Introduction

Citrus, which have been cultivated globally, is one of the most important fruit species1. Shiranuhi [Citrus reticulata × (Citrus reticulata × Citrus sinensis)] and Huangguogan (Citrus reticulata × Citrus sinensis) are relatively new hybrid citrus cultivars grown in China2. Etiolation, which occurs in many angiosperms, refers to the phenomenon that leaves appear yellow under dark conditions. Seedlings undergo skotomorphogenesis in lightless culture condition, and leaves take on the color of carotenoids. This phenomenon results in a rapid elongation of the hypocotyl3. Changes in plant morphology and growth are the ultimate reflection of plant damages caused by etiolation. This phenomenon leads to decreased leaf and optical areas, the production of dwarf plants, a weakened growth potential, and even death. A stable supply of chlorophyll, which is the main photosynthetic pigment of plants, is a key requirement for the normal development of photosynthesis4. Analyses of light regulation5, ethylene responses6, riboflavin biosynthesis7, endogenous abscisic acid levels3, phospholipid hydroperoxide glutathione peroxidase activity8, and proteomics analysis9 have been used to examine plant growth and development induced by etiolation. Many studies have focused on the expression analysis of related genes during photosynthesis in greening, and the regulation of the corresponding protein levels10. However, there have been relatively few transcriptome-level studies of variegation in citrus seedlings.

Variegated plants, which have both green and white or yellow areas on the same leaf, are considered as the invaluable materials for studying chloroplast biosynthesis, development and maintenance11. The white sectors of var2, which is one of the Arabidopsis (Arabidopsis thaliana) mutants showing leaf variegation, are active tissues that are formed by viable cells with undifferentiated plastids12. Chloroplast development and chlorophyll accumulation are inhibited at the albinism growth stage of albino tea cultivars13. In photosynthetic organisms, chlorophyll and carotenoid are the main pigments of plants that capture light energy. Earlier studies revealed that the etiolated growth of seedlings or leaves considerably affects chloroplast development and chlorophyll metabolism14,15,16. Whether the seedlings undergo etiolation or exhibit variegation, both lead to color changes of leaves.

Next-generation sequencing (NGS) technology is a rapid and cost-effective approach to analyzing a large number of protein-coding genes17,18. This technology can be used to address questions related to ecological comparative and evolutionary genomics in non-model organisms19. Based on the similar expression pattern, using transcriptomic data to group differentially expressed genes (DEGs) is one of the most effective methods to explore the relationships and predict functions of candidate genes20,21. Similarly expressed genes are often functionally related22. In the present study, we identified variegation -related genes and examined the pathways associated with photosynthesis and chlorophyll biosynthesis using RNA-sequencing (RNA-seq) combined with the analysis of chlorophyll contents to characterize the variegation in citrus seedlings. Our findings may help clarify the molecular basis of variegation.

We recently analyzed the transcriptome of etiolated citrus seedlings, there were variegated and green seedlings in both Shiranuhi and Huangguogan under the same conditions of seedling germination23. Although variegated plants exist widely in nature, the mechanism of citrus leaf variegation and the mechanisms regulating the expression patterns of well-characterized genes involved in photosynthesis and chlorophyll biosynthesis are still unknown. In order to study the color difference of citrus leaf after germination under the same culture condition, transcriptome sequencing was performed on the leaves of citrus seedlings. In the present study, we compared the leaf biomass and chlorophyll contents of variegated and green Shiranuhi and Huangguogan seedlings. In addition to the differentially expressed photosynthesis-related genes, the expression profiles of chlorophyll biosynthesis-related genes were analyzed to reveal the molecular mechanism underlying variegation.

Results

Biomass accumulation of variegated and green seedlings

Variegated and green Shiranuhi and Huangguogan seedlings were sampled at 20 days after germination to measure dry weight, length, and chlorophyll contents. We observed a significant difference in shoot and root dry weight between the variegated and green seedlings. Additionally, the roots of variegated seedlings were longer than those of green seedlings for both analyzed cultivars, while the opposite trend was observed for the shoots. Furthermore, the shoot lengths of green seedlings were longer than those of variegated seedlings in both Shiranuhi and Huangguogan, while the roots were shorter (Fig. 1). These results indicated that the variegation of citrus leaves had different effects on seedling root and shoot lengths.

