Analysis of Variation inMYB1 andMYB10 in Several Malus Plants

For the study of the polymorphism and evolutionary characteristics of MYB1 and its allele MYB10 involved in the regulation of anthocyanin biosynthesis in genus Malus. The MYB1 and MYB10 gene sequences amplified by RT-PCR technology in several Malus plants were sequenced and predicted for their protein structures to detect the differences. Six MYB1 sequences were obtained and the full-length CDS were 1 239 bp except for those of Malus cv. Eleyi and Malus sieversii (1 209 bp). While nine MYB10 were sequenced and the full-length CDS were 732 bp (Malus micromalus was 717 bp). The result from the comparison of protein sequences of MYB1 and MYB10 showed that the polymorphism of MYB1 was significantly higher than MYB10 and the polymorphism difference between the alleles may be related to the differentiation of gene function. Two polymorphism sites, the interchange of alkaline arginine and non-polar leucine, located on functional domain which may affect the function of MYB1 in different Malus plants. The result suggested that sequence conservatism of the allele MYB10 and MYB1 significantly differed from each other, and the genetic function responsible for evolution was differentiated. The result can provide further theoretical basis for the study of the gene function and regulation mechanism of apple fruit color.

MYB1 and its allele MYB10 (Wang et al., 2010), located in the same locus of apple 9 linkage group (Wang et al., 2010), belong to MYB transcription factors, and their proteins all have a double DNA binding domain named R2R3 at the N-terminal (Allan et al., 2008). MYB1 and MYB10 play a key role in the regulation of anthocyanin accumulation in apple peel by binding the promoter region of gene in anthocyanin synthesis pathway (Takos et al, In 2006). MYB transcription factors involved in anthocyanin regulation have been identified in many fruit trees, including strawberry, grape, sweet cherry, peach and Litchi (Zhu Tingting et al., 2016).
In the previous study of MYB1 in apple, the researchers found that there were at least three alleles with slight differences, namely MdMYB1-1, MdMYB1-2 and MdMYB1-3. Among them, the coding regions of MdMYB1-1 and MdMYB1-2 were the same, but the difference sites were located in the promoter region. The differential sites of MdMYB1-1 and MdMYB1-3 were located in the third exon, causing the conversion of arginine and serine near the C-terminal of the protein, but no significant change in function was detected between the two alleles (Takos et al., 2006). A promoter mutation MYB10 gene was found in red-fleshed apples (Espley et al., 2009;Mahmoudi et al., 1 Results

Cloning of MYB1 and its allele MYB10
The PCR results of 9 Malus plants ( Figure 1) showed that actin fragments (positive control) were successfully amplified, the PCR products were about 250 bp and the size was in accordance with the expected design size. MYB10 were amplified in 9 Malus plants and fragments were about 850 bp in size. However, MYB1 were amplified in 6 Malus plants except Malus×domestica Borkh. cv. Golden Delicious, Malus micromalus Makino and Malus×domestica Borkh. cv. Delicious (The amplification did not achieve after refinement of multiple amplification conditions and primer). The fragments were about 1200 bp. These results suggested that MYB10 gene may be widely existed in Malus, but its allele MYB1 might not appear in every Malus plants.

Obtaining sequences of MYB1 and its allele MYB10
Sequencing results showed that the amplified fragments of MYB10 in nine materials were 817 bp except for Malus micromalus Makino which was 802 bp. Among them, the open reading frame length of Malus micromalus Makino was 717 bp, encoding 238 amino acids, and the rest of the materials were 732 bp, encoding 243 amino acids. The comparison of the amplified MYB10 with NCBI database showed that the sequences were highly similar to the MdMYB10 from NCBI database with a similarity between 97% to 100%. The similarity between nine materials MYB10 was high, with a sequence consistency of 99.63%. There were seven gene differential sites, and five differential sites were located in the open reading frame, including a 15 bp deletion in Malus micromalus Makino. Malus plants were highly similar to those of cultivated apples, but there were still some polymorphism loci in MYB1 and MYB10 in different Malus plants. The polymorphism of MYB1 was higher than that of MYB10, and there were obvious differences in the conservation between the alleles.

Obtaining of amino acid sequences in MYB1 and allele MYB10
The sequences were translated into amino acid sequences by machine, and clustering was carried out with the sequences of NCBI database. It was found that MYB1 of Malus robusta, Malus domestica subsp. chinesis var. binzi and Malus domestica Borkh were similar to those of Malus baccata in the database, and MYB1 of Malus cv. Radiant was highly similar to that of apple in the database, clustering into a small branch with Pyrus bretschneideri and Pyrus usssuriensis. The MYB1 of Malus cv. Eleyi and Malus sieversii were similar to Malus×domestica Borkh. cv. Golden Delicious (Figure 2A). The translating results were highly similar to MYB1 of plants such as Malus×domestica Borkh. cv. Golden Delicious and Malus baccata in the database, indicating that the amplifications were indeed MYB1. The MYB10 of the nine Malus plants clustered together with the Royal Gala, red-fleshed apples and Malus baccata in the database and were also similar to those of the pear species ( Figure 2B), indicating that the amplifications were indeed MYB10. Cluster analysis of MYB1 and MYB10 showed that the obtained sequences were clustered into one group with Maloideae plants and were more closely related to those of Liliaceae.

