Welcome to Acta Agronomica Sinica，

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).

RichHTML378

45

PDF

3044

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
[1] Nuss E T, Tanumihardjo S A. Quality protein maize for Africa: closing the protein inadequacy gap in vulnerable populations. Adv Nutr, 2011,2:217-224.
doi: 10.3945/an.110.000182 pmid: 22332054
[2] Berger F. Endosperm development. Curr Opin Plant Biol, 1999,2:28-32.
doi: 10.1016/s1369-5266(99)80006-5 pmid: 10047564
[3] Guo Z F, Wang , Tao J J, Ren Y H, Xu C, Wu K S, Zou C, Zhang J A, Xu Y B. Development of multiple SNP marker panels affordable to breeders through genotyping by target sequencing (GBTS) in maize. Mol Breed, 2019,39:37-49.
[4] Sun X, Shantharaj D, Kang X, Ni M. Transcriptional and hormonal signaling control of Arabidopsis seed development. Curr Opin Plant Biol, 2010,13:611-620.
doi: 10.1016/j.pbi.2010.08.009 pmid: 20875768
[5] 任雪梅. 玉米籽粒发育基因Emp11及ZmSmk3的克隆及功能分析. 华中农业大学博士学位论文, 湖北武汉, 2018.
Ren X M. Cloning and Functional Analysis of Emp11 and ZmSmk3 Controlling Kernel Development in Maize PhD Dissertation of Huazhong Agricultural University, Wuhan, Hubei, China, 2018 (in Chinese with English abstract).
[6] 李见坤. 玉米籽粒发育基因UBL1的克隆与功能分析. 中国农业大学博士学位论文, 北京, 2016.
Li J K. Cloning and Characterization of UBL1 Controlling Kernel Development in Maize PhD Dissertation of China Agricultural University, Beijing, China, 2016 (in Chinese with English abstract).
[7] Neuffer M G, Sheridan W F. Defective kernel mutants of maize: I. Genetic and lethality studies. Genetics, 1980,95:929-944.
pmid: 17249053
[8] Wang G, Sun X, Wang G, Wang F, Gao Q, Sun X, Tang Y, Chang C, Lai J, Zhu L, Xu Z, Song R. Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm. Genetics, 2011,189:1281-1295.
doi: 10.1534/genetics.111.133967
[9] 姚东升. 玉米opaque10突变体基因的图位克隆和功能分析. 上海大学博士学位论文, 上海, 2016.
Yao D S. The Map-based Cloning and Functional Analysis of Maize opaque10 Gene PhD Dissertation of Shanghai University, Shanghai, China, 2016 (in Chinese with English abstract).
[10] Yao D, Qi W, Li X, Yang Q, Yan S, Ling H, Wang G, Wang G, Song R. Maize opaque10 encodes a cereal-specific protein that is essential for the proper distribution of zeins in endosperm protein bodies. PLoS Genet, 2016,12:e1006270.
doi: 10.1371/journal.pgen.1006270 pmid: 27541862
[11] Holding D R, Otegui M S, Li B, Meeley R B, Dam T, Hunter B G, Jung R, Larkins B A, Larkins B A. The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation. Plant Cell, 2007,19:2569-2582.
doi: 10.1105/tpc.107.053538 pmid: 17693529
[12] Mertz E T, Bates L S, Nelson O E. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science, 1964,145:279-280.
pmid: 14171571
[13] Schmidt R J, Burr F A, Aukerman M J, Burr B. Maize regulatory gene opaque-2 encodes a protein with a“leucine-zipper”motif that binds to zein DNA. Proc Natl Acad Sci USA, 1990,87:46-50.
doi: 10.1073/pnas.87.1.46 pmid: 2296602
[14] Schmidt R J, Ketudat M, Aukerman M J, Hoschek G. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. Plant Cell, 1992,4:689-700.
