cluster not only had similar intron/exon number and arrangement pattern, but also had very similar composition pages of conserved motifs. lv et al.[45] also found that the number of exons and introns in the GAox gene was similar.

Tandem repetition and fragment replication are the main ways of gene family expansion in plant[46]. Han et al.[47] found that 75% of GAox genes in the soybean genome undergo fragment replication events. In cucumber, the tandem or fragment repeat events are crucial in the expansion of the GA2ox and GA3ox gene families[48]. In this research, five pairs of fragment duplication genes were determine, it is suggested that fragment duplication is one of the main reasons for the evolution of the GAox gene family in potato. it is worth noting that replication events have been reported in other plants, but different amounts, such as eight segment duplicate pairs were found in soybean, three pairs of fragment replicating genes and two pairs of fragment replicating genes were found in rice and Arabidopsis thaliana, respectively[47].

In the case of gene duplication, some members of evolution may lose their original functions and gain new functions to enhance adaptations of plant[49]. In our work, we found that several segmental repeat genes displayed different expression patterns under abiotic stresses. For instance, PG0024249 and PG0027963 were a pair of duplication genes. PG0024249 was up-regulated in drought-tolerant cultivar Q at flower-falling stage under drought stress, but PG0027963 was not expressed under drought stress (FPKM = 0). Similar reports have also been reported in Arabidopsis thaliana and rice[50]. AtGA3ox2 and AtGA3ox4 are a pair of tandem repeating genes in Arabidopsis Thaliana, AtGA2ox2 was mainly expressed in flowers, whereas AtGA2ox4 was mainly expressed in roots[51, 52]. OsGA20ox2 and OsGA20ox4 in rice were a pair of fragment repeating genes, OsGA20ox2 was up-regulated under salt stress, but OsGA20ox4 was not expressed[53, 54]. In conclusion, gene duplication provides a large genome with multiple functional genes for potato.

Fragment repeat genes have similar expression patterns[55]. GmGA2ox5 and GmGA2ox8 were a pair of fragment repeating genes, both of which are expressed specifically in roots[56]. OsGA20ox1 and OsGA20ox3 in rice were a pair of fragment repeating genes, OsGA20ox1 is expressed specifically in flowers and OsGA20ox3 is expressed specifically in carpels[57]. PG0002068 and PG0011254 were a pair of fragment. duplication genes located in the same cluster of GA2ox-Ⅰ, and they were specifically expressed in mature flowers and petioles. PG0027963 and PG0024249 are a pair of fragment duplication genes located in the same cluster of GA20ox, which were specifically expressed in petioles. Based on the above complaints, the GAox gene of fragment replication event has similar expression patterns in different tissues.

Genes in the same cluster have similar functions[58]. AtGA2ox1 gene was a highly expression level in roots and stems in Arabidopsis thaliana, which was the key to the growth of Arabidopsis thaliana plants[59]. PG0027645, which was divided into the same subfamily as AtGA2ox1, was specifically expressed in mature flowers and immature fruits of potato. This indicates that PG0027645 have to do with the growth and development of potatoes.

Under hormone (BAP, ABA, ABA and GA₃) treatments, there were 7, 6, 4, and 10 StGAox genes differentially expressed, respectively. among them, PG0021383 of the GA2ox subfamily and PG0024249 of the GA20ox subfamily were down-regulated under four hormone treatments.It is worth noting that under BAP stress, none of the StGAox genes were up-regulated. It is interesting that some genes exhibit different expression patterns under the action of different hormones, but the amplitude of changes was similar.For example, PG0009427 of the GA2ox subgroup and PG0016516 of the GA3ox subgroup were up-regulated under the action of IAA, while down-regulated under the action of GA₃, which increased (decreased) by 1.2 and 1.5 times compared to the control, respectively. CsGA2ox3 and CsGA3ox2 are up- or down-regulated at least four times under the action of ABA and GA₃, which is consistent with the results of this study[60]. Similar conclusions have been reported in species such as Arabidopsis and tomato[61, 62]. Furthermore, the GAox gene plays a crucial role in response to various abiotic stresses [17, 20, 63]. In Arabidopsis thaliana, the double mutants of AtGA3ox1 and AtGA3ox2 enhanced plants tolerance to salt stress[14]. PG0016516 and PG0005698 are a pair of duplicated genes in GA3ox, which were also up-regulated in salt and mannitol stress, suggesting that two StGAox genes may have similar capabilities as AtGA3ox1. Mutants of AtGA20ox1, AtGA20ox2 and AtGA20ox3 in Arabidopsis showed significantly increased under drought tolerance[64]. The expressions of MaGA3ox2 and MaGA3ox5 were up-regulated under drought stress[65]. Under drought stress, OsGA20ox5 and OsGA20ox6 exhibit high expression levels[66]. In Arabidopsis, the expression of six AtGA2ox genes (AtGA2ox1, AtGA2ox2, AtGA2ox4, AtGA2ox6, AtGA2ox7 and AtGA2ox8) was up-regulated under drought stress[67]. In our work, the expression level of PG0024249 (GA20ox member) was up-regulated under mannitol and salt stress in DM potato, and also up-regulated under drought stress in drought-tolerant variety Q, indicating that PG0024249 might be related to drought stress response. PG2003479 and PG0032156 in GA2ox-Ⅰ clade was up-regulated in DM and Q under drought, while PG0021292 and PG0002068 in GA2ox-Ⅲ clade was also up-regulated in Q under drought stress, declaring that these genes may be related to drought stress response. In conclusion, StGAox gene may play an important role in potato growth and development and resistance to abiotic stress, which is worth further investigation.

