Complete chloroplast genome studies of different apple varieties indicated the origin of modern cultivated apples from Malus sieversii and Malus sylvestris

Sampling and DNA extraction

In this study, 24 samples of four representative Malus varieties (Red Delicious, Golden Delicious, Red Fuji, and Ralls) were collected from different provinces in China. All samples were immediately frozen with silica gel and stored at −20 °C (Yang et al., 2020). Red Fuji varieties were selected from three provenances with three individuals in each region; for other cultivars, three different provenances were selected for each cultivar (Table 1). We extracted DNA from 24 samples using the new Tiangen plant genomic DNA extraction kit (Tiangen, Urumqi, Xinjiang, China).

Chloroplast genome sequencing, assembly and annotation

The complete chloroplast genome of 24 cultivated apple accessions were sequenced using the next-generation sequencing. The tested DNA samples were randomly interrupted by Covaris ultrasonic crusher, and the whole library preparation was completed via end repair, addition of polyA-tail, addition of sequencing adapters, purification, PCR amplification. The constructed libraries were sequenced using the Illumina MiSeq 2,000 platform. After they passed the library inspection, the different libraries were pooled according to the effective concentration and target downstream data volume and then sequenced using Illumina HiSeq/MiSeq. The raw image data files obtained from high-throughput sequencing were converted into raw sequences (sequenced reads) by CASAVA base identification analysis, which we called raw data or raw reads, and the results were stored in FASTQ file format, which contains sequence information (reads) and their corresponding sequencing quality information. For each sample, approximately 50.0 GB of raw data were generated, which were assembled using the programme NOVOPlasty (Dierckxsens, Patrick & Guillaume, 2017) with M. sieversii (MH890570.1) as the reference. The focus of the assembly is on the NOVOPlasty configuration file, also known as config.txt; the settings included config.txt parameter values, K-mer = 39, The reference sequence was uploaded; forward reads, and reverse reads uploaded that need to be assembled. The best quality of the assembly appeared as a circular assembly file; however, in general, there are two options, both of which are correct owing to the indeterminate gene order of the two reverse repeat regions of the chloroplast. When one wants to determine which one is correct, sequence comparisons or gene annotations must be performed. All annotated genes were manually curated using Sequin. 16.0 (Wyman, Jansen & Boore, 2004), and start/stop codons and intron/exon boundaries were adjusted using Geneious v4.8.5. Finally, we used the online OGDRAW software to generate a circular genome map (Lohse, Drechsel & Bock, 2007).

Characterization of chloroplast genomes in Malus

The total length of the genome and the length of each region, including LSC, SSC, and inverted repeats (IRs; a pair of reverse complementary repeat regions), gene composition (protein-coding genes, transcriptional RNA genes, ribosomal RNA genes, introns, and exons), base composition, and GC (AT) content were analysed using the Geneious v4.8.5 software.

Analysis of the LSC, SSC, and IR border regions

The IRscope (http://irscope.shinyapps.io/irapp/) online analysis software was used to upload the manually annotated Genbank file and generate a boundary map (Ali, Jaakko & Peter, 2018). Finally, the length differences and related genes of the four regions in different apple varieties were analysed.

Repeat sequence and simple sequence repeats (SSRs)

Four repeats of the chloroplast genome, which included repeats, forward repeats, complement repeats, and palindromic repeats, were identified using the REPuter (https://bibiserv.cebitec.unibielefeld.de/reputer) online software (Kurtz et al., 2001). The setting parameters were as follows: minimum sequence length of 30 bp, edit distances of three bp, minimum alignment score of 50, and sequence identity > 90%. Finally, repetitive sequences of the chloroplast genome were obtained.

MISA (https://webblast.ipk-gatersleben.de/misa/index.php) software was used to detect SSR loci in the genome sequences of wild and cultivated apples (Biswas et al., 2011). The repeat units are mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides. Default: definition (unit_size, min_repeats): 1-10 2-6 3-5 4-5 5-5 6-5; interruptions (max_difference_for_2_SSRs): 100.

Codon usage analysis

All protein-coding genes were used for determining the codon usage. We further analyzed the codon usage frequency and relative synonymous codon usage (RSCU) based on sequences of 83 protein-coding genes in the Malus chloroplast genomes. Avoiding the influence of the amino acid composition, we examined the RSCU using MEGA v7.0, and the results are presented as charts (Sharp & Li, 1987).

Variation analysis

Although chloroplast genomes are highly conserved with regard to composition and sequence, there are some internal mutations and structural variations among the chloroplast genomes of different varieties. In this study, the online software mVISTA (http://genome.lbl.gov/vista/index.shtml) was used for sequence alignment and variation analysis of the apple chloroplast genome (Frazer et al., 2004). The degree of variation in the complete sequence can be evaluated by comparing the similarities between the coding region, non-coding region, introns, and exons of the input sequence with the other sequences. To identify SNPs or short insertion/deletions (InDels), we used the Geneious. v4.8.5 software.

