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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (3): 603-612.doi: 10.3724/SP.J.1006.2024.33035

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

Mapping and functional analysis of maize inflorescence development gene AFP1

XUE Ming1(), WANG Chen-Chen1, JIANG Lu-Guang2, LIU Hao1, ZHANG Lu-Yao1, CHEN Sai-Hua1,*()   

  1. 1Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding / Key Laboratory of Plant Functional Genomics of the Ministry of Education / Jiangsu Key Laboratory of Crop Genetics and Physiology, Agricultural College of Yangzhou University, Yangzhou 225009, Jiangsu, China
    2MOE Key Laboratory of Crop Heterosis and Utilization, National Maize Improvement Center of China, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
  • Received:2023-05-26 Accepted:2023-09-13 Online:2024-03-12 Published:2023-10-08
  • Contact: *E-mail: chensaihua@yzu.edu.cn
  • Supported by:
    Jiangsu Province Government(JBGS[2021]002);Key Research and Development Program of Jiangsu Province(BE2022343)

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Abstract:

The normal development of maize inflorescence is the foundation of yield. Mining genes and metabolic pathways can reveal the molecular mechanism of inflorescence development, which will provide a theoretical guidance for maize yield improvement. In this study, a mutant with altered flower pattern 1 (named afp1) was screened from a Mo17 EMS mutant library. The afp1 mutant showed excrescent branches with no silk in the ear, while it increased the lateral spikelet number in the tassel. Genetic analysis revealed that the afp1 phenotype was controlled by a single recessive gene. The gene was initially restricted between M150 and M176 on chromosome 7 by linkage analysis in the afp1×B73 F2 population. It was further narrowed into a 300 kb region between M1722 and M1725 with 14 newly developed molecular markers. Within the mapping region, a known gene related to inflorescence development, BD1 (Branched silkless 1), was located. Due to a C-T mutation in afp1, the 67th conserved arginine (R) in the ERF/AP2 domain of the BD1 protein was altered into tryptophan (W). A total of 274 differentially expressed genes (DEGs) were identified between young ears in afp1 and the wild-type by RNA-seq. Among these genes, 56.20% were down-regulated and 43.80% were up-regulated, which were enriched in multiple metabolic pathways by KEGG analysis. Among them, 83.3% of DEGs in phytohormone signaling pathways were significantly down-regulated, suggesting that afp1 may modulate inflorescence development via a hormone-dependent pathway.

Key words: maize, inflorescence development, BD1, hormone signaling

Table 1

Primers used in this study"

引物名称
Primer name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 备注
Note
M150 GACATTCCGTCCTTGAAATC AGGGATCGGCGTTTGATG 图位克隆 Map-based cloning
M158 ATGACTCACGATGGGTATTG CCAAGCAGGTCAGGGTTC 图位克隆 Map-based cloning
M168 TCACGAGTCACGCGACAA GGAAGACGACAGCGAGGA 图位克隆 Map-based cloning
M1690 AGGAGTAGTAGCGGGTGG CACCTGATTTGTGCAAGGG 图位克隆 Map-based cloning
M1696 GTTGAGACGGCGACGACA AAGTGGAGGACGAAAGGA 图位克隆 Map-based cloning
M1702 GTATGGTTTGGGCGTTTG TGCTGGCAAGAATCCACT 图位克隆 Map-based cloning
M1705 CTTGTTTTGAGTTTGCTGCTTC TAACTGCTGGCTATGTCGAGC 图位克隆 Map-based cloning
M171 GTAGCGGCGGTGGAGAAT CCGTGGCTGACAAAGGGA 图位克隆 Map-based cloning
M1718 GCGTATGCGTCTTGGTTG TCGGCTTTAATACTTGCTATCT 图位克隆 Map-based cloning
M1721 GATTCTCGTAGAAGCGGACTG CTGGAGCAATGGCTGTCG 图位克隆 Map-based cloning
M1722 CTGTACATGTGCAGTGTCC TGCTACTGGTCGATCTATC 图位克隆 Map-based cloning
M1725 CGCCGCTGCTTTCTACTCAT TCAATCGTTTCTGGTGGG 图位克隆 Map-based cloning
M173 GCGTCGCTTTCGGGTCTT ATCGGCCTGAAATCGTGT 图位克隆 Map-based cloning
M176 AGGGTGGAACGGATAGGG TGTCGTGGCTGGCTCACT 图位克隆 Map-based cloning
BD1-promoter AGTGGGCCGATTGACGTTGC AATGCCGCTCCTTGGTCGTG 测序 Sequencing
BD1-gene TAAACAGCAGGGATCGGAAGA CAGGCCACCCAGCACTCA 测序 Sequencing
Zm00001d039958 ATTGATATGGCGTTCGCTGAG CTCGTTGTTGACCTCGTAGTGC 实时定量PCR qRT-PCR
Zm00001d014840 TCGTCACCGAGCAGTGGACC CGCCAAGGATGTCGCAGAA 实时定量PCR qRT-PCR
Zm00001d047736 GCCCGTCAACAAGGAGG GGAGTCGTGGGAGGAGATG 实时定量PCR qRT-PCR
Zm00001d007464 CTTGCCGAGCAGAGGAG TTCGTTGTTAGCAGGGAC 实时定量PCR qRT-PCR
Zm00001d037737 ACAACCATGAGCCACAA CAACTCCATTTCCCTTCT 实时定量PCR qRT-PCR
Zm00001d028216 GAGATTCAGCGCATCAAAG CAAAGCGTGGCTCAGTT 实时定量PCR qRT-PCR
Zm00001d049641 GTCACCGTCTTTGGCATCAG AGCAGCCTTGTCCTTGTCAGT 实时定量PCR qRT-PCR