In this study, 33 StGAox genes were analyzed at the genome-wide level, unevenly distributed across 10 chromosomes. Based on highly conserved gene structure and motif, the StGAox gene is delimited five subgroups. Collinearity analysis suggested that the fragment duplication events play a pivotal role in the expansion of the StGAox genes. According to the Ka/Ks ratio, these gene pairs evolve under purification selection. In addition, 5 candidate genes related to potato drought resistance were identified. These findings provide important information for the study of the GAox gene family in potato.

GA₃: gibberellin 3 GA20ox: GA20-oxidases

GA2ox: GA2-oxidases GA3ox: GA3-oxidases

Ks: synonymous substitution rate NJ: Neighbor–Joining

Ka: non-synonymous substitution rate

PG: PGSC0003DMG40 qRT-PCR: quantitative real-time PCR;

HMM: hidden Markov model AA:amino acid

MW: molecular weight PI: Isoelectric point

TF: transcription factor DM: doubled monoploid

ABA: Abscisic Acid BAP: 6-Benzylaminopurine

IBA: 3-Indolebutyric acid

Data Availability

RNA-seq data were derived from the NCBI database using accession number PRJNA541096(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA541096/). All datasets generated in this study are included in the published article/Additional Files. Websites used for analyses in this study are as follows: TAIR (http://www. arabidopsis.org/), Pfam (http://pfam.xfam.org/), HMMER3.1 (http://hmmer.org/download.html), SMART (http://smart.embl-heidelberg.de), NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), ExPASy (http://web.expasy.org/protparam/), GSDS2.0 (http://gsds.cbi.pku.edu.cn/), ExPASy (http://web.expasy.org/protparam/), MCScanX (http://chibba.pgml.uga.edu/mcscan2/), KaKs Calculator 2.0 (https://sourceforge.net/projects/kakscalculator2/), Access to these databases or websites is open. No new sequence data was generated in this study.

Acknowledgements

We are grateful to the providers of genomic and transcriptome data. We would also like to thank the anonymous reviewers for constructive suggestions that helped to improve the manuscript.

Funding

The Research Program was sponsored by the Major S&T Special Projects of Gansu Province (22ZD6NA009), the Key Program of Gansu Natural Science Foundation (22JR5RA834), the Director Fund of State Key Laboratory of Aridland Crop Science (GSCS-2021-Z02), the Earmarked Fund for China Agriculture Research System (CARS-09-P14), Central government guidance for local scientific and technological development projects (23ZYQJ304), and the Fuxi Talent Project of Gansu Agricultural University (Gaufx-02Y04).

Author information

Shujuan Jiao, Zhen liu, Yuhui Liu contributed equally to this work.

Authors and Affiliations

College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China

Shujuan Jiao, Zhen Liu, Yichen Kang, Junlian Zhang, Yuhui Liu & Shuhao Qin

State Key Laboratory of Aridland Crop Science/Agronomy College, Gansu Agricultural University, Lanzhou 730070, China

Shujuan Jiao, Zhen Liu, Junlian Zhang & Yuhui Liu

Contributions

S. J. conceived the project and revised the manuscript. Z. L. conducted most of the experiments and analyzed the data. J.Z., and S.Q. funding acquisition and project administration. Y. K. helped in data analysis. Y. L., and S. J. wrote the first draft of the manuscript. All authors have read and agreed to the submitted version of the manuscript. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Yuhui Liu or Shuhao Qin.

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Ethics approval and consent to participate

Experimental research and field studies on plants including the collection of plant material are compliant with relevant guidelines and regulations. The authors confirm that all the experimental research done on cultivated plants and carried out in accordance with relevant institutional, national, and international guidelines, standards, and legislation. The plants used were cultivated under controlled conditions and thus posed no threat to other plant species, and no specific permission was required to collect the required sample material used in this study.

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The authors declare no competing interests.

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No competing interests reported.

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Identification and expression profiling of GAox gene family in potato (Solanum tuberosum L.)

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Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and Methods

Plant materials and treatments

Identification of GAox in potato

Sequence analysis and structure characterization of StGAox genes in potato

Chromosome distribution and gene duplication analysis

Evolutionary analysis and classification of StGAox

RNA isolation and quantitative real-time RT-PCR (qPCR)

Expression pattern analysis of StGAox in potato

Results

Evolutionary analysis and classification of StGAox proteins

Physicochemical properties and subcellular localization of StGAox proteins

Structure and conserved motifs of StGAox

Chromosomal location and gene duplication of StGAox genes

Expression profiles of StGAox genes in different tissues of DM potato

Expression profiles of StGAox genes under abiotic stress and exogenous hormone treatment

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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