Phylogenetic analysis

To study the phylogenetic position of the chloroplast genomes of cultivated apples and other genera of the Rosaceae family, phylogenetic analysis was performed based on the chloroplast genome sequences for four cultivated apples and 16 Rosaceae species obtained from NCBI (National Center for Biotechnology Information (nih.gov))—M. hupehensis (MK020147.1), M. prunifolia (KU851961.1), M. baccata (KX499859.1), M. micromalus (MF062434.1), M. sylvestris (MK434924.1), M. sieversii (MK434920.1), M. yunnanensis (NC_039624.1), M. florentina (NC_035625.1), M. trilobata (NC_035671.1), Pyrus hopeiensi (MF521826.1), Pyrus ussuriensis (MF521826.1), Pyrus pyrifolia (NC_015996.1), Crataegus kansuensis (MF784433.1), Prunus pedunculata (MG869261.1), Prunus persica (HQ336405.1), and Prunus yedoensis (KU985054.1). We aligned the 20 corresponding sequences using MAFFT 7.017 and adjusted them manually (Katoh et al., 2005). The complete chloroplast genome sequences were used to construct the phylogenetic topology using Bayesian inference (BI) (Huelsenbeck, 2012). Based on the results of the phylogenetic analysis, the phylogenetic relationships between different apple germplasms were further clarified.

Features of the chloroplast genome

The chloroplast genome structure and length of four apple cultivars and two wild ancestors were compared, and the results are summarised in Table 2. The complete chloroplast genome maps of M. domestica and wild apples as shown in Fig. 1. Subtle differences were observed among the chloroplast genome sequences. The results showed that the length of the chloroplast genome of four apple cultivars and two wild ancestors was approximately 160 kb. Among them, the genomes of Red Delicious and M. sieversii were the longest, with their lengths ranging from 160,225–160,290 bp, while those of others were shorter, with lengths ranging 160,067–160,069 bp. The chloroplast genome structures consisted of four stable parts: two IR regions (26,323–26,337 bp), which were separated by an LSC region (88,240–88,440 bp) and an SSC region (19,174–19,340 bp).

M. sieversii and M. sylvestris contained 128 genes, including 83 coding sequence (CDS), 35 tRNA, 8 rRNA, and infA and ycf1 genes. Among the 130 genes annotated in cultivated apple varieties, there were 84 CDS, 8 rRNA, 37 tRNA, and ycf1 genes. The LSC region of M. sieversii and M. sylvestris contained 83 genes, accounting for 64.8% of the chloroplast genome, including 21 tRNA genes, 61 protein-coding genes, and infA gene, whereas the SSC region contained 11 CDS genes, accounting for 8.6% of the chloroplast genome. Eight rRNA genes, 12 CDS genes, and 14 tRNA genes were located in the IR region, accounting for 26.6% of the genome. The LSC region of 24 cultivated apples contained 61 CDS genes and 22 tRNA genes, while 11 CDS genes and 1 tRNA gene were located in the SSC region. All eight rRNA genes, 13 CDS genes, and 13 tRNA genes were located in the IR region. The CDS genes in the LSC, SSC, and IR regions accounted for 64.1%, 9.9%, and 26.0% of the total chloroplast genome, respectively. The GC content of the complete chloroplast genome of Malus ranged from 36.5% to 36.6%, with an average content of 36.56%. Specifically, the GC content in the IR region (42.7%) was higher than that in the LSC region (34.1–34.2%) and SSC region (30.3–30.4%) (Table S1).

All genes were divided into three categories according to their functions: photosynthesis-related genes, transcription and translation-related genes, and other genes (Table 3). Among the protein-coding genes of cultivated apples and wild ancestors, 20 genes contained one intron each (trnI-GAU, rps12, ndhB, trnK-UUU, rpL2, rps16, trnG-UCC, atpF, rpoC1, trnL-UAA, trnV-UAC, petB, petD, rpl16, trnI-GAU, trnA-UGC, ndhA, rps12, ndhB, rpL12, and trnA-UGC), and two (clpP and ycf3) had two introns each. The gene with the largest intron (2,497 bp) was trnK-UUU, and the matK gene was included in this gene.

