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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (2): 285-293.doi: 10.3724/SP.J.1006.2021.03015

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

Phenotype analysis and gene mapping of small kernel 7 (smk7) mutant in maize

JIANG Cheng-Gong1,2, SHI Hui-Min2, WANG Hong-Wu2, LI Kun2, HUANG Chang-Ling2, LIU Zhi-Fang2, WU Yu-Jin2, LI Shu-Qiang2, HU Xiao-Jiao2,*, MA Qing1,*   

  1. 1National Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, National Engineer Laboratory of Crop Molecular Breeding, Beijing 100081, China
  • Received:2020-03-11 Accepted:2020-08-19 Online:2021-02-12 Published:2020-09-10
  • Contact: HU Xiao-Jiao,MA Qing
  • Supported by:
    National Natural Science Foundation of China(31500984);Agricultural Science and Technology Innovation Program of CAAS, and the Storing Grain in Technology for Maize (CAAS-ZDRW202004).

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Abstract:

In this study, a stable small kernel mutant, named small kernel 7 (smk7), was isolated from ethylmethane sulfonate (EMS) mutagenesis of maize inbred line B73. Compared with wild type, the smk7 mutants showed smaller kernel size, defective embryo and endosperm development and a significant decrease in 100-kernel weight. The smk7 kernels showed a low level of germination rate at 10% and cannot grow into normal plants. No significant changes were detected in protein, starch and oil content between mature wild type and smk7 kernels, but the starch grains became significantly smaller and irregular in smk7 kernels compared with wild type. The smk7 kernels could be clearly distinguished from the wild type as early as 12 days after pollination (DAP), on the basis of their smaller and emptier phenotype. Microscopic inspection of the paraffin sections revealed that the development of embryo and endosperm were delayed, and the cell wall in growth in basal endosperm transfer layers (BETL) were arrested in smk7 compared with wild type. The F2 populations with multiple backgrounds were constructed by crossing heterozygous plants (+/smk7) with several other inbred lines. Genetic analysis showed that the mutant phenotype was controlled by a single recessive gene. Based on genotyping by target sequencing (GBTS) strategy, the SMK7 was initially mapped on the short arm of chromosome 2. The fine mapping results suggested that SMK7 was located between markers RM1433917 and RM1535316, with a physical distance of 120 kb. There were eight protein-coding genes in this region. This study laid a foundation for further genes cloning and research of the SMK7 function in regulating maize kernel development.

Key words: maize, small kernel mutant, genetic analysis, gene mapping

Fig. 1

Morphological phenotypes of the smk7 A: maize ear of M4 generation, B: comparison of the phenotype between smk7 and WT kernels; C: comparison of the phenotype between smk7 and WT seedlings."

Fig. 2

Hundred-kernel weight of wild type and smk7 kernels ** represents significant differences between the smk7 mutant and wild type at the 0.01 probability level."

Fig. 3

Scanning electron microscopy (SEM) of mature smk7 and WT kernels A: wild type endosperm; B: smk7 endosperm; SG: starch granule; PB: protein body; Bar = 20 μm."

Table 1

Primer sequences for gene mapping used in the study"

引物
Primer		 正向序列
Forward sequence (5°-3′)		 反向序列
Reverse sequence (5′-3′)
In4.3 AACGCATCATCCTATGTCCAAC GGGTGAAGCCAGCCATTATTT
In1.9 ATGGTACGATCAACATAAAGGGAA GGCGTCACCGAAGAAATACAC
In0.83 GCAAGAAGCACCAGCCCT CGAGCGAAAGAAAGGAATGT
In1.16 ACATGACCCACGATCCAGACA TGCAGCCACTCTCCTTATGGT
SNP1 TTAGGGTGGAGTTGCTTCGC TTATGAATGAAGCACGGAAATGA
SNP2 TTAGGGTGGAGTTGCTTCGC TTATGAATGAAGCACGGAAATGA
SNP3 AAGGAATGGAGGCTTGGGTT ACAGCCGCCTTCGGATTC
SNP4 AGCGGTCCTTGACTTTATTTGA CATTACACGACCAATACAGCCA
SNP5 TGGCTTTTCATACCCTCCTCC CCTTCGCTGTGACTTGGATGT

Fig. 4

Determination and analysis of WT and smk7 kernel components ** represents significant differences between smk7 mutant and wild type at the 0.01 probability level."

Fig. 5

Observation of embryo and endosperm of WT and smk7 at different development stages A: the observation of embryo, endosperm and whole seed of wild type at 12, 15, 18, 21, 25, and 30 DAP; Bar =3 mm. B: the observation of embryo, endosperm and whole seed of smk7 at 12, 15, 18, 21, 25, and 30 DAP; DAP: days after pollination; Bar = 3 mm."

Fig. 6

Observation of paraffin sections of wild type and smk7 kernels on different days after pollination A: wild type kernels at 12 DAP; Bar = 1000 μm. B: smk7 kernels at 12 DAP; Bar = 1000 μm. C: the basal endosperm transfer layer of WT endosperm at 12 DAP; Bar = 150 μm. D: the basal endosperm transfer layer of smk7 at 12 DAP; Bar = 150 μm; BETL: basal endosperm transfer layers."

Table 2

Genetic segregation test of different populations"

世代
Populations		 总粒数
Total kernels		 正常籽粒数
Normal kernels		 突变籽粒数
Mutant kernels		 实际比例
Actual ratio		 理论比例
Theoretical ratio		 卡方值
χ2
M2 6372 4742 1630 2.91:1 3:1 1.1151
M3 9821 7412 2409 3.08:1 3:1 1.1367
F2 (Mo17×smk7) 5123 3870 1253 3.09:1 3:1 0.7730
F2 (Z58×smk7) 2016 1512 504 3:1 3:1 0.1120

Fig. 7

Number of SNP markers"

Fig. 8

Distribution profile of SNP index on whole genome"

Fig. 9

Fine mapping of SMK7 the number of individuals; Recombinants: the number of recombinants."

Table 3

Gene information in the candidate region"

基因名称
Locus name		 基因注释
Gene annotation
ZM00001D001818 Probable polyol transporter 4
ZM00001D001819 N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase family protein
ZM00001D001820 Protochlorophyllide reductase1
ZM00001D001821 Unknown
ZM00001D001822 Unknown
ZM00001D001823 Anthranilate synthase beta subunit 1 chloroplastic
ZM00001D001824 Dof zinc finger protein DOF1.6
ZM00001D001825 Transducin/WD40 repeat-like superfamily protein
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