Figure 1
figure 1

Effect of etiolation on growth of variegated and green seedlings of Huangguogan and Shiranuhi.

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Chlorophyll content and photosynthesis of variegated seedlings

The chlorophyll a (Chl a) concentration of variegated Shiranuhi and Huangguogan seedlings were 0.56 and 0.71 mg g−1 fresh weight, respectively (Fig. 2), while the corresponding concentrations in green seedlings were 1.86 and 2.22 mg g−1 fresh weight, respectively. Similarly, chlorophyll b (Chl b) and carotenoid contents as well as the composition and proportion of photosynthetic pigments exhibited the same trends as the Chl a content.

Figure 2
figure 2

Effect of etiolation on the photosynthetic pigment contents of variegated and green seedlings of Huangguogan and Shiranuhi.

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The photosynthetic parameters differed between the Huangguogan and Shiranuhi variegated and green seedlings (Fig. 3). For example, the net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were lower in variegated seedlings than in green seedlings. Additionally, the photosynthetic parameter values were lower for Huangguogan than for Shiranuhi seedlings, except for the Ci.

Figure 3
figure 3

Photosynthetic gas exchange parameters of variegated and green seedlings of Huangguogan and Shiranuhi.

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Sequencing and assembly of expressed genes

To investigate the genome-wide expression patterns of variegated seedlings, more than 10 leaves harvested from each seedling type for each cultivar at 20 days after germination were analyzed by RNA-seq. We constructed four cDNA libraries, namely R_V and R_G, which represent variegated and green Shiranuhi seedlings, respectively, and Y_V and Y_G, which correspond to variegated and green Huangguogan seedlings, respectively. After removing the sequencing adapters and low-quality data, we obtained 567.07 million clean reads and 85.05 Gb of RNA-seq data. More than 94.19% of the reads had a quality score of Q30 (sequencing error rate = 0.1%). The sequencing data statistics are listed in Table 1. The raw data were deposited in the NCBI Gene Expression Omnibus database (accession number GSE90935).

Table 1 Overview of the sequencing results.
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Evaluation of gene expression levels

The number of fragments per kilobase of exon per million fragments mapped (FPKM) was used to quantify the unigene expression levels, which were detected by RNA-seq with high sensitivity. Hierarchical cluster analyses were conducted with 66 DEGs identified for variegated and green Shiranuhi and Huangguogan seedlings. Genes with the same or similar expression profiles were clustered to present the gene sets exhibiting different expression patterns under various experimental conditions (Fig. 4).

Figure 4
figure 4

Hierarchical cluster analysis of DEGs in four libraries. Hierarchical cluster analysis was carried out with 66 significantly differentially expressed genes in green and variegated seedlings of Shiranuhi and Huangguogan. Value = log2 FPKM.

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Identification and functional annotation of differentially expressed genes

During the screening of DEGs, we used P value < 0.00524 and |log2 (fold change)| > 1 as the threshold criteria to determine the significance of the differences in gene expression levels. A total of 4,786 and 7,007 DEGs between variegated and green Shiranuhi and Huangguogan seedlings were classified into the gene ontology (GO) categories biological process (GO-BP), molecular function (GO-MF), and cellular component (GO-CC) (Figs 5–6). For the R_V library, significant differences in the enrichment of 30 biological processes were detected in the GO-BP class, including photosynthesis (79 DEGs), cellular hormone metabolic process (44 DEGs), hormone metabolic process (44 DEGs), regulation of hormone levels (44 DEGs), oxidation-reduction process (485 DEGs), and other processes. Additionally, ADP binding (79 DEGs), oxidoreductase activity (482 DEGs), 3-beta-hydroxy-delta 5-steroid dehydrogenase activity (39 DEGs), and 19 other processes were significantly enriched in the GO-MF class. Furthermore, photosystem (72 DEGs), photosystem II oxygen-evolving complex (25 DEGs), chloroplast (40 DEGs), chloroplast thylakoid membrane (14 DEGs), chloroplast thylakoid (14 DEGs), photosystem II (51 DEGs), and photosystem I (22 DEGs) were significantly enriched in the GO-CC class (Fig. 6). For the Y_V library, significant differences in enrichment were observed for 83 sub-categories in the GO-BP class, 28 in the GO-MF class, and 32 in the GO-CC class. A comparison of both libraries indicated that 15 sub-categories were related to the GO-BP class, such as oxidation-reduction process (GO: 0055114), single-organism metabolic process (GO: 0044710), and photosynthesis (GO: 0015979). Meanwhile, 13 sub-categories were related to the GO-CC class, such as photosystem I (GO: 0009522), chloroplast thylakoid (GO: 0009534), chloroplast thylakoid membrane (GO: 0009535), photosynthetic membrane (GO: 0034357), photosystem (GO: 0009521), and chloroplast (GO: 0009507). In contrast, only oxidoreductase activity (GO: 0016491) was related to the GO-MF class (Fig. 6).