Obtaining of the protein physical and structural informations of MYB1 and its allele MYB10
According to the translated amino acid sequence, the protein prediction tool was used for analysis of isoelectric point, molecular weight, hydrophilicity and other parameters with the reference of three cultivated apple sequence in NCBI database. The results showed that the MYB1 protein isoelectric point and hydrophilicity of Malus sieversii and Malus cv. Eleyi decreased compared with other proteins (Table 1; Table 2). The basic properties of MYB10 protein showed that the protein properties of Binzi, Delicious, Golden Delicious, Malus domestica Borkh and Malus sieversii were the same, the isoelectric point and hydrophilicity of Malus cv. Eleyi and Malus cv. Radiant decreased, and the hydrophilicity of Malus robusta increased. The results suggested that the difference of amino acid sequence did affect the hydrophilicity of MYB1 and MYB10 in different materials. The isoelectric point CV (coefficient of variation) of MYB10 was 0.015 for 9 plants of Malus, and hydrophilicity standard deviation (SD) of MYB10 was 0.010. While isoelectric point CV and hydrophilicity SD of MYB10 were respectively 0.061 and 0.038 for 6 plants of Malus. From the numerical differences of the coefficient of variation and the standard deviation, it can be seen that the isoelectric point (the proportion of positive and negative residues) of MYB1 fluctuated more than MYB10, which may reflect the change of their function. Note: * shows the sequence from the NCBI Further domain prediction of the protein was carried out to analyze the effect of polymorphism sites on the functional domain. The MYB1 had 6 amino acid polymorphism sites in the predicted major functional domains ( Figure 3A). Among them, the 121st amino acid polymorphisms (130th for Malus cv. Eleyi and Malus sieversii) were located in the key sites of DNA binding domain, and the polymorphic amino acids were non-polar amino acids glycine and alanine. The polymorphic amino acids of 46th and 113rd (55th and 122nd for Malus cv. Eleyi and Malus sieversii) were basic arginine and non-polar leucine. The polymorphic amino acids of 39th and 90th (48th and 99th for Malus cv. Eleyi and Malus sieversii), 45th (54th for Malus cv. Eleyi and Malus sieversii) were polar neutral serine and threonine. The analysis showed that the protein sequence of MYB10 was more conservative than that of MYB1 ( Figure 3B). The three polymorphic sites of MYB10 were all located at the downstream of the functional domain, but some of polymorphic sites were located at the functional domain of MYB1, which may affect the function of the gene, especially the two polymorphic sites of basic arginine and non-polar leucine. The two polymorphic sites were likely to lead to the change of protein spatial conformation to affect the recognition of downstream genes.

