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A splice site mutation in shrunken1-m causes the shrunken 1 mutant phenotype in maize

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Abstract

Endosperm development plays an important role in the determination of grain weight in maize. In this context, a spontaneous recessive shrunken kernel mutant, shrunken1-m (sh1-m), identified from improved maize inbred line Zheng58 (Gai-Z58) was studied. Physiological and microscopic analysis revealed that the sh1-m mutant significantly increased the content of amylose, and its starch granules showed distinctive morphology. Utilizing 1877 recessive sh1-m individuals from a BC1 segregating population of B73/sh1-m and 19 molecular markers, the sh1-m gene was limited to a 2.1 kb interval within the GRMZM2G089713 gene. A 5-bp substitution in sh1-m covering the 3′ end of the thirteenth exon and the 5′ splicing site of the thirteenth intron led to a missense mutation and a mis-splicing site that resulted in early translational termination. sh1 reference mutant sh1-912A (shrunken-912A) had a single transversion at the 3′ splice junction of the second intron resulting in a mis-splicing site causing the 13-bp spliced out in the third exon and the initiation codon to shift to 118-bp downstream. Further genetic allelism tests between them confirmed that sh1-m is a novel allele of the Sh1 locus. Sh1 was constitutively expressed in all tested tissues, and its expression pattern/level was not significantly changed in developing seeds of the sh1-m mutant. Transcript mRNA patterns/levels of some other genes involved in starch biosynthesis was significantly altered in the sh1-m mutant. These results provide direct evidence that sh1-m plays a key role in starch biosynthesis and provide valuable information for grain quality research and breeding in maize.

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Abbreviations

AGPase:

ADP-glucose pyrophosphorylase

SS:

Starch synthases

GBSS:

Granule-bound starch synthase

SBE:

Starch branching enzymes

DBE:

Debranching enzyme

CTAB:

Cetyl trimethyl ammonium bromide

SSR:

Simple sequence repeat

HTGS:

High throughput genomic sequences

PCR:

Polymerase chain reaction

InDel:

Insertion/deletion

SNP:

Single nucleotide polymorphism

ORF:

Open reading frame

US:

United States

NCBI:

National Center for Biotechnology Information

R:

Root

S:

Stem

L:

Leaf

E:

Ear

T:

Tassel

DAP:

Days after pollination

EMS:

Ethyl methane sulfonate

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Acknowledgements

We thank Marty Sachs in the Maize Genetics COOP Stock Center for furnishing seeds of sh1-912A (Stock 912A), and Guoqi Yao (colleague) for furnishing SSR primers. This work was supported by Natural Science Foundation of Shandong Province (ZR2016CB52), National Natural Science Foundation of China (31701443), Major Science and Technology Projects of Shandong Province (2015ZDJS03001), Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2017B01), National Key Research and Development Plan (2017YFD0101204) and Research on the foundation and advanced technology of the science and Technology Department of Henan (162300410179).

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  1. Haiying Guan and Yongbin Dong have contributed equally to this work.

Authors and Affiliations

  1. Maize Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory of Wheat and Maize/Key Laboratory of Biology and Genetic Improvement of Maize in Northern Yellow-huai River Plain, Ministry of Agriculture, P.R. China, Jinan, 250100, China

    Haiying Guan, Chunmei He, Chunxiao Liu, Qiang Liu, Rui Dong, Tieshan Liu & Liming Wang

  2. College of Agronomy, Henan Agricultural University, Collaborative Innovation Center of Henan Grain Crops, National Key Laboratory of Wheat and Maize Crop Science, 95 Wenhua Rd, Zhengzhou, 450002, China

    Yongbin Dong & Yuling Li

  3. Maize Research Institute, Southwest University, Chongqing, 400715, China

    Chaoxian Liu

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  1. Haiying Guan
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Corresponding authors

Correspondence to Tieshan Liu or Liming Wang.

Electronic supplementary material

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10725_2017_309_MOESM1_ESM.tif

Supplementary material 1 Sequence analysis of Sh1 ORFs. The start (ATG) or stop (TAG/TAA) codons are enclosed with yellow and red boxes, respectively. The blue box indicates three nucleotide (CAG to ATA) substitution at 1,993-1,995 bp in the sh1-m coding region. The black box indicates a single base transversion from C to G at 2,183 bp in sh1-912A. (TIF 9163 KB)

10725_2017_309_MOESM2_ESM.tif

Supplementary material 2 Schematic diagram of Sh1 genomic structure. a B73; b Gai-Z58, SNPs and InDels indicate the allelic variation compared with B73, the red asterisk indicates the SNP causing a missense mutation Arg (615) Lys; c sh1-m, SNPs and InDels indicate the allelic variation compared with wild type lines B73 and Gai-Z58, the gray box indicates the thirteenth intron that did not splice out, nucleotides with green and blue font indicate the difference in the exon and intron, respectively, CC with red star indicates the mis-splicing site at the 5′ splice junction of the thirteenth intron and the TAA with red font indicates stop codon; d sh1-912A, SNPs and InDels indicate the allelic variation compared with wild type lines (B73 and Gai-Z58) and sh1-m, nucleotides with blue font and red star indicate the mis-splicing site at the 3′ splice junction of the second intron and the 13-bp nucleotides with green font indicate the sequence at the start of the third exon that spliced out resulted from a new splice site (AG with green font and green star) in this exon. The red asterisk indicates the SNP causing a missense mutation Ala (728) Gly. Black boxes and numbers above and below them indicate exons, their order and their length, respectively. Black short lines indicate introns and numbers above them indicate their length. Green and blue vertical lines indicate SNPs in exon and intron, respectively, red vertical lines indicate InDels. The different length of these vertical lines indicates different SNPs or InDels nearby (TIF 2086 KB)

10725_2017_309_MOESM3_ESM.tif

Supplementary material 3 Multiple amino acid sequence alignment of sh1-m, sh1-912A, B73 and Gai-Z58. The orange line indicates the GT1_Sucrose_synthase region. The red box indicates an Ala to Gly missense mutation in the sh1-912A encoded protein. Dark blue and pink colors indicate 100% and 75% homology levels, respectively (TIF 7600 KB)

10725_2017_309_MOESM4_ESM.tif

Supplementary material 4 Segregation of InDel marker P20 in an F2 population of B73/sh1-m. M DL-2000 marker, P1 B73, F1 B73×sh1-m, P2 sh1-m, 1-4 Homozygous Sh1Sh1 plants in F2, 5-8 Heterozygous Sh1sh1 plants in F2, 9-12 Homozygous sh1sh1 plants in F2 (TIF 1184 KB)

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Supplementary material 9 (DOCX 18 KB)

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Guan, H., Dong, Y., Liu, C. et al. A splice site mutation in shrunken1-m causes the shrunken 1 mutant phenotype in maize. Plant Growth Regul 83, 429–439 (2017). https://doi.org/10.1007/s10725-017-0309-9

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