doi: 10.1105/tpc.4.6.689 pmid: 1392590
[15] Cord-Neto G, Yunes J A, da Silva M J, Vettore A L, Arruda P, Leite A. The involvement of opaque 2 on beta-prolamin gene regulation in maize and coix suggests a more general role for this transcriptional activator. Plant Mol Biol, 1995,27:1015-1029.
doi: 10.1007/BF00037028 pmid: 7766871
[16] Li Q, Wang J, Ye J, Zheng X, Xiang X, Li C, Fu M, Wang Q, Zhang Z, Wu Y. The maize imprinted gene FLOURY3 encodes a PLATZ protein required for tRNA and 5S rRNA transcription through interaction with RNA polymerase III. Plant Cell, 2017,29:2661-2675.
doi: 10.1105/tpc.17.00576 pmid: 28874509
[17] Silva-Sanchez C, Chen S, Li J, Chourey P S. A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1) seed mutant and its wild type Mn1 in maize. Front Plant Sci, 2014,5:63-77.
doi: 10.3389/fpls.2014.00063 pmid: 24616729
[18] Zhu C, Jin G, Fang P, Zhang Y, Feng X, Tang Y, Qi W, Song R. Maize pentatricopeptide repeat protein DEK41 affects cis-splicing of mitochondrial nad4 intron 3 and is required for normal seed development. J Exp Bot, 2019,70:3795-3808.
doi: 10.1093/jxb/erz193 pmid: 31020318
[19] Tian Q, Olsen L, Sun B, Lid S E, Brown R C, Lemmon B E, Fosnes K, Gruis D F, Opsahl-Sorteberg H G, Otegui M S, Olsen O A. Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1. Plant Cell, 2007,19:3127-3145.
pmid: 17933905
[20] Yuan N, Wang J, Zhou Y, An D, Xiao Q, Wang W, Wu Y. EMB-7L is required for embryogenesis and plant development in maize involved in RNA splicing of multiple chloroplast genes. Plant Sci, 2019,287:110203.
doi: 10.1016/j.plantsci.2019.110203 pmid: 31481208
[21] Shen Y, Li C, McCarty D R, Meeley R, Tan B C. Embryo defective12 encodes the plastid initiation factor 3 and is essential for embryogenesis in maize. Plant J, 2013,74:792-804.
doi: 10.1111/tpj.12161 pmid: 23451851
[22] Ding S, Liu X Y, Wang H C, Wang Y, Tang J J, Yang Y Z, Tan B C. SMK6 mediates the C-to-U editing at multiple sites in maize mitochondria. J Plant Physiol, 2019,240:152992.
pmid: 31234031
[23] Wang H C, Sayyed A, Liu X Y, Yang Y Z, Sun F, Wang Y, Wang M, Tan B C. SMALL KERNEL4 is required for mitochondrial cox1 transcript editing and seed development in maize. J Integr Plant Biol, 2019.
pmid: 33215867
[24] Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J, Hoffmann B, Lecharny A, Le Ret M, Martin-Magniette M L, Mireau H, Peeters N, Renou J P, Szurek B, Taconnat L, Small I. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell, 2004,16:2089-2103.
doi: 10.1105/tpc.104.022236 pmid: 15269332
[25] Wang Y, Liu W, Wang H, Du Q, Fu Z, Li W X, Tang J. ZmEHD1 is required for kernel development and vegetative growth through regulating auxin homeostasis. Plant Physiol, 2019,182:1467-1480.
doi: 10.1104/pp.19.01336 pmid: 31857426
[26] Li X J, Zhang Y F, Hou M, Sun F, Shen Y, Xiu Z H, Wang X, Chen Z L, Sun S S, Small I, Tan B C. Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa). Plant J, 2014,79:797-809.
doi: 10.1111/tpj.12584 pmid: 24923534
[27] Yang Y Z, Ding S, Wang Y, Li C L, Shen Y, Meeley R, McCarty D R, Tan B C. Small kernel2 encodes a glutaminase in Vitamin B6 biosynthesis essential for maize seed development. Plant Physiol, 2017,174:1127-1138.
pmid: 28408540
[28] Pan Z, Ren X, Zhao H, Liu L, Tan Z, Qiu F. A mitochondrial transcription termination factor, ZmSmk3, is required for nad1 intron4 and nad4 intron1 splicing and kernel development in maize. G3: Genes Genom Genet, 2019,9:2677-2686.
