Mutation in a PHD-finger protein MS4 causes male sterility in soybean

Thu et al. BMC Plant Biology (2019) 19:378 https://doi.org/10.1186/s12870-019-1979-4 RESEARCH ARTICLE Open Access Mutation in a PHD-finger protein MS4 causes male sterility in soybean Sandi Win Thu1†, Krishan Mohan Rai1†, Devinder Sandhu2*, Alex Rajangam3, Vimal Kumar Balasubramanian1, Reid G. Palmer4ˆ and Venugopal Mendu1* Abstract Background: Male sterility has tremendous scientific and economic importance in hybrid seed production. Identification and characterization of a stable male sterility gene will be highly beneficial for making hybrid seed production economically feasible. In soybean, eleven male-sterile, female-fertile mutant lines (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS, and msp) have been identified and mapped onto various soybean chromosomes, however the causal genes responsible for male sterility are not isolated. The objective of this study was to identify and functionally characterize the gene responsible for the male sterility in the ms4 mutant. Results: The ms4 locus was fine mapped to a 216 kb region, which contains 23 protein-coding genes including Glyma.02G243200, an ortholog of Arabidopsis MALE MEIOCYTE DEATH 1 (MMD1), which is a Plant Homeodomain (PHD) protein involved in male fertility. Isolation and sequencing of Glyma.02G243200 from the ms4 mutant line showed a single base insertion in the 3rd exon causing a premature stop codon resulting in truncated protein production. Phylogenetic analysis showed presence of a homolog protein (MS4_homolog) encoded by the Glyma.14G212300 gene. Both proteins were clustered within legume-specific clade of the phylogenetic tree and were likely the result of segmental duplication during the paleoploidization events in soybean. The comparative expression analysis of Ms4 and Ms4_homologs across the soybean developmental and reproductive stages showed significantly higher expression of Ms4 in early flowering (flower bud differentiation) stage than its homolog. The functional complementation of Arabidopsis mmd1 mutant with the soybean Ms4 gene produced normal stamens, successful tetrad formation, fertile pollens and viable seeds, whereas the Ms4_homolog was not able to restore male fertility. Conclusions: Overall, this is the first report, where map based cloning approach was employed to isolate and characterize a gene responsible for the male-sterile phenotype in soybean. Characterization of male sterility genes may facilitate the establishment of a stable male sterility system, highly desired for the viability of hybrid seed production in soybean. Additionally, translational genomics and genome editing technologies can be utilized to generate new male-sterile lines in other plant species. Keywords: Fertility, Mapping, mmd1, ms4, Plant homeodomain, Soybean, Sterility Highlight Fine mapping of soybean ms4 resulted in identification of male sterile phenotype causal mutation. The MS4 is a PHD-finger protein which is functionally characterized using Arabidopsis mmd1 mutant. * Correspondence: ; † Sandi Win Thu and Krishan Mohan Rai contributed equally to this work. 2 US Salinity Laboratory (USDA-ARS), Riverside, CA 92507, USA 1 Fiber and Biopolymer Research Institute, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USA Full list of author information is available at the end of the article Background Soybean (Glycine max) is one of the most important food crops in the world known for its high seed protein and oil content [1]. In general, the crop performance and yield can be increased by generating hybrid plants which outcompete pure lines due to heterosis or hybrid vigor [2]. As soybean is a self-pollinating crop, producing large quantities of hybrid seed by manual emasculation and cross-pollination is difficult and economically nonviable. Hence, identification of a stable male sterility system similar to rice and maize is much needed in soybean [3]. Male sterility resulting from mutations in genes © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Thu et al. BMC Plant Biology (2019) 19:378 involved in microsporogenesis and/or microgametogenesis has been described in various plant species [4]. Mutations in the genes involved in synapsis (proper chromosomal pairing and gamete formation) are known to be responsible for male-sterile female-sterile, malesterile female-fertile or male-fertile female-sterile phenotypes in plants [5]. In soybean, so far, a total of 11 malesterile, female-fertile (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS and msp) mutants have been identified and mapped on to different chromosomes [6– 8]. Further, genetic inheritance and allelism tests, revealed that these 11 soybean male-sterile mutants are genetically independent of each other [7]. Cytological studies on these mutants suggested various mechanisms governing the male-sterile phenotype, which include cytokinesis failure during telophase II, failure of tetrad formation during microsporogenesis, microspore and pollen degeneration, abnormality/lack of vacuoles, and low callose level [4, 7, 9–14]. Molecular characterization of the male sterility genes will be the key to establish a sterility system for commercial application in hybrid seed production. Among the male-sterile mutant lines, the ms4 mutant was an outcome of a spontaneous mutation in cultivar “Rampage” that was identified at the Iowa State University in 1973 (Fig.1) [15]. Phenotyping of homozygous Page 2 of 12 ms4/ms4 plants showed that the anthers were slightly smaller in size and lighter in color compared to the control [15]. The ms4 mutant forms clumps of degenerated empty microspores due to the failure of cytokinesis after telophase II resulting in coenocytic microspores [10]. Genetic analysis of the ms4 mutant line revealed that the male-sterile phenotype is governed by a single recessive gene that mapped to a 694 kb region containing 88 predicted protein-coding genes on soybean chromosome 2 [6]. Fine mapping and/or causal gene identification of ms4 will help in designing molecular markers to screen male-sterile plants for hybrid seed production. The process of hybrid seed production requires planting pure male-sterile plants next to the donor soybean genotypes in field for cross pollination to occur. However, a malesterile line cannot be maintained in homozygous condition as it does not produce seed, hence, is maintained in heterozygous condition. For hybrid seed pro (...truncated)