Discussion
The fruit color of Malus depends on the base color and cover color. The base color is related to the content of chlorophyll and carotene, while the red color of cover color is mainly related to the pigmentation of anthocyanin (Jaakola, 2013). The accumulation of anthocyanin is affected not only by genetic factors, but also by environmental factors, especially the change of light, such as paper-bagging (Chang bo et al, 2018) can make the fruit red. At present, it has been confirmed that many R2R3-MYB transcription factors can participate in the regulation of anthocyanin synthesis in Malus, including MYB1 and MYB10 (Jaakola, 2013). However, in previous studies, most of the related MYB transcription factor genes were amplified from cultivated apples, such as Cripps' Red (Takos et al., 2006), Golden Delicious, Gala, Jonathan and Australian green apple (Mahmoudi et al., 2012). The amplification of the related MYB transcription factor genes in Begonia was mainly concentrated in some varieties of Begonia in North America (Li et al., 2018). There was a lack of comparative study on genetic diversity among species and evolutionary characteristics of this locus gene.
In this study, the MYB10 sequence of 9 Malus plants and the MYB1 sequence of 6 Malus plants were obtained. The polymorphic level of MYB1 was significantly higher than that of MYB10, and the variation range of isoelectric point and hydrophilicity of MYB1 were larger than those of MYB10. In addition, two polymorphic sites in the functional domain of MYB1which were basic arginine and non-polar leucine were polar transformation of amino acid. The changes were likely to affect the function of MYB1 transcription factors. While the MYB10 polymorphism sites were only three, and all of them were located in the non-functional domain. This suggests that the MYB10 transcription factor was more conservative in evolution than the MYB1 on the same locus in the detected Malus plants. This indicated that the genes of the same locus of MYB1 and MYB10 may be subject to different selection pressures in evolution, and also suggests that MYB1 and MYB0 were involved in different gene regulation in fruit color control.
It has been shown in the previous study that MYB1 and MYB10 have different emphasis on the regulation of fruit color. A mutant of MYB10 gene, due to an insertional mutation of a 23 bp microsatellite sequence in its promoter region, caused the whole apple plant to turn red, including the peel, pulp and leaves (Espley et al., 2009). According to the phenotypic analysis, it can be inferred that the MYB10 gene was dominant and constitutively expressed (Espley et al., 2009;Wang et al., 2010). However, MYB1 was shown to be expressed only in the peel but not in the pulp (Mahmoudi et al., 2012), and MYB1 expression was regulated by light (Takos et al., 2006) and also by epigenetic regulation induced by paper-bagging (Bai et al., 2016;Ma et al., 2018), which indicates that MYB10 itself may have the potential to express in different tissue, but no similar expression pattern has been reported for MYB1 induced by a variety of in vitro factors. This tendency of different expression regulation patterns may be related to the differences in the conservativeness of MYB10 and MYB1 sequences in this study, and may be related to the functional differentiation of this locus among alleles in artificial cultivation and domestication. In addition to its involvement in the regulation of anthocyanin synthesis, MYB10 may also be involved in some other physiological and biochemical processes, while MYB1 may be more specifically responsive to environmental signals to regulate fruit color changes. The MYB1 sequence varied more in the genus Malus, but the expression tissue was single, while the MYB10 sequence is conserved and can express in more tissues.
Gene mutations, especially those caused by extensive bud mutation in fruit trees, are common phenomena of fruit trait-related variation in fruit trees, especially when mutations occur in transcription factor genes, which may lead to a series of trait changes. The polymorphic sites of MYB1 detected in this study, especially the polarity changes of amino acid in the differential sites of functional domains, may lead to the influence of transcription factors on the regulation of anthocyanin synthesis genes, thereby affecting the regulation of anthocyanin synthesis. Previous studies have demonstrated that there are indeed examples of gene mutations that alter fruit color, such as reddening of red meat umbilical orange which is thought to be associated with the accumulation of lycopene caused by a mutation of the lycopene β-cyclase gene (non-polar methionine changed into alkaline arginine) (Xu, 2002). Red-fleshed apples were associated with mutations in the MYB10 promoter region (Espley et al., 2009). However, the differences detected in MYB1 of functional domains or other non-functional domains, especially two polymorphic sites with changes between basic arginine and non-polar leucine in functional domains, were likely to cause changes in gene function. Although some of the samples collected from Malus plants have been studied, there may be a limitation of small coverage for the entire genus Malus. Whether the mutations in the polarity changes of the two amino acids do affect the function of transcription factors needs further validation and analysis. MYB1 was only amplified from six of the nine Malus plants except for Golden Delicious, Malus micromalus Makino and Delicious, even after several replacements of primer design sites (other primers were not listed in this paper). However, the amplification results of other materials were stable. It may be related to the variation of amplification sites or amplification conditions. MYB1 was not amplified from two cultivars and a In this study, the obvious differences in evolutional conservation were observed in the alleles MYB10 and MYB1 in Malus plants. There are many variations in the sequence of MYB1 within Malus, but the expression site is single, while the sequence of MYB10 is conserved, which can express in more tissues. The two polymorphism sites of MYB1 may affect the regulation of anthocyanin synthesis after domain prediction, thus affecting fruit pigmentation, and there is differentiation in the genes function responsible for allele evolution. The results can provide a certain data for further exploring the fine regulation of fruit color in Malus and provide theoretical reference for the study of the evolution of fruit color in Malus. There are many variants in this locus. Further study of the functional differentiation of these variants from an evolutionary view may be a new direction in the study of fruit color in Malus.

Experimental materials
The leaves of plants more than three years old entering the flowering and fruiting period were frozen and stored in a refrigerator at -80℃,

RNA extraction
RNA was extracted by modified CTAB method (Kiefer et al., 2000). DNA was digested and removed by DNase I (Takara). The total RNA was detected by electrophoresis and then frozen in refrigerator at -80℃.

RT-PCR
The total RNA was reversed by the primer oligo(dT)18 and the reverse enzyme M-MLV (Promega). The protocol was followed by the instructions of M-MLV. The Premix ExTaq TM HotStart Version (Takara) was employed to process the PCR with reverse transcripts. The amplification system and procedures were carried out according to the instructions. The amplification of actin gene was used to detect the quality of cDNA.
The amplification primer of MYB1 was based on the sequence of EF016490.1 in NCBI database, and the primer of MYB10 was based on NM_001328848.1. The primer premier 5.0 was used to design on the both sides of open reading frame, and actin primer was designed with sequence NM_001301116 (Table 3). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Cloning and sequencing
After the PCR products have been detected by electrophoresis, T-A clone is carried out by using the pEASY-T1 vector according to the instructions. After the confirmation by the original amplified PCR primers, the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and each gene was sequenced by two-ways, with three biological repetitions to eliminate the error.

Sequence analysis and alignment
The sequencing results were compared with the NCBI database by blast program, and then DNAman 6.0 software was used for cluster analysis (Non experimental sequence came from NCBI database) after machine translating. The Protparam was used for basic information analysis of protein chemistry (https://web.expasy.org/protparam/), and domain information collected from NCBI database.