[29] Pan Z, Liu M, Xiao Z, Ren X, Zhao H, Gong D, Liang K, Tan Z, Shao Y, Qiu F. ZmSMK9, a pentatricopeptide repeat protein, is involved in the cis-splicing of nad5, kernel development and plant architecture in maize. Plant Sci, 2019,288:110205.
doi: 10.1016/j.plantsci.2019.110205 pmid: 31521217
[30] Handa N, Terada T, Kamewari Y, Hamana H, Tame J R, Park S Y, Kinoshita K, Ota M, Nakamura H, Kuramitsu S, Shirouzu M, Yokoyama S. Crystal structure of the conserved protein TT1542 from thermus thermophilus HB8. Protein Sci, 2003,12:1621-1632.
doi: 10.1110/gad.03104003 pmid: 12876312
[31] 王家利, 刘冬成, 郭小丽, 张爱民. 生长素合成途径的研究进展. 植物学报, 2012,47:292-301.
Wang J L, Liu D C, Guo X L, Zhang A M. Research progress of auxin synthesis pathway. Chin Bull Bot, 2012,47:292-301 (in Chinese with English abstract).
[32] Bashline L, Li S, Zhu X, Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis. Proc Natl Acad Sci USA, 2015,112:12870-12875.
doi: 10.1073/pnas.1509292112 pmid: 26417106
[1] Liang Jin-Yu, Yin Jia-De, Wang Hong-Li, Zhang Guo-Ping, Hou Hui-Zhi, Dong Bo, Ma Ming-Sheng. Estimation of leaf nitrogen content in dryland forage maize using UAV-based hyperspectral imaging and machine learning [J]. Acta Agronomica Sinica, 2026, 52(6): 1788-1801.
[2] Zhang Hong-Rong, Wang Fei-Er, Li Pan, Qiu Hai-Long, Zhu Jing, Zhao Lian-Hao, Nan Yun-You, He Wei, Fan Zhi-Long, Hu Fa-Long, Chai Qiang, Yin Wen. Photosynthetic characteristics of 20% reduced irrigation combined with 25% organic substitution for chemical fertilizer in increasing silage maize yield [J]. Acta Agronomica Sinica, 2026, 52(5): 1487-1500.
[3] Yang Yang, Chang Shi-Hui, Tian Hong-Li, Yi Hong-Mei, Wang Lu, Ren Jie, Fan Ya-Ming, Liu Ya-Wei, Wang Feng-Ge, Zhao Jiu-Ran. Genetic diversity analysis of nationally approved maize varieties in different ecological regions [J]. Acta Agronomica Sinica, 2026, 52(5): 1352-1364.
[4] Yang Xin-Yu, Cui Wen-Tao, Dilinigeer Alimu, Wang Kai-Xiang, Wu Peng-Hao, Ren Jiao-Jiao. Genome-wide association and genomic selection analysis of the number of leaves above the ear in maize [J]. Acta Agronomica Sinica, 2026, 52(5): 1573-1590.
[5] Han Ya-Xin, He Guan-Hua, Zhang Xiao-Qiong, Zhang Deng-Feng, Li Yong-Xiang, Liu Xu-Yang, Wang Tian-Yu, Li Yu, Zou Hua-Wen, Li Chun-Hui. Identification of maize lateral root density genes resources through integrated RNA-seq and BSA-seq analyses [J]. Acta Agronomica Sinica, 2026, 52(5): 1341-1352.
[6] Sun Shu-Feng, Xu Zhen-Nan, Huang Jia-Xin, Weng Jian-Feng, Li Xin-Hai. Genome-wide identification of the maize MAPK gene family and its response to Fusarium verticillioides infection [J]. Acta Agronomica Sinica, 2026, 52(5): 1291-1308.
