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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (11): 3122-3130.doi: 10.3724/SP.J.1006.2023.23076

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Map-based cloning and transcriptomic analysis of a maize miniature kernel mutant mn-Mu

DING Meng-Li(), WANG Ru-Yin, SHI Dong-Sheng, LI Ying-Bo, LEI Jie, CHEN Hong-Yu, SHEN Qing-Wen(), WANG Gui-Feng   

  1. College of Agronomy, Henan Agricultural University / State Key Laboratory of Wheat and Maize Crops Science / CIMMYT-China (Henan) Joint Research Center of Wheat and Maize, Zhengzhou 450046, Henan, China
  • Received:2022-11-14 Accepted:2023-05-24 Online:2023-11-12 Published:2023-05-31
  • Supported by:
    National Natural Science Foundation of China(32001562);National Natural Science Foundation of China(U22A20466);Science and Technology Innovation Fund of Henan Agricultural University(KJCX2020A04)

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Abstract:

Kernel is the main storage organ, which accumulates nutritional compounds and determinates maize yield. In this study, we identified a miniature kernel mutant, mn-Mu, caused by a random mutator’s insertion. Compared with wild type, mn-Mu had smaller kernels, shrunken pericarp, and decreased significantly kernel weight. Compared with wild type, starch content of mn-Mu kernels decreased slightly, while zein content increased. Cytological observation showed that the development of the central endosperm and the basal endosperm transfer layer (BETL) was impaired in mn-Mu mutant compared with the wild type. Genetic analysis revealed that mn-Mu was a single gene-controlled recessive mutant. Map-based cloning indicated that the mutant gene was positioned in 57.83-61.91 Mb on chromosome 2. In this interval, Zm00001d003776 gene, which encoded CELL WALL INVERTASE 2 (INCW2, Miniature 1, Mn1) previously reported, was found that a 1442 bp transposon derived from Bronze 1 locus inserted into its 5th exon. Allelic test validated that mn-Mu was a new allelic mutant of mn1. Furthermore, transcriptomic analysis revealed that the loss of Mn1 affected gene expression in the processes of carbohydrate metabolism, storage material accumulation, glycosyltransferase activity, cell wall biosynthesis, and cell cycle regulation. In conclusion, a new allelic mutant of maize Mn1 (mn-Mu) was identified and transcriptomic analysis was further performed, which providing a novel maize germplasm and clues toward comprehensive understanding of molecular regulation mechanism of Mn1 on kernel development.

Key words: maize, kernel development, mn-Mu, gene mapping, cell wall invertase, transcriptomic analysis

Table 1

Primers for gene mapping and Mn1 gene amplification in this study"

引物名称
Primer name		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
Indel-26.11 ACTTGCCTGCTGCGACTGG TCCTGCCCGTCCCGACAG
Indel-34.89 TCGTGGCTGGCTGGAGTG CGGGAAATCGGGTTGTTGATTG
Indel-43.19 TGGTAGGCAGGGCGTTGG TGTCCGTGTAGACTGTAGGAAC
Indel-53.27 GGGAGAGCATCGACAGATACTG GACAATGACTTCGTACCTTCGG
Indel-55.89 GCCGCACACACCCTATGAAG AGCGTGCATGTATCTGGAAGTG
Indel-57.83 GCAATCCCCTTGTCCTGAAAAG CTTCCAACGCAGCAGCAATG
Indel-61.91 AGGTCAACCATCGAATCGAAGC ATCTGCATGGTTCAGAGTCTCC
Indel-83.16 TCCATCTATGGGGAGCGAGTAC GCAGTCCGTGACGACAACC
PDC1R/2F GCAACGGTGCAAGTGAACAA GCGGTACGACTACTACACCG
PDC1F/2R GTCGTAAGTTTCGCTTCGGC TCGTAGAACGTCTTGGACGC

Fig. 1

Phenotypic features of maize mn-Mu kernels A: the typical self-cross mature ear of F1 plant; B-C: the comparison of kernel length (B) and kernel width (C) of the wild type (the upper row) and mn-Mu (the lower row) kernels; D: phenotype of the wild type (the left one) and mn-Mu (the right one) kernels; E: the longitudinal-sectional view of of the wild type (left) and mn-Mu (right) kernels; F: the comparison of 100-kernel weight of randomly selected mature wild type and mn-Mu kernels in the segregated ears. Values are means ±SDs. (**: P< 0.01, Student’s t-test). Bar: 1 cm."

Table 2

Segregation ratio of wild-type and mutant kernels in F2 ears"

果穗
Ear		 正常籽粒数
Wild type		 突变籽粒数
Mutant		 χ2
1 330 89 2.9602
2 295 100 0.0076
3 297 92 0.3093
总计 Total 922 281 1.6428

Fig. 2

Histological observation of mn-Mu mutant kernels A-B: the longitudinal paraffin sections observation of developing wild-type (A) and mn-Mu mutant (B) kernels, Bar: 1 mm; C-D: the embryos of the wild-type (C) and mn-Mu mutant (D) kernels, Bar: 1 mm (C) and 500 μm (D); E-F: the basal endosperm transfer layer of the wild-type (E) and mn-Mu mutant (F) kernels, Bar: 200 μm. Samples were collected at 18 days after pollination."

Fig. 3

Biochemical component of mn-Mu mutant A: starch contents of the wild type and mn-Mu mutant kernels; B-C: zein (B) and non-zein (C) accumulation pattern of wild type and mn-Mu mutant kernels by 15% SDS-PAGE. M: marker; WT: wild type; Re1/Re2/Re3 represent three biological replicants."

Fig. 4

Map-based cloning of mn-Mu gene in maize A: gene mapping of mn-Mu. n: the number of individuals for gene mapping, the number under each InDel marker indicates recombinants in the population. B: schematic diagram of Mn1 gene with the indicated mutator insertion site in mn-Mu by a triangle. C: PCR amplification of Mn1 gene using genomic DNA to detect the mutator insertion in mn-Mu mutant. M: marker; WT: wild type; Z58: the inbred line Zheng 58 as a control."

Fig. 5

Allelism test between mn-Mu and mn1 A-B: the self-pollinated ears of the reciprocally crossed ears between heterozygous mn-Mu and mn1segregate defective kernels at a ratio of 25%. Bar: 1 cm."

Fig. 6

Transcriptome of mn-Mu mutant A: the volcano plot for differentially expressed genes (DEGs) (Fold change > 2 and adjusted P-value < 0.05) between the wild type and mn-Mu mutant; B: significant KEGG pathways in DEGs; C: significant GO terms in DEGs."

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