Figure 5
figure 5

Differential expression analysis of unigenes in the variegated seedlings (M) versus the green seedlings (G) of Shiranuhi (R) and Huangguogan (Y). The log2(fold change) is used as the x-axis and −log10(pdaj) is used as the y-axis. 2273 and 3880 unigenes were up-regulated in R_V and Y_V (highlighted in green), and 2513 and 3127 unigenes were down-regulated (highlighted in red), respectively.

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Figure 6
figure 6

Gene Ontology functional classification analysis of genes with different expression patterns of variegated seedlings of Shiranuhi and Huangguogan (P-value < 0.01). Gene ontology biological process (GO-BP), gene ontology cellular component (GO-CC), and gene ontology molecular function (GO-MF).

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To investigate the biological pathways important for the variegation in Shiranuhi and Huangguogan seedlings, the DEGs between variegated and green seedlings were further annotated based on the reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Fig. 7). These DEGs in the R_V and Y_V libraries were assigned to 117 and 119 KEGG pathways, respectively. The pathway search results were sorted based on the number of hits, and photosynthesis (44 and 63 DEGs), photosynthesis-antenna proteins (14 and 29 DEGs), and flavonoid biosynthesis (16 and 29 DEGs) were the most highly represented pathways in both libraries. Genes associated with cysteine and methionine metabolism (47 DEGs), monobactam biosynthesis (9 DEGs), and ABC transporters (15 DEGs) were also common in the R_V library (Fig. 7). In contrast, genes related to carbon fixation in photosynthetic organisms (80 DEGs), ribosome (154 DEGs), and porphyrin and chlorophyll metabolism (41 DEGs) were common in the Y_V library (P value < 0.05).

Figure 7
figure 7

Statistics of the top 20 KEGG pathway enriched with genes with different expression patterns of variegated seedlings of Shiranuhi and Huangguogan.

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Analysis of chlorophyll biosynthesis and photosynthesis-related transcripts

Chlorophyll is a key photosynthetic pigment in plant chloroplasts, and chlorophyll metabolism is an important factor influencing crop yield25. To investigate whether photosynthesis pathway genes are involved in this decrease, we analyzed the expression patterns of genes encoding regulatory enzymes associated with chlorophyll biosynthesis (Fig. 8). Fifteen enzymes are required for the chlorophyll biosynthesis from glutamyl-tRNA to Chl b26. Magnesium (Mg) chelation is the first step in the cholorophyll branch of porphyin metabolism, forming magnesium-protoporphyrin (MgP, EC: 2.1.1.11), which is followed by a SAM-dependent methylation of the 6-propionate side chain to form Mg-protoporphyrin monomethyl ester (MgPME)27. We observed that the expression of the gene encoding magnesium chelatase subunit I (MgCE I, EC: 6.6.1.1) was down-regulated from 580.67 to 15.05 FPKM (Cluster-2274.70486) in the Y_V library. Magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase (MgPMEC, EC: 1.14.13.81) is another key enzyme involved in chlorophyll biosynthesis. Our RNA-seq data revealed a considerable difference in the abundance of MgPMEC transcripts (Cluster-2274.8338), with 46.77 FPKM in the Y_G library and 0 FPKM in the Y_V library, this was consistent with previous research about variegated Epipremnum aureum11. Meanwhile, protochlorophyllide reductase (POR, EC: 1.3.1.33) catalyzes a reaction with divinyl-protochlorophyllide as a substrate to synthesize divinyl-chlorophyllide a25. The expression levels of the gene encoding POR (Cluster-2274.3601) were down-regulated, with 19.73 FPKM in the Y_G library and 0 FPKM in the Y_V library. Furthermore, in the chlorophyll biosynthesis pathway, 7-hydroxymethyl chlorophyll a reductase (HCAR, EC: 1.17.7.2) converts Chl b to 7-hydroxymethyl Chl a and ultimately to Chl a28. We observed no changes in the expression levels of the gene encoding HCAR in the Y_V library, but it was down-regulated from 51.14 to 10.30 FPKM (Cluster-2274.20724) in the R_V library.