[7] Zhang Ning-Ning, Teng Yu-Fei, Ren Na-Na, Wei Xing-Zhuo, Yan Shu-Hao, Fan Ke-Xin, Wang Yong-Hong, Chen Wen-Kang, Zhang Xing-Hua, Zhu Wan-Chao, Xu Shu-Tu, Xue Ji-Quan. Phenotypic evaluation and plasticity analysis of drought resistance in 201 maize inbred lines [J]. Acta Agronomica Sinica, 2026, 52(5): 1309-1325.
[8] Cai Hong-Wei, Yu Ai-Zhong, Jiang Ke-Qiang, Wang Peng-Fei, Wang Yu-Long, Huo Jian-Zhe, Pang Xiao-Neng, Yin Bo, Shang Yong-Pan. Key mechanisms underlying the enhancement of sweet maize yield through partial substitution of chemical fertilizers with organic manure in arid irrigation districts [J]. Acta Agronomica Sinica, 2026, 52(4): 1166-1180.
[9] Tian Hong-Li, Yang Yang, Fan Ya-Ming, Yi Hong-Mei, Guo Dan-Dan, Wang Feng-Ge, Zhao Jiu-Ran. A novel set of tri-allelic variant SNP loci suitable for maize variety identification [J]. Acta Agronomica Sinica, 2026, 52(4): 993-1005.
[10] Guo Xiang-Yang, Tu Liang, Wang Dong, Liu Peng-Fei, Wang An-Gui, Yi Qiang, Ren Hong, Li Gang, Zhu Yun-Fang, Wu Xun, Jiang Yu-Lin, Tian Feng, Chen Ze-Hui. Application and prospects of Suwan germplasm in maize breeding in China [J]. Acta Agronomica Sinica, 2026, 52(3): 655-664.
[11] Ma Liang, Ma Lu, Zhang Shu-Yu, Zhang Hui-Min, Wang Ren-Ming, Song Xu-Dong, Zhang Zhen-Liang, Mao Yu-Xiang, Lu Hu-Hua, Chen Guo-Qing, Hao De-Rong, Zhou Guang-Fei. Transcriptome analysis and identification of candidate genes associated with husk number in maize [J]. Acta Agronomica Sinica, 2026, 52(3): 790-801.
[12] Meng Cheng, Wang Zhe. Genome-wide identification and expression analysis of the ZmPFK gene family under biotic and abiotic stresses in maize [J]. Acta Agronomica Sinica, 2026, 52(3): 764-779.
[13] Li Xin-Hao, Xing Meng-Ke, Zhou Zi-Hui, Li Si-Ye, Ren Hao, Wang Hong-Zhang, Lai Hua-Jiang. Exogenous melatonin enhances heat tolerance of maize at the seedling stage by coordinating light and dark reactions [J]. Acta Agronomica Sinica, 2026, 52(3): 839-856.
[14] Liu Ji-Chang, Li Si-Ye, Li Xue-Ting, Wang Hong-Zhang, Liu Peng, Zhang Ji-Wang, Zhao Bin, Ren Bai-Zhao, Ren Hao. Effects of salt stress on root growth and nutrient absorption efficiency of different salt-tolerant summer maize varieties [J]. Acta Agronomica Sinica, 2026, 52(2): 565-577.
[15] Lin Zi-Qing, Zhong Xing-Yu, Liu Fan, Ren Zi-Ao, Ma Rui, Deng Xiu-Feng, Wang Dong-Wei, Liu Shao-Peng, Chen Kang, Zhang Ming-Cai, Li Zhao-Hu, Zhou Yu-Yi, Duan Liu-Sheng. Development of ultra-high-yield technology for a wheat-maize double cropping system achieving a 2-ton annual grain yield per mu in the coastal plain of Northern Shandong peninsula, China [J]. Acta Agronomica Sinica, 2026, 52(2): 631-643.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd