Abstract
Key message
The duplicated male sterile genes ms5m6 in cotton were map-based cloned and validated by the virus-induced gene silencing assays. Duplicate mutations of the GhCYP450 gene encoding a cytochrome P450 protein are responsible for the male sterility in cotton.
Abstract
The utilization of male sterility in cotton plays a vital role in improving yield and fiber quality. A complete male sterile line (ms5ms6) has been extensively used to develop hybrid cotton worldwide. Using Zhongkang-A (ZK-A) developed by transferring Bt and ms5ms6 genes into the commercial cultivar Zhongmiansuo 12, the duplicate genes were map-based cloned and confirmed via the virus-induced gene silencing (VIGS) assays. The duplicate mutations of GhCYP450 genes encoding a cytochrome P450 protein were responsible for producing male sterility in ms5ms6 in cotton. Sequence alignment showed that GhCYP450-Dt in ZK-A differed in two critical aspects from the fertile wild-type TM-1: GhCYP450-Dt has three amino acid (D98E, E168K, G198R) changes in the coding region and a 7-bp (GGAAAAA) insertion in the promoter domain; GhCYP450-At appears to be premature termination of GhCYP450 translation. Further morphological observation and cytological examination of GhCYP450-silenced plants induced by VIGS exhibited shorter filaments and no mature pollen grains. These results indicate that GhCYP450 is essential for pollen exine formation and pollen development for male fertility. Investigating the mechanisms of ms5ms6 male sterility will deepen our understanding of the development and utilization of heterosis.
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Data availability
Data supporting the findings of this work are available within the paper and its Supplementary Information files. The plant materials and datasets generated and analyzed during the present study are available from the corresponding authors upon reasonable request.
Change history
23 March 2023
A Correction to this paper has been published: https://doi.org/10.1007/s00122-023-04323-z
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Acknowledgements
We would like to thank the Bio-ultrastructure Analysis Lab. of the Analysis center of Agrobiology and Environmental Sciences, Zhejiang Univ. for the TEM and SEM assays. We thank Prof. Zhu Jun from College of Life and Environment Sciences, Shanghai Normal University, for providing the Arabidopsis mutant SALK_119582.
Funding
This study was financially supported in part by grants from the NSFC (32130075), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2019R01002), and the Fundamental Research Funds for the Central Universities (226-2022-00100).
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Data analysis and writing of the manuscript were performed by YM. Study conception and design were performed by TZ, and YM carried out the experiments. FD conducted the bioinformatic analysis. ZS, YM planted and scored the phenotypes. YM, TZ, and LF wrote the manuscript. All authors read and approved the final manuscript.
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Supplementary file 1
: Table S1. Primers used in this study. Table S2. χ2 test for F2 populations. Table S3. Candidate genesS1 in the 646 Kb interval of A12 chromosome. Table S4. Candidate genes in the 309 Kb interval of D12 chromosome. Table S5. Expression levels of candidate homologous genes between A12 and D12 candidate regions. Table S6. Variations in coding sequence of candidate genes on chromosome A12. Table S7. Variations in coding sequence of candidate genes on D12. Table S8. Information of GhCYP450 and its orthologs in 8 species used in Phylogenetic analysis. Table S9. DEGs between ZKA and WT anthers in the tetrad pollen (TTP) stage. Table S10. DEGs between ZKA and WT anthers in the binucleate pollen (BNP) stage. Table S11. DEGs between ZKA and WT anthers in the mature pollen (MP) stage. Table S12. Male sterility genes related to pollen development.
Supplementary file 2
: Fig. S1. Expression of candidate genes during the different development stages of ovules between ZK-A and TM1 based on RNA-seq analyses. Fig. S2. The qRT-PCR analyses of candidate genes. Fig. S3. The alignment of the cDNA sequence of GhCYP450 in ZK-A and TM1. Fig. S4. The alignment of GhCYP450 protein in ZK-A and TM1. Fig. S5. The alignment of the GhCYP450_D12 promoter sequence between the TM-1 and ZK-A. Fig. S6. Promoter activity assay of GhCYP450_D12 by histochemical GUS staining in Nicotiana transiently expressing leaves. Fig. S7. Phenotypes were observed during the seeding period after vaccination with the TRV virus. Fig. S8. The glandular phenotypes of different tissues in the flowering stage were observed. Fig. S9. Functional enrichment of the differential expressed genes (DEGs) with decreased and increased expression during the different development stages of ovules. Fig. S10. The expression levels of other male fertility genes associated with fatty acid transportation, pollen wall and anther cuticle construction.
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Mao, Y., Dai, F., Si, Z. et al. Duplicate mutations of GhCYP450 lead to the production of ms5m6 male sterile line in cotton. Theor Appl Genet 136, 2 (2023). https://doi.org/10.1007/s00122-023-04296-z
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DOI: https://doi.org/10.1007/s00122-023-04296-z