Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (15): 2678-2685.doi: 10.3864/j.issn.0578-1752.2019.15.011

• HORTICULTURE • Previous Articles     Next Articles

Fine Mapping and Candidate Genes Analysis for Regulatory Gene of Anthocyanin Synthesis in Red-Colored Tuber Flesh

XU YunMei1,LI YuMei1,3,JIA YuXin2,ZHANG ChunZhi2,LI CanHui1,HUANG SanWen2,ZHU GuangTao1()   

  1. 1 School of Life Science, Yunnan Normal University/Joint Academy of Potato Sciences, Kunming 650500
    2 Agricultural Genomics Institute as Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120
    3 Faculty of Information Engineering & Automation, Kunming University of Science and Technology, Kunming 650500;
  • Received:2019-03-04 Accepted:2019-05-13 Online:2019-08-01 Published:2019-08-06
  • Contact: GuangTao ZHU E-mail:zhuguangtao@caas.cn

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Abstract:

【Objective】 The color of tuber flesh is an important agronomic trait, which directly affects the nutritional and commercial value of potatoes. Accordingly, it is an important goal for potato genetic research and breeding improvement. In this study, we mapped the locus controlling red flesh color by bulked segregant analysis (BSA) and fine mapping, and predicted the candidate genes aided by expression analysis, which provides a foundation for gene function characterization, regulation analysis as well as the molecular breeding of colored potatoes. 【Method】 We crossed a red flesh diploid clone with the Sli gene donor to produce F1, and the F1 was backcrossed with the red flesh clone, and finally generated a BC1S1 diploid population. From the BC1S1 segregation population with 300 individuals, the genomic DNA of 18 red-colored flesh and 21 yellow-colored flesh individuals were extracted respectively and used for sequencing, and the locus controlling red flesh was preliminarily mapped by BSA-seq. The locus was fined mapped through genotyping and phenotyping of the recombination plants from 796 BC1S1 individuals. Based on reference genomic annotation and qRT-PCR expression analysis, candidate genes were predicted. 【Result】 In this study, BC1S1 segregating population with red and yellow-colored tuber flesh was constructed. The major locus which regulates related anthocyanin synthesis in potato flesh was mapped on chromosome 10 within a physical distance between 48.70 Mb and 52.20 Mb. Finally, molecular markers were used to locate the gene into a 377 kb genomic interval from 51.47 Mb to 51.85 Mb. There are five annotated genes in this genomic region based on reference genome annotation information, two of them were MYB transcription factors. We predicted that the two genes, PGSC0003DMG400013966 and PGSC0003DMG400013965, were candidate genes controlling red tuber flesh. 【Conclusion】 In this research, the major locus which regulates accumulation of anthocyanin in potato tuber flesh finally was mapped to chromosome 10 with an interval from 51.47 Mb to 51.85 Mb and predicted the candidate genes, PGSC0003DMG400013966 and PGSC0003DMG400013965.

Key words: red-colored flesh, anthocyanin metabolism, BSA-seq, fine mapping, candidate genes

Table 1

Molecular markers information for fine mapping"

引物名称
Primer name		 Indel位置
Indel position		 正向引物
Forward (5′-3′)		 反向引物
Reverse (5′-3′)
P1 48710951 GAAGAATGGTAGCTGGAGTG CCGAATATGCATGTATTAGC
P2 49255127 TGTGTCAACGCTAACAACAT TACCCCAGACAGAATGAGTC
P3 49962534 CCATGGCCTCTAACTTTAGTC GTAGTTGCAAATGGGTGATT
P4 51413788 AACTCAATAACGGACCAGTG GATCGGAGAACAAGTTGAAA
P5 52271249 TGGAGGGACTAAAAGTGCTA TACGTACGTTTCCCTATGCT
P6 51427985 TCCCAGAGACTCCATTAAAA GTTTGATGAAATGTGCAGTG
P7 51460414 ATTAGCGGTGAAAGAAGACA AGTAAATGAGAGAGTAGTGCGA
P8 51469924 GTTGTTGAATTATGTTCCTCATACAACA GGAAAATTGGAAAGAATTTGAGGAA
P9 51473988 AGGAAGAGAGTTCCTTCACCACG GCTTCTAGCTACTTCTATAATGACGCAG
P10 51723735 GGATCATCATTAGTGGATCG GAAAGTGTCACGACTTGGAT
P11 51725907 TGTGAAGAACTATTGGAACACTCACTT CCGAGGTTGAGGTCTTATTA
P12 51732426 GCGCCGAAAGGTTTATATT CTATGTAAGTCGATTTGGGC
P13 51744513 GGATTATGACGAACCTAATAACCCAA TTCACCATTCTTGAAGCTAAATGTAA
P14 51845484 TGTTGTTGAACATGCACTCT AGAAAATAATATAAGTTTAATCCGTTCACT
P15 51850970 ATTTTTCCTACCGTAATCCC TCACTCTTCAAGATCAATGC

Table 2

Primers information for candidate genes"

引物名称 Primer name 正向引物 Forward (5′-3′) 反向引物 Reverse (5′-3′)
R2R3-MYB (CDS) CCTTGCACGGGCACCCTTATCTA GCAAATTCATCCCAACCACCATCAC

Table 3

Primers information for qRT-PCR"

引物名称 Primer name 正向引物 Forward (5′-3′) 反向引物 Reverse (5′-3′)
Actin GGGATGGAGAAGTTTGGTGGTGG CTTCGACCAAGGGATGGTGTAG
MYB GGCTAAATTATCTAAGGCCACATATCAAGAGA CGAGGAGGAGGAGGAGGAGCAA
R2R3-MYB GGAGTAATAAGGAAAGGTTCATGGACTGAAG AGCTTCCTTAGAAGGTTAGTGTTCCAATAGTTT

Fig. 1

The phenotype of the tuber flesh color and BSA-seq result A: The tuber flesh color of the parent (BC1) and typical BC1S1 individuals, n: the individual number of the corresponding phenotype; B: SNP-index graphs of two mixed pools and Δ(SNP-index) graph from BSA-seq result respectively, X-axis: the physical location of the chromosome, Y-axis: SNP corresponding to SNP-index and Δ(SNP-index) values, The red arrow points to the target region"

Fig. 2

Fine mapping of major effects gene which regulates tuber flesh anthocyanin synthesis"

Table 4

Genes information in the candidate regions"

基因编号 Gene ID 基因注释 Gene annotation
PGSC0003DMG400013964
PGSC0003DMG400041702
PGSC0003DMG400013966
PGSC0003DMG400045858
PGSC0003DMG400013965		 未知功能基因 Gene of unknown function
未知功能基因 Gene of unknown function
MYB转录因子 MYB transcription factor
未知功能基因 Gene of unknown function
R2R3-MYB转录因子 Transcription factor R2R3-MYB

Fig. 3

The expression of candidate genes in red/yellow-colored flesh Using qRT-PCR, the expression levels of candidate genes were detected in red and yellow colored flesh. Three biological replicates were repeated for each phenotype, values are means ± SD, using the students t-test, ** indicated P<0.01"

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