Identification of the MYB Gene Family in Pomelo (Citrus grandis) and Functional Analysis in Chlorosis of Graft Incompatibility

doi:10.13560/j.cnki.biotech.bull.1985.2025-0873

Abstract

Abstract:

Objective Grafting is commonly used in citrus propagation, while scion-rootstock incompatibility often occurs in orchards and leads to economic losses. This study is aimed to investigate the role of MYB family genes in leaf chlorosis associated with scion-rootstock incompatibility, and to elucidate the molecular mechanisms underlying scion-rootstock interaction in Citrus. Method We performed a genome-wide identification and systematic analysis of the MYB gene family in pummelo (Citrus grandis). Based on transcriptome data and RT-qPCR from leaves at different yellowing stages of compatible/incompatible graft combinations, key MYB genes were screened. By employing transient overexpression in tobacco leaves, the function of the candidate gene CgMYB53 in leaf chlorosis associated with scion-rootstock incompatibility was preliminarily validated. Result A total of 143 MYB family members were identified in the pomelo genome, classified into four subfamilies, R1-MYB (35 members), R2R3-MYB (104 members), R3-MYB (3 members), and R4-MYB (1 member). Promoter sequences of MYB members primarily contained cis-acting elements responsive to light (53.23%), hormones (27.94%), and stress (16.07%). Comparative transcriptome analysis of leaves from compatible and incompatible graft combinations, 10 differentially expressed MYB genes were identified. RT-qPCR results showed that the relative expression of CgMYB53 was relatively low in the green leaves, but relatively high in the yellow leaves. Transient overexpression of CgMYB53 in tobacco leaves resulted in significant decrease in chlorophyll a and chlorophyll b content, accompanied by significant increase in amylose, amylopectin, and total starch content,starch synthesis-related genes CgGBSS2、CgISA2 and CgBE3 were highly expressed. Conclusion This study identified 143 MYB family members in pomelo and screened 10 MYB TFs potentially involved in leaf chlorosis associated with scion-rootstock incompatibility. Preliminary functional validation indicates that CgMYB53 may participates in starch synthesis related genes expression, inducing leaf chlorosis by regulating starch accumulation.

Key words: citrus, graft compatibility, starch metabolism, chlorophyll degradation, genome-wide analysis, MYB family gene, promoter analysis, transient overexpression

GUO Dao-xiang, SU Quan, LI Ao-ting, WANG Yan, CHEN Qing, WANG Xiao-rong, HE Wen. Identification of the MYB Gene Family in Pomelo (Citrus grandis) and Functional Analysis in Chlorosis of Graft Incompatibility[J]. Biotechnology Bulletin, 2026, 42(4): 170-181.

