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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (03): 369-375.doi: 10.3724/SP.J.1006.2018.00369

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

Genetic Analysis and Molecular Characterization of a New Allelic Mutant of Vp15 Gene in Maize

Rui WANG1,2,**(), Xiu-Yan ZHANG3,**(), Yang-Song CHEN2, Yi-Cong DU2, Ji-Hua TANG1, Guo-Ying WANG2, Jun ZHENG2,*()   

  1. 1 College of Agriculture, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 School of Life Science, China Agricultural University, Beijing 100193, China
  • Received:2017-08-07 Accepted:2017-11-21 Online:2018-03-12 Published:2017-12-18
  • Contact: Rui WANG,Xiu-Yan ZHANG,Jun ZHENG E-mail:18612261636@163.com;xyzhang0818@163.com;zhengjun02@caas.cn
  • Supported by:
    This study was supported by the National Key Research and Development Program of China (2016YFD0101002) and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

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Abstract:

We identified a new maize viviparous mutant during seed reproduction, designated as vp-like4.This mutant phenotype was steadily inherited and genetically regulated by a single recessive gene. Using an F2 segregation population derived from vp-like4 and inbred line Mo17, we mapped the target gene in an interval from 173.8 to 175.6 Mb on chromosome 5 by the BSR-Seq strategy. Using genomic sequence database, we found that viviparous gene Vp15 is located in this mapping region. The maize Vp15 gene encodes the molybdopterin synthase small subunit, which is required in the process of catalyzing the reaction from carotenoid to ABA. The heterozygous plants from two independent vp15 mutants, vp15-umu1 and vp15-DR1126, were used to cross with vp-like4 heterozygous plants, showing a 3:1 segregation ratio for normal and viviparous kernels. The genomic sequence analysis revealed that vp-like4 mutant had a 60-bp deletion in the second exon and 3’-untranslated region of Vp15 gene, which is different from vp15-umu1 and vp15-DR1126 both mutated from a Mutator transponson inserting in the second exon of Vp15 gene. Further RT-PCR analysis revealed that the expression level of vp15 was significantly lower in vp-like4. Taken together, these evidences suggest that vp-like4 is a new allele mutant from vp15.

Key words: maize, viviparous, mutant, Vp15, gene mapping

Table 1

Primers used in this study"

引物
Primer		 正向序列
Forward sequence (5°-3°)		 反向序列
Reverse sequence (5°-3°)
VP15-G-F1/R1 AAGGACGCGAGAGTTTTTGA GGTGAGCGAGTAACCCATGT
VP15-G-F2/R2 ACATGGGTTACTCGCTCACC CTTCCTGGTAGCGAGACGTG
VP15-G-F3/R3 GCTCGTTCACCATCCAGAAT AGCAAAGCCCTTTCTGAACA
VP15-G-F4/R4 GGGCTTTGCTAAGATGTCTCC GGACAATACATAACTGCTCCCTAAA
VP15-G-F5/R5 TCTTCCACTCTTCCTACCAGCTA AGGAGCCAACAACTGCTGAT
VP15-G-F6/R6 GAACAATCGAGCCTTCTTCG TCGTCCTCCTTGACCACTTC
VP15-G-F7/R7 CACCAACTGCAAGATCGAAA CCAACAGACGTCAACACCAG
VP15-CDS-F1/R1 GGAACTCTCTGATACCTGGCTTT CAGTTCCGTGATTAGCCGC
Zm-GAPDH-F/R CCCTTCATCACCACGGACTAC AACCTTCTTGGCACCACCCT

Fig. 1 Viviparous phenotype of vp-like4 mutant A: normal and viviparous kernels on a vp-like4 heterozygous ear at 30 days after self-pollination; B: normal and viviparous kernels on a vp-like4 heterozygous ear at 60 days after self-pollination; C: mature normal (WT) and viviparous kernels (vp). Bar = 1 cm."

Table 2

Segregation of normal and viviparous kernels on vp-like4 self-pollinated heterozygous ears"

年度
Year		 地点
Location		 植株基因型
Plant genotype		 籽粒表型Kernel phenotype
正常籽粒 Normal 穗发芽籽粒 viviparous 总数 Total χ2 (3:1)
2014 海南Hainan vp-like4/+ 355 127 482 0.398
325 111 436 0.027
2016 北京Beijing vp-like4/+ 129 47 176 0.189
225 70 295 0.190

Fig. 2

Gene mapping of the vp-like4 mutant by the BSR-Seq strategy"

Fig. 3

Allelism test of vp-like4 with vp15 by heterozygous mutants A, B: viviparous kernels were respectively emerged on vp15-umu1 (A) and vp15-DR1126 (B) heterozygous ear crossed by the mixed pollen of vp-like4 heterozygous plants; C, D: viviparous kernels were emerged on vp-like4 heterozygous ear crossed by the mixed pollens of vp15-umu1 (C) and vp15-DR1126 (D) heterozygous plants."

Fig. 4

Gene structure of Vp15 and mutation site of three mutants"

Table 3

Allelism test of vp-like4 with vp15"

父母本基因型
Parental genotype		 籽粒表型 Kernel phenotype
正常籽粒Normal 穗发芽籽粒viviparous 总数Total χ2 (3:1)
vp-like4/+ × vp15-umu1/+ 217 68 285 0.141
vp-like4/+ ×vp15-DR1126/+ 123 40 163 0.002
vp15-DR1126/+ ×vp-like4/+ 262 83 345 0.116
vp15-umu1/+ ×vp-like4/+ 239 84 323 0.124

Fig. 5

Gene expression levels of Vp15 in the normal (WT) and viviparous (vp) kernels of three mutants, vp-like4, vp15-umu1, and vp15-DR1126, by quantitative real-time PCR analysis"

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