Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (23): 4729-4741.doi: 10.3864/j.issn.0578-1752.2023.23.014

• HORTICULTURE • Previous Articles     Next Articles

Analysis of Genetic Effect of Fruit Color Controlled by Epistatic Genes in Eggplant

CHEN JinRong1,2(), LÜ ZiJian1,3, FAN LiSha1,2, YOU Qian1, LI Tao1, GONG Chao1, SUN GuangWen2, LI ZhiLiang1(), SUN BaoJuan1()   

  1. 1 Vegetable Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory for New Technology Research of Vegetables, Guangzhou 510640
    2 College of Horticulture, South China Agricultural University, Guangzhou 510642
    3 Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070
  • Received:2023-06-05 Accepted:2023-10-04 Online:2023-12-04 Published:2023-12-04
  • Contact: LI ZhiLiang, SUN BaoJuan

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Abstract:

【Objective】Fruit color is an important trait that affects the commercial value of eggplant fruit. By analyzing the causes for the special segregation ratio of individual plants with purple red peel and with white peel in the F2 population constructed by crossing between two white-fruit parents, this paper could lay the foundation for elucidating the mechanism of epistatic gene interaction on regulating eggplant fruit coloration.【Method】The white-flower and white-peel female parent 19141 with mutation at the structural gene ANS involving in the anthocyanin biosynthesis pathway, white-flower and white-peel male parent 19142 with unknown mutation genes involving in the anthocyanin biosynthesis pathway, and their F1 population with purple red peel and F2 population with separate peel colors were used to explore the epistatic inheritance of eggplant fruit coloration. Genes and their mutation patterns were studied by cloning the known genes, D (SmMYB1) and Y (SmDFR) related to peel color in male parent 19142. Molecular basis of peel color-controlling epigenetic genes was analyzed by developing molecular markers based on genetic variations of peel color genes, analyzing the relationship between genotype and phenotype in E4450F2 population, and crossing with other eggplant parents without anthocyanin pigmentation in peel.【Result】The segregation ratio of plants with purple red fruit and white fruit in E4450 F2 progeny was consistent with the segregation ratio of 27:37 controlled by three pairs of epistatic genes, that is, mutations occurred at the P gene locus in 19141, with genotype DDppYY, and mutations occurred at both D and Y gene loci in 19142, with genotype ddPPyy. The results of cloning and sequencing showed that alternative splicing occurred in SmMYB1 of 19142, which led to the second exon skipping. In 19142, SNP (C→G) in the promoter region’s -326 bp upstream of the start codon resulted in the absence of a CAAT-box cis-acting element in SmDFR gene. An SNP, G to C, at the last base of the second exon, was annotated as splicing mutation, which might cause abnormal function of SmDFR gene in 19142, resulting in the eggplant peel’s inability to synthesize anthocyanin. Based on the genetic variation of SmMYB1, SmANS and SmDFR, the functional molecular markers were developed, and the progenies of E4450F2 were genotyped. The results showed that genotype and phenotype were completely consistent. D_P_Y_ corresponded to phenotypes of purple flower and purple red peel, ddP_Y_ corresponded to phenotype of purple flower and white peel, D_ppY_, D_P_yy, D_ppyy, ddppY_, ddP_yy and ddppyy genotypes corresponded to white flower and white peel phenotypes. When 19142 was crossed with white-peel inbred line 19147 (dtdtPPYY), and green-peel inbred line 19144 (DDPPyy), it was found that the fruit color of the two F1 progenies were white and green, respectively, and there was no anthocyanin pigmentation in the peel, which further proved that 19142 was a double mutant in SmMYB1 and SmDFR.【Conclusion】When two eggplant parents without anthocyanin pigmentation in the peel were crossed, the peel of the F1 generation had anthocyanidin pigmentation, and the segregation ratio of plants with anthocyanin pigmentation and non-anthocyanin pigmentation in F2 population was 27:37, it was because one of the parents had a mutation at a gene locus in the anthocyanin biosynthesis pathway, and the other parent had mutations at two other loci in the anthocyanin biosynthesis pathway. When two parents without anthocyanin pigmentation were crossed, the peel of F1 was able to synthesize anthocyanin and present purple red color, which was due to the simultaneous dominance of three epistatic gene loci D, P and Y, and anthocyanin biosynthesis was restored. Mutation of the structural gene of SmANS or SmDFR inhibited anthocyanin biosynthesis in all parts of the plant. The regulation of transcription factor mutation SmMYB1 was tissue specific, and its mutation inhibited anthocyanin biosynthesis in the peel, but did not inhabit anthocyanin biosynthesis in the flower.