IR expansion and contraction

Different species have different gene sequences in the four junction regions, and the contraction or expansion of the IR region usually leads to length changes in the chloroplast genome. To further analyse the boundary differences between the two wild species and cultivated apple varieties, four apple samples from different varieties were selected. It can be seen from the results that structural variation was still found in the IR-SSC and IR-LSC boundary in Malus. The IR regions of M. sieversii, M. sylvestris, Red Delicious, Golden Delicious, Red Fuji, and Ralls are shown in Fig. 2. The rps19 gene had an extension of 85 bp in the IRa region and 114 bp in M. sieversii and M. sylvestris. Because the ycfl gene was located at the boundary between SSC and IRb, a 1,074 bp long pseudogene fragment was generated in the IRa region. The ndhF gene crossed the IRa/SSC boundary and was located in the IRa region with a length spanning 12 bp. In addition, the trnH-GUG genes in the four cultivated apple varieties and two wild species were all located in the LSC region, and its distance from the IRb/LSC boundary was between 32 and 82 bp. In general, the genus Malus showed similar characteristics in the IR/SC boundary region.

Repeat sequence and SSR analyses

In this study, Malus-related sequences (n = 24) were narrowed to 11 representative sequences and two progenitor sequences for comparative analysis. In these chloroplast genomes, the number of the four repeat types was similar, and their overall distribution in the chloroplast genome was highly conserved. A total of 633 repeats were detected in the 11 chloroplast genomes, including 337, 220, 60, and 16 forward repeats, palindromic repeats, reverse repeats, and complementary repeats, respectively (Fig. 3A), and four repeats were observed in 52.8%, 35.3%, 9.9%, and 2%, respectively (Fig. 3B). The length of the repeat sequence was 30–65 bp and mainly 30–40 bp among Malus (Fig. 3C). Most of these repeats were located in the intergenic spacer and gene region, accounting for 66% and 27.1%, respectively, while only 6.9% were located in the intron region (Fig. 3D).

The types, distribution and number of SSRs in the chloroplast genome of Malus were counted (Table S2). A total of 2,279 SSRs of single nucleotides, dinucleotides, and complex polynucleotides were detected in the chloroplast genome of Malus. Two wild and 11 cultivated apple sequences were selected. The number of mononucleotide, dinucleotide, and complex nucleotide repeats ranged 45–60, 2–3, and 8–9, respectively, in four cultivated apples and two progenitor speices (Fig. 4A). The most common mononucleotide repeats were A/T repeats. SSRs were broadly similar across genomes, compared with SSC and IR regions, most of which were located in the LSC region, and most SSRs were located in the intergenic spacer region (Fig. 4B, Table S3).

Codon usage

A total of 61 codons encoding 20 amino acids were detected (Fig. 5). The minimum value of Relative synonymous codon usage (RSCU) was 0.47 and the maximum value was 1.85. AGA had the highest RSCU value, ranging from 1.85 to 1.97. Methionine and tryptophan were the least common amino acids, whereas leucine, arginine, and serine were the most common amino acids. The RSCU values of 33 and 34 codons exceeded one for M. sieversii and M. sylvestris, respectively; 31 codons of Red Delicious exhibited greater preference (RSCU > 1), and 32 codons of Golden Delicious, Red Fuji, and Ralls showed greater preference (RSCU < 1). Half of the frequently used codons had RSCU value > 1, and all codons ended in A/U, which was consistent with the rich A/T characteristics of the angiosperm chloroplast genome (Table S4).

Comparative chloroplast genomic analysis

Considering the chloroplast genome sequence of M. sieversii as reference, mVISTA was used to compare the differences in the chloroplast genome sequences of 13 Malus materials. The results are shown in Fig. 6. The whole sequences of 13 chloroplast genomes were highly similar. The sequence variation was mainly concentrated in the non-coding region, and the non-coding region was less conserved than the coding region. In general, the intergenic region was a highly divergent region among these chloroplast genomes, such as psbI-atpA, psbM-psbD, and ndhC-atpE.

In the four-part structure, 135 SNP sites and 107 indel sites were identified (Table S5, S6). Among all SNP sites, the number of highly variable regions located in the IR region was the lowest, and among 26 InDels, large InDels (>50 bp) were found in the trnT-trnL, psaI-ycf4, rpoB-trnC-GCA, trnR-UCU-atpA, psbA-trnK intergenic spacer and trnG-GCC, trnV-UAC, clpP, and trnV-UAC introns.

Phylogenetic analyses

Phylogenetic analyses revealed strong bootstrap support for each node. All Malus species formed a branch with strong guidance support that the four cultivated apples had a close phylogenetic relationship with M. sieversii and M. sylvestris, in which the species M. sieversii and Red Delicious formed a cluster, while the Golden Delicious, Red Fuji, Ralls and M. sylvestris clustered into another branch (Fig. 7). Therefore, M. sieversii was not the unique parent source of the modern cultivated apple; however, it was a major progenitor.