Fig. 1

Ear and tassel of Mo17 and afp1 (A): schematic of branching in maize inflorescence; AM: axillary meristem; IM: inflorescence meristem; SPM: spikelet-paired meristem; BM: branch meristems; SM: spikelet meristem; FM: floral meristem; (B): female inflorescence[8]; (C): male inflorescence[8]; (D): ear of Mo17; (E): ear of Mo17 without husk; (F): ear of afp1; (G): ear of afp1 without husk; (H): tassel and spikelet of Mo17; (I): tassel and spikelet of afp1."

Table 2

Genetic analysis of the afp1 locus"

群体
Population		 总植株数
Total number		 突变体植株数
Mutant plants		 野生型植株数
Wild type plants		 突变体/野生型(1:3)
Mutant plants/wild type plants (1:3)		 χ2 χ2(0.05)
B73×afp1 F2 171 45 126 1:2.8 0.69 3.84

Table 3

Data analysis identified by SNP array"

样本
Sample		 缺失数
Number of missing genotypes		 缺失率
Missing rate (%)		 杂合数
Number of heterogeneities		 杂合率
Heterogeneity rate (%)
WM1 2865 4.6956 26,377 43.2304
WM2 3056 5.0086 26,126 42.819
MM1 2709 4.4399 26,426 43.3107
MM2 2826 4.6316 26,143 42.8468
Mo17 777 1.2735 4351 7.131
B73 243 0.3969 238 0.3888
afp1 1117 1.8307 5356 8.7782

Fig. 2

Fine mapping of afp1 and analysis of candidate gene (A): fine mapping of afp1. (B): structure of BD1 (Branched silkless 1) and mutant loci. Single-nucleotide mutations and the corresponding amino acid changes in afp1 are shown beneath the schematic illustration of BD1, red boxes indicate the positions of the AP2/ERF domain. (C): the conservation of AP2/ERF domain was examined among gramineous species."

Table 4

Overview of the sequence assembly after Illumina sequencing"

样品
Sample		 过滤后序列
Clean reads		 Q20比例
Q20 (%)		 Q30比例
Q30 (%)		 GC含量
GC (%)		 比对到基因组比例
Total mapping ratio (%)		 单一位置序列比例
Unique mapping ratio (%)
CK1 48,448,700 96.84 91.62 55.74 86.61 84.16
CK2 71,004,610 96.79 91.57 56.21 85.73 83.44
CK3 53,127,902 96.76 91.46 55.26 86.03 83.73
601-1 51,585,326 96.85 91.74 55.60 85.41 82.82
601-4 56,660,540 96.67 91.24 55.40 85.29 82.75
600-3 57,037,362 96.81 91.59 55.13 85.51 83.17

Fig. 3

Volcano plot of differentially expressed genes (DEGs) and the relative expression patterns of genes (A): Volcano plot of DEGs. (B): qRT-PCR analysis."

Fig. 4

KEGG analysis based on the differentially expressed genes (DEGs)"

Table 5

Genes involved in phytohormone signaling pathway"

激素
Hormones		 基因编号
Gene ID		 差异倍数
log2 (Fold Change)		 基因注释
Gene description
生长素信号 Auxin signaling Zm00001d039513 -1.33 生长素响应蛋白 Auxin-responsive protein IAA
Zm00001d043350 -6.38 生长素响应GH3基因家族 Auxin responsive GH3 gene family
水杨酸Salicylic acid Zm00001d018738 6.07 病害相关蛋白1 Pathogenesis-related protein 1
Zm00001d004966 -2.08 调节蛋白NPR1 Regulatory protein NPR1
Zm00001d010658 -5.12 转录因子TGA Transcription factor TGA
乙烯信号Ethylene signaling Zm00001d021687 -2.59 乙烯受体 Ethylene receptor

Fig. 5

Relative expression pattern of differentially expressed genes (DEGs) (A): the relative expression pattern of reported genes involved in SM and FM development; (B): the relative expression pattern of the MADS-box family; (C): the relative expression pattern of genes specifically expressed in inflorescences of afp1; (D): the relative expression pattern of low-expressed genes in the ear of afp1."

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