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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (3): 572-579.doi: 10.3724/SP.J.1006.2022.13005

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

Genetic analysis and molecular characterization of a novel maize Bt2 gene mutant

XU Ning-Kun(), LI Bing, CHEN Xiao-Yan, WEI Ya-Kang, LIU Zi-Long, XUE Yong-Kang, CHEN Hong-Yu*(), WANG Gui-Feng   

  1. College of Agronomy, Henan Agricultural University/Key Laboratory of Wheat and Maize Crops Science, Zhengzhou 450002, Henan, China
  • Received:2021-01-19 Accepted:2021-06-16 Online:2022-03-12 Published:2021-07-19
  • Contact: CHEN Hong-Yu E-mail:15839300945@163.com;chenhongyu@henau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(U1804235);National Natural Science Foundation of China(31771800);Science and Technology Innovation Fund of Henan Agricultural University(KJCX2020A04)

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Abstract:

The research of the molecular mechanism underlying maize kernel development is particularly important for the genetic improvement of maize yield and quality traits. In this study, we characterized a new shrunken kernel mutant 5601Q, which was generated by a random transposon insertion. Genetic analysis indicated that the kernel phenotype was stably controlled by a single recessive gene. F2 segregating population was constructed by crossing 5601Q into B73 inbred line, and the mutant gene was located in the genetic interval of 60.19-62.58 Mb on chromosome 4. Sequence annotation showed that the BRITTLE ENDOSPERM2 (Bt2) gene, previously reported to be involved in maize kernel development, was located in this region. Maize Bt2 gene encoded the small subunit of ADP-glucose pyrophosphorylase (AGPase), the first rate-limiting enzyme in the starch biosynthetic pathway of higher plants. Compared with wild type, 100-grain weight and starch content of mutant 5601Q decreased significantly, but the soluble sugar content increased dramatically 4.67 times. We confirmed that 5601Q was a new allele mutant of Bt2 by allelic test of Bt2 mutant 1774 and 5601Q. Sequencing analysis revealed that Mutator 19 transposon was inserted in the 2nd exon of Bt2 gene. In summary, our results indicated that the shrunken kernel in 5601Q was caused by the loss-of-function of Bt2 gene, which provided a new germplasm resource to elucidate the mechanism of maize Bt2 gene in endosperm storage substance accumulation.

Key words: maize, defective kernel mutant, gene mapping, AGPase, Bt2

Table 1

Primers for gene mapping in this study"

引物
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
K5 ATCCGGTACCACCAATTCCT CGTCTGCAGCATGAAACACT
K10 CACACTCCACCTGCATCACT GGCACCAACGAGGATTAGTC
YF100 GACCAACACTGCCACTTCCTTC AGTTGTAGTTGCTGCTCGTTCG
A3 TCCAAAAACAGAACGCAGTG ACTCAATGATCGGGCATAGG
A4 AGATGGCAATGGCTATGAGG CCAAGCAACAAGAGCAACAA
K10-15 CCAACTTGTAGCCACGAAAAGG GCCGATACCGTGACCCAAATC
A12 CCATGATAATTGCGCCTTTT ATTTGGGACATCAACATGCA

Table 2

Primers used in this study"

引物
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
Bt2-F1/R1 TCTTGCCTCCCTCGTCCTTCCTC GGACAAGGACAGCTCTGAAACTGA
Bt2-F2/R2 GTTATTGGTTTCCGTGGAGCTA TTTGCCAGATGCCAGCCATTGA
Bt2-F3/R3 CTCTCTTCCCTTCCAAAGAAAGGC TTCATAGTCCATCCGGTACAGG
Bt2-F4/R4 ACCTGTACCGGATGGACTATGA TTCCTTCCATCCTGCCTTGAGACA
Bt2-F5/R5 TGTCTCAAGGCAGGATGGAAGGAA GACCGTTGGAACATCAGTTTGG
Bt2-F6/R6 TGAATAGGCTTGCATGCTCT AATCCGCACTCCAAACCAGCAA
Tir 8.2 CGCCTCCATTTCGTCGAATCCSCTT

Fig. 1

Kernel phenotype of maize 5601Q mutant A: the self-cross mature ear of F1 plant; B: the comparison of single wild-type and mutant kernel; C: the longitudinal-sectional view of single wild-type and mutant kernel; D: the overall comparison of wild-type and mutant kernels. Bar: 1 cm."

Fig. 2

Kernel component and germination for WT and 5601Q A: 100-kernel weight of wild-type and mutant kernels; B: the starch content of wild-type and mutant kernels; C: the soluble sugar content of wild-type and mutant kernels; D: the statistical analysis of wild-type and 5601Q seed germination tests; E: the comparison of the phenotype between wild-type and 5601Q mutant seedings; * and ** represent significant difference between the mutant and wild-type at the 0.05 and 0.01 probability levels, respectively. Bar: 1 cm．"

Table 3

Segregation ratio of mutant kernels in four F2 ears"

果穗
Ear		 野生型籽粒数
Wild-type		 突变体籽粒
Mutant		 χ2
1 418 144 0.08600
2 485 140 2.25300
3 176 142 1.61100
总计 Total 1279 426 0.00019

Fig. 3

Fine mapping of 5601Q mutant and structure schematic diagram of Bt2 gene A: the fine mapping of 5601Q mutant; N: the number of individuals for gene mapping; Recombinant: the number of recombinants; B: schematic diagram of Bt2 gene structure and mutation sites of two mutants."

Table 4

Allelism test of 5601Q with bt2"

父母本基因型
Parental genotype		 籽粒表型 Kernel phenotype χ2
正常籽粒 Normal kernel 突变体籽粒 Mutant kernel 总数 Total
5601Q/+ × bt2/+ 155 55 210 0.102
bt2/+ × 5601Q/+ 162 55 217 0.015

Fig. 4

Allelism test of 5601Q with bt2 by heterozygous mutants A: 5601Q/+ × bt2/+ shrunken kernels appear on the ears of hybrids; bar: 1 cm. B: bt2/+ × 5601Q/+ shrunken kernels appear on the ears of hybrids; bar: 1 cm."

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