Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (17): 3573-3586.doi: 10.3864/j.issn.0578-1752.2021.17.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Genome-Wide Identification of WOX Family and Expression Analysis of Callus Induction Rate in Tartary Buckwheat

HOU SiYu1(),WANG XinFang1,DU Wei1,FENG JinHua1,HAN YuanHuai1,LI HongYing1,LIU LongLong2,SUN ZhaoXia1()   

  1. 1College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi
    2Center for Agricultural Genetic Resources Research, Shanxi Agricultural University, Taiyuan 030031
  • Received:2021-02-18 Accepted:2021-05-08 Online:2021-09-01 Published:2021-09-09
  • Contact: ZhaoXia SUN E-mail:bragren123@163.com;18636071356@163.com

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Abstract:

【Objective】 This study aimed to identify the whole genome WOX (WUSCHEL-related home obox) gene family in Tartary buckwheat and reveal the correlation with sequence characteristics of its gene family members, gene expression pattern and the rate of callus induction. It provides a theoretical basis for breaking through the regeneration and genetic transformation problem of Tartary buckwheat. 【Method】 The protein and nucleic acid sequence of the WOX gene family members in Tartary buckwheat were obtained by homology blast and the sequence of Arabidopsis WOX genes were served as reference. Based on protein homology and conserved domain analysis, all members of Tartary buckwheat WOX gene family were identified. The TBtools software was used to further demonstrate the characteristics of the WOX genes in Tartary buckwheat, including gene structure, conserved domain and cis-acting element. Genomic collinearity of WOX gene family members between Tartary buckwheat and Arabidopsis thaliana was analysed. Based on proximity method, the MEGA X software was used to perform phylogenetic tree of these WOX genes in Tartary buckwheat, Arabidopsis and rice. The hypocotyl explants of 70 Tartary buckwheat varieties were cultured with MS+2,4-D 3.0 mg·L -1+6-BA 1.0 mg·L-1 for callus induction and the callus emergence rate of different genotypes was evaluated. The FtWOX gene expression level was performed by qPCR to compare the different Tartary buckwheat varieties with high and low callus yield. The correlation between callus rate and FTWOXS gene family members was analysed based on Pearson correlation coefficient. 【Result】 A total of 30 WOX genes were identified in Tartary buckwheat and they were unevenly distributed on 8 chromosomes. The 30 Tartary buckwheat WOX genes could be divided into three groups by phylogenetic tree. The WOX genes contained different conserved domains in different groups, and the main conserved domains were HD(Homeodomain), START and MEKHLA. The conserved motif analysis showed that the conserved motif number of FtWOX genes may contain 2 to 10 motifs, and the gene structure analysis showed that the number of exons contained in the genes between 2 to 18. Promoter elements analysis showed 26 different kinds of cis-acting elements in the 30 WOX genes. The phylogenetic analysis showed that 30 Tartary buckwheat, 15 Arabidopsis thaliana and 12 rice WOX gene family members could be divided into three categories, of which the third group is unique to Tartary buckwheat. The collinearity analysis showed that six WOX genes were genomic collinearity between Tartary buckwheat and Arabidopsis thaliana. Expression pattern and correlation analysis show that the expression level of FtWOX1/FtWOX12/FtWOX22/FtWOX23/ FtWOX24 has positive correlation with the callus induction. 【Conclusion】 Collectively, these data suggest that the Tartary buckwheat FtWOX members showed abundant sequence variation characteristics. The expression level and callus rate of WOX gene in different Tartary buckwheat genotypes were significantly different and correlated to some extent, suggesting that different Tartary buckwheat WOX genes had potential functional diversity.

Key words: Tartary buckwheat, WOX gene family, callus induction, gene expression

Table 1

The varieties used for callus induction"

来源Origin 品种Variety name
中国湖北Hubei, China ZNQ171, ZNQ164, ZNQ165, ZNQ166, ZNQ167, ZNQ168, ZNQ169, ZNQ170
中国贵州Guizhou, China ZNQ183, ZNQ184, ZNQ185, ZNQ186, ZNQ187, ZNQ188, ZNQ189
中国广西Guangxi, China ZNQ194, ZNQ195, ZNQ196, ZNQ197, ZNQ198
中国安徽Anhui, China ZNQ161, ZNQ162, ZNQ163
中国云南Yunnan, China ZNQ192, ZNQ190, ZNQ191
中国甘肃Gansu, China ZNQ154, ZNQ155, ZNQ156
中国青海Qinghai, China ZNQ158, ZNQ159
中国湖南Hunan, China ZNQ176, ZNQ177, ZNQ178
美国USA PI647612, PI476852, PI658431, PI658429, PI658439, PI647613
不丹Bhutan PI481673, PI481675, PI481658, PI481672
尼泊尔Nepal PI427240, PI481666, PI658430
未知Unknown ZNQ172, ZNQ174, ZNQ180, PI673868, PI673869, PI673870, PI673874, PI673858, PI673876, PI673845, PI673856, PI673857, PI673855, PI673862, PI673854, PI673846, PI673873, PI673863, PI673847, PI481673, PI673865, PI658438, PI673852

Table 2

The primer sequence for qRT-PCR"

引物名称
Primer name		 正向引物
Forward sequences (5'-3')		 反向引物
Revered sequences (5'-3')		 扩增长度
Amplification length (bp)
FtWOX1 CAGGCGGTGTCTTCTCGATT AACTGACCGGAAGATGGGTG 94
FtWOX2 GATGAAGGACGACGAGCCAG AGAACTCGGTAGGGTTGCAG 137
FtWOX3 CGAGGGGAACGATAAGGTGG GACTCAACTTTGCCATGCTCC 142
FtWOX4 CTCAAGCGCCTTACTCCCTC GCTTTCAAACGCCCTACTCG 102
FtWOX5 AGGACCTGCAAATGGGAAGC TCTGAGACGGCTGCTACTCT 138
FtWOX6 AAGGTGCTCTAATGGCCGC TTGGTCAACGCTTAAACGCC 137
FtWOX7 TTCAAGTCCAAGCCCTGCAA CTCCGCTGTCGATCCATCTC 108
FtWOX8 ACACGACTCTCTCCTGCTTG TGTCGTTGGACTGTTGTGGT 142
FtWOX9 AGTGGACAGTTCGTTGCTGA GGACTCCTGACCGTCGTAAC 133
FtWOX10 ACTACATTGGCACCTGGTCG CCACTAAGCTGCCATCCTCC 124
FtWOX11 AGGCGTGCTAGATTGAAGAGG CTTGTGGCTGGGAATCCTCG 124
FtWOX12 AGAGGACCACCATAGACTCG ACTGAACCGGACCCTCATTC 98
FtWOX13 ACCTTCCAATCAGTTCGCGG ACTTCAAGTTGCTCGTCGGT 117
FtWOX14 GGTGGAACCCTACGCAAGAG CTGAGTTGCGTCGTGATGTG 106
FtWOX15 GGTAGTCCGACTTGAAGGGC CAACCGCCAACTGCATTCTC 111
FtWOX16 AGGCGTGCTAGATTGAAGAGG CTTGTGGCTGGGAATCCTCG 124
FtWOX17 CCTCGAGAAGAGCTTCGACA AACCAGATAGCAACCTGCCG 102
FtWOX18 ACCAAGCTTGAACCACGTCA AGCTGCTTCGACTTCCATCG 113
FtWOX19 ACGGAGATGGAGTGCGAGTA GTAGAGCTCGGCACAGTCAA 128
FtWOX20 TCTCACAGAGCAGAATCGCTG GTCCTTGAATGGCAGGAGGC 129
FtWOX21 GCGGCATTGCGTCATCTAAG TTCAACCTCTGGCTGAGTGC 107
FtWOX22 TCTTGCCCGTTGGATCACTC GCAACACAAGATGGCATCCG 127
FtWOX23 CGTGATTGCCGAGCTGTAGA AGCCGATGCCAAAGTCGTAA 108
FtWOX24 TGAGGAGAAGTCTCCAGGGT CCGGTTCAGGCCTCATCATA 104
FtWOX25 CGTGAACAGGAGATGCCGAT TACTGAACTTGCGTCGGTGT 120
FtWOX26 TGTGAAGAAAGCTCCCTCGG TTCTGTATTTGCTGCGCCGT 111
FtWOX27 CATACGATGACTCTCCAGGGC ATCTAGCGGCGGATGTTCG 116
FtWOX28 TCAGCGATGAGCAGATCAGG CATCTGGCTCGTCGGTTCTG 145
FtWOX29 AAGCTTGCTCGTGAGCTTGG ATGGAGACGGCATCGAACTC 134
FtWOX30 TACGCTCCGGTTGACATTCC AGGCAACAGAGAGAAGCGAC 130
FtHis ATCGACTGGAGGAAAGGCTC GCGGTATCTGTGGGACTTCT 106

Fig. 1

The distribution location of FtWOXs on chromosomes"

Fig. 2

FtWOXs gene family Phylogenetic analysis (A), conserved domain (B) and gene structure analysis (C) in Tartary buckwheat"

Fig. 3

The phylogenetic tree of WOX protein in Arabidopsis thaliana (At), Oryza sativa (Os) and Fagopyrum tataricum (Ft) AtWOX1-13C and AtWUX: The WOX protein of Arabidopsis thaliana; OsWOX3-12B, OsWUX and OsNS1-2: The WOX protein of rice; FtWOX1-30: The WOX protein of Tartary buckwheat, numbers in brackets are the corresponding protein ID"

Fig. 4

The analysis of FtWOXs gene promoter elements in Tartary buckwheat"

Fig. 5

Collinearity analysis of WOX gene family between Fagopyrum tataricum (Ft) and Arabidopsis thaliana (At) "

Fig. 6

The callus growth situation (A) and cluster diagram of induction rate (B) of different Tartary buckwheat varieties Ⅰ: The varieties with callus induction rate of 92% to 100%;Ⅱ: The varieties with callus induction rate of 66% to 78%; Ⅲ, Ⅳ: The varieties with the callus induction rate of 30% to 64% and less than 30%, respectively"

Fig. 7

The heat map of gene expression pattern of FtWOXs in callus among different Tartary buckwheat varieties In black box, the expression tendency of five genes was consistent with inducing rate"

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