Morphological characterization and genetic mapping of shrunken endosperm mutant sh2021 in maize
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摘要:目的
分析玉米籽粒皱缩突变体的表型特征并进行籽粒相关基因的精细定位，为揭示该基因调控玉米籽粒发育的分子机制奠定基础。
方法以玉米自交系‘B73’种植过程中籽粒自发突变个体为材料，命名为shank2021(sh2021)，对其形态学和细胞学特征进行观察；构建分离群体，通过混合群体分离分析法(Bulked segregant analysis，BSA)对基因进行初步定位，筛选交换单株进一步缩小定位区间，最后结合转录组测序及基因功能注释推测控制籽粒缺陷性状的候选基因。
结果与野生型相比，sh2021籽粒凹陷皱缩、颜色加深、籽粒排列不规则，且百粒质量降低。扫描电镜观察发现，与野生型相比，sh2021胚乳细胞和淀粉粒均显著变小，且淀粉粒大小不均匀。遗传分析结果表明，sh2021是由单个隐性基因突变所致。利用BSA分析方法将目的基因定位在3号染色体末端约13.25 Mb区域。进一步扩大分离群体筛选交换单株，将目的基因定位在标记ID5与ID9之间的529.60 kb范围。通过sh2021和野生型的转录组测序，结合基因功能注释结果，预测Zm00001d044119、Zm00001d044120、Zm00001d044122、Zm00001d044129、Zm00001d044142这5个基因可能是影响玉米籽粒发育的重要候选基因。
结论sh2021的发现与鉴定为进一步深入解析玉米籽粒发育的遗传机制提供了丰富的试验材料，同时也为后续相关基因的克隆和功能解析奠定了基础。
Abstract:ObjectiveTo analyze the phenotypic characteristic of maize shrunk endosperm mutant and fine mapping of related genes, and lay a foundation for further understanding molecular mechanism of maize kernel development.
MethodThe spontaneous shrunk endosperm mutant shank2021(sh2021) was isolated from maize inbred line B73. Morphological and cytological characteristics were observed. A segregating population was developed, and the bulked segregant analysis (BSA) was used for preliminary gene mapping. The recombinant plants were screened for further narrowing the mapping interval. Finally the candidate genes controlling grain defect traits were speculated by transcriptome sequencing combined with gene function annotation analysis.
ResultCompared with wild type, sh2021 displayed sunken and shrunken kernels, darker color, irregular grain arrangement, and lower 100-grain weight. The scanning electron microscope observation revealed that sh2021 had smaller endosperm cells and starch granules, and the starch granules were varied in size. The genetic analysis results indicated that sh2021 was caused by a single recessive gene mutation. The BSA indicated that the target gene was located on a 13.25 Mb fragment at the end of chromosome 3. By further expanding the segregating population and screening recombinant plants, the target gene was narrowed down to an interval of 529.60 kb between markers ID5 and ID9. Transcriptome sequencing and gene annotation of sh2021 and wild type indicated that Zm00001d044119, Zm00001d044120, Zm00001d044122, Zm00001d044129, and Zm00001d044142 mighted be candidate genes controlling maize kernel development.
ConclusionThe identification of sh2021 provides abundant experimental materials for the study of kernel development, and lays a foundation for further map-based cloning and functional analysis of sh2021.