Key words: eggplant, peel color, epistatic gene, anthocyanin, genetic law

Table 1

Primer sequences for target gene cloning"

用途
Usage		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 退火温度
Anneal temperature (℃)		 延伸时间
Extending time (s)
克隆SmANS全长
Cloning of the full length SmANS 		ANS-B1 F: CCAGTCGTTTGTTCCCACAT
R: GGACATTACCAGAGGGTTGA		 55 30
ANS-B2 F: TACCTTCTTTGATCTGCCTGTT
R: GCTCTTGTACTTTCCGTTGC		 60 60
ANS-B3 F: TCATCCTCCACAATATGGTGC
R: CTGTGTTGCTCCTTCACAAAGG		 60 30
克隆SmMYB1全长
Cloning of the full length SmMYB1		 MYB1-D1 F: TCCGTACAAGGTGGCAGGATAGGTTATTAGG
R: CGGAAGTCTACCAGCAATAAGTGACCATCTA		 60 60
MYB1-C2 F: GTGCAATGATGACGCTCACA
R: GGACCTATGCCGAAAACAGA		 55 30
克隆SmMYB1 cDNA
Cloning of cDNA of SmMYB1		 MYB1-CDS1 F: ATGAATAATCCTCCTATAATCTGTACGTC
R: CTGCTCTAGCAGGAACAAGAT		 53 30
MYB1-CDS2 F: TAATCTGTACGTCTGTGCGAGTGA
R: GTTATTTCTTTGCAGCGTTCTTCC		 55 30
MYB1-CDS3 F: TGGAACACTAACCTTCTAAGG
R: TTAAACAAGTAGATTCCATAAATCAAT		 50 30
克隆SmDFR全长
Cloning of the full length SmDFR		 65-1036 F: AGGTCTCCCAAACCCACAAC
R: CGCTCCAGCTAGTCTCGTCA		 55 60
DFR-A2 F: CTGTCAGGGAGTATTTCATGTG
R: AGCTTCTTCCCTTGCAGC		 55 30
1060-1900 F: AAGATGACAGGATGGGTTCG
R: CCTTGCCACTTGCATAGTTT		 53 60
DFR-A4 F: GTATACTTGGCTCTTTCATAGAGTG
R: CTGCCTGAAATTAAGCAAGATGAG		 53 30
克隆SmDFR启动子
Cloning of the promoter of SmDFR		 DP2 F: GGCTTCCTTCGTAGTCAA
R: CCCGCAAGTCTAATCTCAC		 53 60
3-403 F: CCGTAACAAATATCAGCATTTC
R: GAGAGAATCGTATTTTTGAAAGC		 50 30
DFR-qA4 F: CTGATGTTCATGGATAGACGGA
R: TGCTTGTACGTACATGATGTTAGG		 53 30

Table 2

Primer sequences for InDel markers of SmMYB1 and SNP markers of SmDFR and SmANS"

标记 Marker 引物名称 Primer name 引物序列 Primer Sequence (5′-3′)
SmMYB1-26bpInDel SmMYB1 FF2 TATAATCTGTACGTCTGTGCGAGTG
SmMYB1 FR2 GCCTTAAATAATTCAACCACCTCA
SmMYB1-52bpInDel MYB1-52FF TAGGCTGAGGTGGTTGAAT
MYB1-52FR TTCGCTAAATATATTATTGTGGAGAC
SBJ-DFR SBJ-DFR-Fg GAAGGTGACCAAGTTCATGCTTCGAGTCCAAGGACCCTGAG
SBJ-DFR-Fc GAAGGTCGGAGTCAACGGATTTCGAGTCCAAGGACCCTGAC
SBJ-DFR-R AGGCAATAAGTATAGATGAGTTAACTCTG
SmANS-Chr08SNP SmANS-Chr08SNP-F CCTAAAGAGTATGTGAGGCCACAAG
SmANS-Chr08SNP-Rt GAAGGTGACCAAGTTCATGCTCCTCAAAGATGTTTCCTATACCATTTT
SmANS-Chr08SNP-Ra GAAGGTCGGAGTCAACGGATTCCTCAAAGATGTTTCCTATACCATTTA

Fig. 1

Phenotype of different E4450 progenies (A and C) and anthocyanin content (B) in peels A: Phenotypes of 19141, 19142and E4450F1; B: Anthocyanin content of 19141, 19142 and E4450F1 (**, P<0.01); C：Phenotypes of E4450F2"

Table 3

Segregation ratios of different progenies"

群体
Population		 总株数
Total plants		 紫红果单株数
Number of purple red peel plants Anthocyanin pigmented		 白果单株数
Number of white peel plants		 卡方检验x2
(27﹕37)		 P值
P value
19141 30 - 30 - -
19142 30 - 30 - -
E4450F1后代 E4450F1 descendants 30 30 0 - -
E4450F2群体 E4450F2 population 247 106 141 0.054 0.817