Figures/Tables 9

Table 1 Primer sequences for RT-qPCR, gene cloning, and plasmid construction

基因 Gene	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	退火温度 Annealing temperature （℃）	产物长度 Primer length （bp）
β-Tubulin	ACATCCCGCCTAAGGGTCTG	TTCCTCCGAAACATAGCCGTA	58	150
q-CgAPL8	ATTGGAGCCCTTCTCTCAGC	AACTGCTCTGTCTCAGCCAG	58	150
q-CgISA2	CCATTACTACACGGCTCTTCT	GCGATTGAGGCTTCTTGATAA	58	150
q-CgISA3	GGCATTGGGTGACTGAGTTT	CCCATCTCTTCCAATGAGGA	58	150
q-CgMYB53	GAGGCACATTCCCAAAGCTG	TGTTCGTCCTGGAAGTCTCC	58	150
q-Cg2g041650	TGGGAGCTCCCAAGTAAGGC	GGCTTATCAGTCCCCGTTGC	58	150
q-CgGBSS2	TAGCCGCTTCAAGCTTTGTCT	CAATCATTAAGGATGGCCCGTTCT	58	150
q-MYB45	TCATGGTGAGGGCAATTGGG	CAATGACCATCGGTTGCCAA	58	150
q-MYB75	ATTCCCAGCAGAGGCACTAG	CCACGCTCTCAAACACAACG	58	150
q-MYB40	ACTCGAAGAGCGACCCATAA	TATCGTTCGACAAGCCTGGT	58	150
q-MYB113	AGAGGACCAGCTATTGCTGA	ATGATCTCGTCTTCGTCGGG	58	150
q-MYB136	AAGCACAGAAAGGGCTTGTG	AATCCATCTCAGTCTGCAGC	58	150
q-MYB129	CGCCCACATCAAAAAACACG	ATCCATCGGAGTCTGCAACT	58	150
q-MYB2	TCGAGTTGGACAGCCAAGAA	CAACGAGCATCTCATACCGC	58	150
q-MYB89	GAATCAAGTTCTGGCCTGCA	TTGGCTTGGGAGCAAAATCG	58	150
q-MYB14	CTTCGAAAGGGCTTGTGGTC	ACAGCTCTTGCCACATCTCT	58	150
q-CgBE3	GACCGTCAACTCCCCTCATA	GCTGATCAACCCTTGGAAAA	58	150
q-CgBE1	ACTTCGCTTCCTTCTGTCCA	CCAGAAACATCTTCGGCAAT	58	150
q-CgAPL3	TCTTAAGAGCGGGGACTTGA	CCACATTTTTGGGATCTGC	58	150
q-CgBAM6	GCTGTTGCAGAGATGGTTGA	GAAGATCATCAACGGCCAAT	58	150
q-CgBAM4	TCTCCGGGCTAAGAGTTCAA	CCAGTGGCAACATCACAAAC	58	150
CgMYB53	ATGGCGGGTAAGCGCAAGA	CAAAATCCCAAATCCGAT	60	753
OE-CgMYB53	GAACACGGGGGACGAGCTCGGTACCATGGCGGGTAAGCGCAAGA	GTGGTGGTGGTCGACGGATCCTCACCAAAATCCCAAATCCGAT	62	799

Fig. 1 Chromosomal localization of CgMYBs

Fig. 2 Co-linearity analysis of CgMYBs genesA: Co-linearity analysis of CgMYBs genes. B: Expression heatmap of genes involved in co-linearity CgMYBs in different graft combinations. Compatible graft combinations: GxPt (‘Guanxi Miyou’ grafted onto trifoliate orange) and HmCj (‘Hongmian Miyou’ grafted onto ‘Shuzhen No.1’). Incompatible graft combination: HmPt (‘Hongmian Miyou’ grafted onto trifoliate orange). S2: Relative chlorophyll content [Soil and Plant Analyzer Development (SPAD) value] between 70-80; S4: SPAD value between 50-60; S8: SPAD value between 10-20. The same below

Fig. 3 Assay result of promoter elements of the R2R3-CgMYBs genesA: Numbers of various promoter elements of the R2R3-CgMYBs. B: Promotor elements in hormone responsive elements of the R2R3-CgMYBs. C: Promotor elements in stress responsive elements of the R2R3-CgMYBs. D: Promotor elements in plant growth-related responsive elements of the R2R3-CgMYBs

Fig. 4 Phylogenetic tree (A), conserved motif (B), and gene structures (C) of R2R3-CgMYBs members

Fig. 5 Phylogenetic tree of R2R3-AtMYBs and R2R3-CgMYBs

Fig. 6 Expression profile analysis of genes involved in CgMYBs in different graft combinationsA: Venn analysis of MYB genes in chlorotic phase of HmPt (incompatible graft). B: Expression heatmap of the ten CgMYBs in different graft combinations [Log10(FPKM+0.01)]

Fig. 7 Expression pattern of MYB in the leaves of HmPt at eight etiolation states

Fig. 8 Transient overexpressions of CgMYB53 in tobacco leavesA: Phenotype and iodine-potassium iodide staining of CgMYB53-transient overexpression tobacco leaves. B: Relative expressions of CgMYB53. C: Starch content in tobacco leaves. D: Chlorophyll and carotenoid content in tobacco leaves (ND stands for not detected). E: Relative expressions of starch metabolism-related genes. * P<0.05; ** P<0.01; *** P<0.001

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