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Keywords:
- Maize /
- Shrunken endosperm mutant /
- Genetic analysis /
- Fine mapping
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图 1 sh2021突变体表型鉴定(F2分离群体)
A：完熟期玉米果穗；B：籽粒表型；C：籽粒百粒质量；“**”表示sh201与野生型在0.01水平差异显著(t检验)
Figure 1. Phenotypic identification of sh2021 mutants (F2 segregating population)
A: Mature ear; B: Grain phenotype; C: 100-kernel weight; “**” indicate significant difference between wild type and sh2021 at the 0.01 level (t test)
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图 2 WT(A)和sh2021(B)籽粒的石蜡切片图
Figure 2. Paraffin sections of mature WT (A) and sh2021 (B) grains
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图 3 野生型与sh2021籽粒扫描电镜观察
A、 B分别为WT、sh2021籽粒胚乳；C、D分别为WT、sh2021籽粒淀粉粒
Figure 3. Scanning electron microscope observation of WT and sh2021 grains
A and B are the endosperms of WT and sh2021 grains respectively; C and D are starch granules of WT and sh2021 grains respectively
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图 4 SNP-index的全基因组分布图
散点为原始值，黑色曲线为拟合值；蓝色阈值线和红色阈值线分别表示各位点以及窗口的ΔSNP-index 95%和99%置信区间；横坐标为染色体的分布，每个点代表ΔSNP-index值
Figure 4. Genome-wide distribution of SNP-index
The scatter points are the original values, and the black curve is the fitted value; Blue threshold line and red threshold line indicate the ΔSNP-index 95% and 99% confidence intervals, respectively; The horizontal coordinate is the distribution of chromosomes, and each dot represents the ΔSNP-index value
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图 5 SNP-index在3号染色体上的分布
散点图为原始值，黑色曲线为拟合值，其他横线为指定阈值位置；横坐标为3号染色体的分布，每个点代表的ΔSNP-index值
Figure 5. Distribution of SNP-index on Chromosome 3
The scatter plot is the original value, the black curve is the fitted value, and the other horizontal lines are the specified threshold positions; The horizontal coordinate is the distribution of Chr 3, and each dot represents the ΔSNP-index value
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图 7 sh2021和WT籽粒在授粉后不同时间的差异表达基因GO (A、B)和KEGG (C、D)富集分析
Figure 7. GO(A,B) and KEGG(C,D) enrichment analyses of differentially expressed genes in sh2021 and WT grains at different time after pollination
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图 8 候选区间内差异表达基因qRT-PCR验证
“**”表示sh2021与WT在0.01水平差异显著(t检验)
Figure 8. Validation of differentially expressed genes in candidate intervals by qRT-PCR
“**” indicates significant difference at the level of 0.01between sh2021 and WT (t test)
表 1 sh2021 基因定位引物
Table 1 Primers used for gene mapping of sh2021
引物名称
Primer name序列(5′→3′)
SequenceID1 F：TACAGCTTGTAAAAATACAGGGCC
R：ATGAGAGTGCATGGTCCGTGID2 F：GGCAGCAGGATCAGAAGAGA
R：GGAACTTGTTGTGCCAAGGID3 F：TCTCGAATCAAGAACCAGCA
R：GGAGGGATTGGGTGAAGATTID4 F：GTCTACAACCGGCTCCTCAA
R；GTGGAAGTGTCGTCCGTTCTID5 F：GCCATCTACAGTGTCAGCCA
R：GTCAACGAGATTCTCAGGGCID9 F：CTTCCCGTCGAGATCCACG
R：GTTGACAGCTTGTGGGGTCTID10 F：GCTAGAGATGGCTATTAGGTGCA
R：AGCGTGTCTAACCTCACTTATAACAID11 F：TAACTGTCTCGCTGGGCTTT
R：GGCGTCCGTCCAAATAAAID12 F：CCATGAGGAGGCATGTGTGG
R：CAGCTGGTTCACGCCACATCID13 F：TGCACTTCGAGAGGTTTGTT
R：TCATAATAGTCTCGTATCCAGCCTID14 F：ACCCAAGGACCCCAAAGAGA
R：GCCCTATGCTTGGGGAGTGID15 F：GCCTGTCTAGCATATGATGTGAA
R：GTCTCATGCCAAACGAGGATC3-102 F：CTAGCCAGCCCCCATTCTTC
R：GCAAGGAGTAGGGAGGACGTG
下载: 导出CSV
表 2 qRT-PCR定量验证引物
Table 2 qRT-PCR quantitative verification primers
引物名称
Primer name序列(5′→3′)
SequenceHX1 F：TTTGAGCGAAACTCCAGTGC
R：ATGTTCATGTGCTGCAGGACHX2 F：GAGTCGGAGATCAGCATCGG
R：AAAGTGTCGGCGAAGAACCTHX3 F：TTGGGAAGACGAAGCCATTG
R：AGGTCGGCTTTCCATATGGACHX4 F：TCACTGAGCAGCCTTCCAAG
R：GACACGTGAGCAGACTCCAAHX5 F：GCCACGGGATCATGAAGGAG
R：TCTCCTCCAACAGACCCTCC
下载: 导出CSV
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