Table 4

Alleles combination in the inbred offspring of F1 with genotype DdPpYy"

F1的配子 Gametes of F1
DPY DpY DPy Dpy dPY dpY dPy dpy
DPY DDPPYY DDPpYY DDPPYy DDPpYy DdPPYY DdPpYY DdPPYy DdPpYy
DpY DDPpYY DDppYY DDPpYy DDppYy DdPpYY DdppYY DdPpYy DdppYy
DPy DDPPYy DDPpYy DDPPyy DDPpyy DdPPYy DdPpYy DdPPyy DdPpyy
Dpy DDPpYy DDppYy DDPpyy DDppyy DdPpYy DdppYy DdPpyy Ddppyy
dPY DdPPYY DdPpYY DdPPYy DdPpYy ddPPYY ddPpYY ddPPYy ddPpYy
dpY DdPpYY DdppYY DdPpYy DdppYy ddPpYY ddppYY ddPpYy ddppYy
dPy DdPPYy DdPpYy DdPPyy DdPpyy ddPPYy ddPpYy ddPPyy ddPpyy
dpy DdPpYy DdppYy DdPpyy Ddppyy ddPpYy ddppYy ddPpyy ddppyy

Fig. 2

Sequence difference of genes related to fruit color and molecular marker development sites A: DNA sequence differences of SmMYB1 between the parents and InDel marker development sites; B: DNA sequence differences of SmANS between the parents and SNP marker development sites; C: DNA sequence differences of SmDFR between the parents and SNP marker development sites"

Fig. 3

Electrophoresis plots of InDel markers based on genetic variations of SmMYB1 A: Electrophoresis plots of MYB1-26bpInDel markers; B: Electrophoresis plots of MYB1-52bpInDel. If the DNA amplification band type of F2 was consistent with the band type in 19141, it was a DD genotype; if it was consistent with the band type in 19142, it was a dd genotype; if it was consistent with the F1 band type, it was a Dd genotype"

Fig. 4

Genotyping plots of SNP markers based on genetic variations of SmANS and SmDFR A: Genotyping plot of SmANS-Chr08SNP marker; B: Genotyping plot of SBJ-DFR marker"

Table 5

Correspondence between genotype and phenotype of E4450F2 population based on genetic variation of SmMYB1, SmANS and SmDFR"

果色
Peel color		 花色
Flower color		 SmMYB1-26bpInDel的基因型
Genotype of SmMYB1-26bpInDel		 SmANS-Chr08SNP的基因型
<BOLD>G</BOLD>enotype of
SmANS-Chr08SNP		 SBJ-DFR的基因型
<BOLD>G</BOLD>enotype of
SBJ-DFR		 基因型
Genotype		 株数
Plant number
紫红色Purplish red 紫色 Purple DD PP YY DDPPYY 3
紫红色Purplish red 紫色 Purple DD Pp YY DDPpYY 2
紫红色Purplish red 紫色 Purple DD PP Yy DDPPYy 2
紫红色Purplish red 紫色 Purple DD Pp Yy DDPpYy 3
紫红色Purplish red 紫色 Purple Dd PP YY DdPPYY 4
紫红色Purplish red 紫色 Purple Dd Pp YY DdPpYY 9
紫红色Purplish red 紫色 Purple Dd PP Yy DdPPYy 8
紫红色Purplish red 紫色 Purple Dd Pp Yy DdPpYy 8
白色White 白色 White DD pp YY DDppYY 2
白色White 白色 White DD pp yy DDppyy 1
白色White 白色 White DD pp Yy DDppYy 4
白色White 白色 White DD PP yy DDPPyy 2
白色White 白色 White DD Pp yy DDPpyy 1
白色White 白色 White Dd pp Yy DdppYy 8
白色White 白色 White Dd pp YY DdppYY 2
白色White 白色 White Dd pp yy Ddppyy 6
白色White 白色 White Dd Pp yy DdPpyy 6
白色White 白色 White Dd PP yy DdPPyy 4
白色White 白色 White dd pp YY ddppYY 0
白色White 白色 White dd pp Yy ddppYy 6
白色White 白色 White dd PP yy ddPPyy 1
白色White 白色 White dd pp yy ddppyy 0
白色White 白色 White dd Pp yy ddPpyy 5
白色White 紫色 Purple dd PP Yy ddPPYy 3
白色White 紫色 Purple dd Pp Yy ddPpYy 7
白色White 紫色 Purple dd PP YY ddPPYY 3
白色White 紫色 Purple dd Pp YY ddPpYY 2

Fig. 5

The flower and fruit phenotypes of male parent 19144, male parent 19147 and their F1 progenies crossed with 19142"

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