Figure 8
figure 8

Chlorophyll metabolism-related gene expression-level changes in the chlorophyll biosynthesis pathways of (A) Shiranuhi and (B) Huangguogan seedlings. Numbers in boxes correspond to the following EC numbers: 4.2.1.24, porphobilinogen synthase. 2.5.1.61, hydroxymethylbilane synthase. 4.1.1.37, uroporphyrinogen decarboxylase. 4.2.1.75, uroporphyrinogen-III synthase. 4.1.1.37, uroporphyrinogen decarboxylase. 1.3.98.3, oxygen-independent coproporphyrinogen III oxidase. 1.3.3.4, protoporphyrinogen oxidase. 6.6.1.1, magnesium chelatase subunit H, D, I. 2.1.1.11, magnesium-protoporphyrin O-methyltransferase. 1.14.13.81, magnesium- protoporphyrin IX monomethyl ester (oxidative) cyclase. 1.3.1.75, divinyl chlorophyllide a 8-vinyl-reductase. 1.3.1.33, protochlorophyllide reductase. 2.5.1.62, chlorophyll synthase. 3.1.1.14, chlorophyllase. 1.14.13.122, chlorophyllide a oxygenase. 1.1.1.294, chlorophyll(ide) b reductase. 1.17.7.2, 7-hydroxymethyl chlorophyll a reductase.

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In comparisons between the R_V and R_G libraries and between the Y_V and Y_G libraries, we detected six common DEGs related to porphyrin and chlorophyll metabolism, namely Cluster-2274.78784 (K08099, 3.1.1.14), Cluster-2274.38688 (K13071, 1.14.15.17), Cluster-2274.8338 (K04035, 1.14.13.81), Cluster-2274.66782 (K03403, 6.6.1.1), Cluster-2274.56424 (K13071, 1.14.15.17), and Cluster-2274.70486 (K03428, 2.1.1.11) (Fig. 8). The comparison between the R_V and R_G libraries revealed 44 DEGs related to the photosynthesis pathway, including 13 genes related to photosystem I (PSI), 15 related to photosystem II (PSII), four related to the cytochrome b6/f complex, three related to photosynthetic electron transport, and nine related to the F-type ATPase. In contrast, the comparison between the Y_V and Y_G libraries detected 63 DEGs associated with the photosynthesis pathway, including 26, 15, 5, 10, and 7 related to PSI, PSII, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase, respectively.

In comparisons between the R_V and Y_V libraries and between the R_G and Y_G libraries, the following nine common DEGs associated with the photosynthesis pathway were identified: Cluster-2274.72645 (PsbQ), Cluster-2274.39503 (PetF), Cluster-2274.81464 (PetF), Cluster-2274.51835 (PetB), Cluster-2274.57179 (F-type ATPase a), Cluster-2274.57012 (PsaA), Cluster-19856.1 (PsaN), Cluster-17851.0 (PsbP), and Cluster-2274.55964 (PsaF), and these DEGs were marked on KEGG imagery (map00195) from Kanehisa laboratories29,30 (Fig. 9). Additionally, 15 genes involved in photosynthetic activities and chlorophyll biosynthesis may be associated with the differences in leaf color between the R_V and Y_V seedlings (Figs 8,9).

Figure 9
figure 9

Differentially expressed genes mapped to photosynthesis pathway. The known pathways were obtained from the KEGG database. Red square denotes up-regulated expression in variegated seedlings of Shiranuhi and Huangguogan compared with that of green seedlings, respectively, while green square denotes both up- and down-regulated genes. We have got the formal permission of KEGG imagery (map00195)29,30 from Kanehisa laboratories.

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qRT-PCR validation

We selected photosynthetic genes (PsbQ, PetF, PetB, PsaA, PsaN, PsbP, and PsaF) and 6 porphyrin and chlorophyll metabolism pathway genes from common DEGs in R_V and Y_V libraries and evaluated their expression profiles using quantitative real-time PCR. Actin (GenBank: XM 006480741.2) was selected for internal controls. The verification results of the 7 photosynthesis-related and 6 chlorophyll metabolism-related DEGs demonstrated that nearly all of the common photosynthetic genes showed similar expression patterns of RNA-seq analysis (Fig. 10). In the porphyrin and chlorophyll metabolism pathway, there were three common DEGs (Cluster-2274.8338, Cluster-2274.38688, Cluster-2274.78784) with a similar expression pattern in R_V and Y_V seedlings. The other three common DEGs showed the opposite expression pattern, up-regulated in R_V and down-regulated in Y_V (Fig. 10).

Figure 10
figure 10

Expression pattern of 7 photosynthesis-related and 6 chlorophyll metabolism-related genes as obtained by RNA-seq and qRT-PCR. (A) qRT-PCR validation for the 7 photosynthesis-related genes of Shiranuhi. (B) qRT-PCR validation for the 7 photosynthesis-related genes of Huangguogan. (C) qRT-PCR validation for the 6 chlorophyll metabolism-related genes of Shiranuhi. (D) qRT-PCR validation for the 6 chlorophyll metabolism-related genes of Huangguogan. R_V, R_G, Y_V and Y_G represent normalized expression levels for the DEGs in the Shiranuhi and Huangguogan libraries, respectively. FC is the ratio of FPKM between variegated and green seedlings.

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Discussion

The purpose of this research was to study the differences in the transcriptome expression of variegated and green Huangguogan and Shiranuhi seedlings, especially regarding photosynthesis and chlorophyll biosynthesis. We identified major transcriptomic features in the variegated seedlings.

Photosynthetic activities are affected by stomatal conductance (Gs), Ci, Tr, and chlorophyll fluorescence31. The Pn directly reflects the photosynthetic capacity32, while Ci is an important indicator of stomatal activity. During photosynthesis, a decrease in Tr caused by a decrease in Gs leads to a significant decrease in transpiration, which can inhibit the absorption and transport of water and nutrients33. In addition to shorter roots, the variegated seedlings also had significantly lower seedling biomass, chlorophyll content, and photosynthetic parameter values (P < 0.05) compared with the green seedlings (Figs 1–3). The identification and functional annotation of DEGs indicated that the photosynthesis pathway genes (44 and 63 DEGs) were the most common genes in the R_V and Y_V libraries (based on the number of hits for DEGs), with the lowest corrected P values (8.17212942029e-11 and 6.07238386484e-12) (Fig. 7).

Transcriptional regulation is important for coordinating the conversion of glutamyl-tRNA to the final product (Chl a and Chl b). There are 15 enzymes encoded by 27 genes in Arabidopsis thaliana in chlorophyll synthesis4. The six-electron oxidation of protogen IX is catalyzed to protoporphyrin IX (proto IX) by Protoporphyrinogen IX (protogen IX) oxidase (PPOX), with flavin as the cofactor34. Previous studies revealed that MgPMT (Mg-protoporphyrin IX methyltransferase) activity is inversely related to the transcriptional activities associated with Mg-chelatase and ALA synthesis, and restricts the formation of chlorophyll4,35. Low expression levels of Cluster-2274.8338 (K04035, 1.14.13.81) lead to a decrease in divinyl protochlorophyllide synthesis, while the up-regulated expression of Cluster-2274.78784 (K08099, 3.1.1.14) induces the transformation of Chl a to Chlorophyllide a. Furthermore, the up-regulated expression of the Cluster-2274.56424 (K13071, 1.14.15.17) gene enhances the synthesis of the chlorophyll catabolite during chlorophyll biosynthesis.

Three common differentially expressed genes of porphyrin and chlorophyll metabolism pathway with the same expression pattern (Cluster-2274.8338, Cluster-2274.38688, Cluster-2274.78784) in the variegated Shiranuhi and Huangguogan seedlings were blasted in Citrus Sinensis Annotation Project (http://citrus.hzau.edu.cn/orange/index.php). The sequence of Cluster-2274.8338 was similar to Cs6g16200 (the e-value was 4e-77, and ident was 83%), which is the ZIP1-like gen of Citrus sinensis. In addition, the sequences of ZIP1 (GenBank: FJ940751.1) of Oryza sativa Japonica Group (Japanese rice) in NCBI have been compared with transcriptome data by sequence alignment. The percentage of identical matches between ZIP1 and Cluster-2274.8338 was 99.92%, the total score was 2261, and the expect value (e-value) was 1.37e-26. On the other hand, Cluster-2274.8338 (ZIP1) was down-regulated in R_V and Y_V, and the log2 FC were −2.7399 and −2.5467, respectively (Fig. 10). Previous studies have found that EaZIP transcripts may not accumulate or become unstable in the early stage of plastid development36, leading to the loss of MPE cyclase, and the low expression results in chlorophyll losing and forming the diverse phenotype of leaf color11. Cluster-2274.38688 was similar to Cs8g15480.1, which encodes protochlorophyllide-dependent translocon component 52 (PTC52). PTC52 is considered to be part of a unique transposon and is most abundant in etiolated plants37. In this study, Cluster-2274.38688 gene was found to be up-regulated in variegated seedlings (Fig. 10). In addition, Cluster-2274.78784 was similar to Cs9g07520 (the total score was 3675, query cover was 78%, e-value was 0, and ident was 99%), which was noted to be involved in chlorophyll catabolic process (GO: 0015996). The up-regulated expression of Cluster-2274.78784 might lead to the decomposition of Chl b to chlorophyllide b. These results indicated that the formation of variegated seedlings may be caused by decreased chlorophyll synthesis and increased catabolism.

Nuclear-encoded genes affecting photosynthesis are co-expressed to assemble PSI and PSII during the de-etiolation of plants34,38. The transcription levels of several photosynthesis-related genes, such as PsbA39, LHCA1–6 and LHCB1–640, as well as LSU and SSU, are regulated by light during chloroplast biogenesis in de-etiolating plants41. In this study, we detected 9 common DEGs involved in the photosynthesis pathways of the variegated Shiranuhi and Huangguogan seedlings. Additionally, we identified 6 common DEGs related to porphyrin and chlorophyll metabolism. The differential expression of these genes may be critical for the leaves variegation in citrus seedlings, but need to be confirmed in future work.

Materials and Methods

Plant materials

The Shiranuhi and Huangguogan hybrid citrus cultivars analyzed in this study (Fig. 11) were provided by the Institute of Pomology and Olericulture, Sichuan Agricultural University, China. Refer to the method of Xiong et al.23 for seed germination and seedling culture. Seeds were presoaked in water for 4 h and then incubated at 25 °C for 3 days, sown in pots (vermiculite: perlite = 1:1) and then transferred to a growth chamber at 25 °C, 50–60% relative humidity, and 12-h light/12-h dark period. After germination (i.e., radicle breaks through the seed coat), seedlings were watered every 2 days. The Shiranuhi variegated seedlings (R_V), Shiranuhi green seedlings (R_G), Huangguogan variegated seedlings (Y_V), and Huangguogan green seedlings (Y_G) sample contained more than 20 seedlings were used for physiological and transcriptome analyses.

Figure 11
figure 11

Variegated and green Shiranuhi and Huangguogan seedlings 20 days after germination. R_V, Shiranuhi variegated seedlings. R_G, Shiranuhi green seedlings. Y_V, Huangguogan variegated seedlings. Y_G, Huangguogan green seedlings.

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Photosynthetic characteristics

Three seedlings of each sample were selected as test materials. The photosynthetic parameters of the second fully unfolded leaves from the base of the seedlings were recorded 20 days after germination. The Pn, Tr, Ci, and Gs were measured using the LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). The photosynthetically active radiation and CO2 concentration were 1,200 µmol m−2 s−1 and 400 µmol m−2 s−1, respectively. Additionally, Gs and Ci were determined at a saturated light intensity of 1,000 µmol m−2 s−1 and 70% relative humidity42.

Chlorophyll and carotenoid contents

All leaves, used for the determination of photosynthetic characteristics, were harvested from variegated and green seedlings for each cultivar at 20 days after germination. Once harvested, plant materials were quick frozen using liquid nitrogen and stored at −80 °C until using. The chlorophyll and carotenoid contents of seedlings 20 days after germination were estimated as previously described using the following epuations43: Chl a = (12.7OD663 − 2.69OD645) × 10/1000 W; Chl b = (22.9OD645 − 4.68OD663) × 10/1000 W; Carotenoid = (1000OD470 − 3.27 Chl a − 104 Chl b)/229 × 10/1000 W.

Root and shoot dry weights and lengths

All seedlings, used for the determination of photosynthetic characteristics, were collected and divided into shoots and roots. The root and shoot lengths were measured as previously described44. After the green removing process at 110 °C for 24 h, the dry weight of root and shoot was measured.

RNA extraction and qualification

Three biological replicates were collected for Shiranuhi and Huangguogan seedlings. Total RNA was extracted from R_V, R_G, Y_V, and Y_G with the PureLink Plant RNA Reagent (Invitrogen, CA, USA). The purity of the extracted RNA was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA), while RNA integrity was assessed using the RNA 6000 Nano Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA)45.

Preparation of cDNA libraries and transcriptome sequencing

We purified mRNA from 1.5 µg total RNA using poly-T oligo-attached magnetic beads. The RNA was fragmented using divalent cations in 5 × NEBNext First Strand Synthesis Reaction Buffer. First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H−). Second-strand cDNA was subsequently synthesized using DNA Polymerase I and RNase H23. The remaining overhangs were converted into blunt ends by exonuclease/polymerase activities. The NEBNext adapter with a hairpin loop structure was ligated to adenylated 3′ ends of DNA fragments for a subsequent hybridization. To preferentially select cDNA fragments that were 150–200 bp long, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). The size-selected, adapter-ligated cDNA sequences were treated with 3 µl USER Enzyme (NEB, USA) at 37 °C for 15 min followed by a 5-min incubation at 95 °C. A PCR was then completed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and an Index (X) Primer. The PCR products were purified using the AMPure XP system, and library quality was assessed using the Bioanalyzer 2100 system46. The index-coded samples were clustered with the cBot Cluster Generation System of the TruSeq PE Cluster Kit v3-cBot-HS (Illumina)47. The resulting libraries were sequenced using the HiSeq. 4000 system (Illumina), which generated paired-end reads.

Quality control

Refer to the previous method, raw data (raw reads) in a fastq format were first processed using in-house perl scripts. Clean data (clean reads) were obtained by removing reads containing the adapter, reads with poly-N sequences, and low quality reads from the raw data. Additionally, the Q20, Q30, GC content, and sequence duplication level were calculated for the clean reads, which were used in all downstream analyses48.

Functional annotation of genes

Gene functions were annotated based on the GO49 and KEGG pathway databases50.

Analysis of differentially expressed genes

Gene expression levels were estimated using the RSEM program51. Clean reads were mapped based on the assembled transcriptome, and the read count for each gene was obtained from the mapping results. Genes differentially expressed between two groups were analyzed using the DESeq R package (1.10.1), which provides statistical algorithms for detecting DEGs using a model based on a negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach to control the false discovery rate52. Genes with an adjusted P value < 0.05 following the DESeq analysis were considered to be differentially expressed between groups52.

Analysis of GO and KEGG pathway enrichment of differentially expressed genes

The GO enrichment analysis of the DEGs was completed using the GOseq R packages based Wallenius’ non-central hypergeometric distribution53, which can adjust for gene length biases among the DEGs. We used KOBAS software to test the significance of the enrichment of DEGs in specific KEGG pathways54.

Validation by qRT-PCR analysis

Leaves harvested from three independent seedlings of variegated and green samples of both Huangguogan and Shiranuhi were used as three biological replicates. Quantitative real-time PCR (qRT-PCR) analysis using the Bio-Rad CFX Manager (Bio-Rad, USA) with SsoFastTM EvaGreen Supermix (Bio-Rad) was employed to verify the DEG expression results23. Primers (Table 2) for specific genes encoding photosynthesis DEGs were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and synthesized by Sangon Biotech44. All primers were amplified with no template control to make sure the amplicons were not primer dimers. Gene expression levels were normalized against the geometric mean of citrus reference gene, Actin (GenBank: XM 006480741.2) and calculated by 2−ΔΔCT method55.

Table 2 Primers for qRT-PCR analysis.
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