Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Concurrent modifications in the three homeologs of <Emphasis Type="Italic">Ms45</Emphasis> gene with CRISPR-Cas9 lead to rapid generation of male sterile bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Hexaploid bread wheat is not readily amenable to traditional mutagenesis approaches. In this study, we show efficient utilization of CRISPR-Cas system and Next Generation Sequencing for mutant analysis in wheat.

Abstract

Identification and manipulation of male fertility genes in hexaploid bread wheat is important for understanding the molecular basis of pollen development and to obtain novel sources of nuclear genetic male sterility (NGMS). The maize Male sterile 45 (Ms45) gene encodes a strictosidine synthase-like enzyme and has been shown to be required for male fertility. To investigate the role of Ms45 gene in wheat, mutations in the A, B and D homeologs were produced using CRISPR-Cas9. A variety of mutations in the three homeologs were recovered, including a plant from two different genotypes each with mutations in all three homeologs. Genetic analysis of the mutations demonstrated that all three wheat Ms45 homeologs contribute to male fertility and that triple homozygous mutants are required to abort pollen development and achieve male sterility. Further, it was demonstrated that a wild-type copy of Ms45 gene from rice was able to restore fertility to these wheat mutant plants. Taken together, these observations provide insights into the conservation of MS45 function in a polyploid species. Ms45 based NGMS can be potentially utilized for a Seed Production Technology (SPT)-like hybrid seed production system in wheat.

     

合成甘蓝型油菜中双隐性雄性核不育材料的发现及遗传规律分析

从甘蓝型油菜与白菜型油菜的种间杂交获得的甘蓝型油菜(Brassica napus)中发现了雄性不育单株,兄妹交株系和不育株与甘蓝型油菜常规杂交F1和F2株系的育性分离分析表明,该不育材料属于双隐性雄性核不育类型.利用育性分离株系的可育株自交和可育株与不育株间兄妹交等方法筛选出7个纯合可育株系,等位测验表明这7个纯合可育株系(B1～B7)中存在两种基因型:Ms1Ms1ms2ms2和ms1ms1Ms2MS2.该材料对油菜核不育基因定位和杂种优势利用研究有重要意义.     

Map-based cloning and characterization of <Emphasis Type="Italic">Zea mays male sterility33</Emphasis> (<Emphasis Type="Italic">ZmMs33</Emphasis>) gene,encoding a glycerol-3-phosphate acyltransferase

Key message

Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize.

Abstract

Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.

     

A single point mutation in Ms44 results in dominant male sterility and improves nitrogen use efficiency in maize

Application of nitrogen fertilizer in the past 50 years has resulted in significant increases in crop yields. However, loss of nitrogen from crop fields has been associated with negative impacts on the environment. Developing maize hybrids with improved nitrogen use efficiency is a cost‐effective strategy for increasing yield sustainably. We report that a dominant male‐sterile mutant Ms44 encodes a lipid transfer protein which is expressed specifically in the tapetum. A single amino acid change from alanine to threonine at the signal peptide cleavage site of the Ms44 protein abolished protein processing and impeded the secretion of protein from tapetal cells into the locule, resulting in dominant male sterility. While the total nitrogen (N) content in plants was not changed, Ms44 male‐sterile plants reduced tassel growth and improved ear growth by partitioning more nitrogen to the ear, resulting in a 9.6% increase in kernel number. Hybrids carrying the Ms44 allele demonstrated a 4%–8.5% yield advantage when N is limiting, 1.7% yield advantage under drought and 0.9% yield advantage under optimal growth conditions relative to the yield of wild type. Furthermore, we have developed an Ms44 maintainer line for fertility restoration, male‐sterile inbred seed increase and hybrid seed production. This study reveals that protein secretion from the tapetum into the locule is critical for pollen development and demonstrates that a reduction in competition between tassel and ear by male sterility improves grain yield under low‐nitrogen conditions in maize.     

PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize

Polyethylene glycol (PEG)-mediated transient gene expression and silencing in protoplasts is widely applied in model plants such as Arabidopsis thaliana and rice. Here, we developed an efficient transient gene expression system based on the PEG-mediated method both in etiolated and green maize mesophyll protoplasts. The results showed that both yellow fluorescent protein encoding gene and glucuronidase encoding gene were efficiently expressed in maize protoplasts. More importantly, double-stranded RNAs (dsRNAs) can also be transfected into maize protoplasts by the PEG-mediated method to specifically silence exogenous and endogenous genes. Our results showed that dsRNA can be used to knockdown both exogenous and endogenous gene expression. Furthermore, bimolecular fluorescence complementation system for the detection of protein–protein interactions in maize protoplasts was developed. We also overexpressed and knockdowned the mitogen-activated protein kinase encoding gene ZmMPK5 to investigate the role of ZmMPK5 in abscisic acid (ABA)-induced antioxidant defense in maize protoplasts. This method here we reported will be valuable for signal transduction study in maize.     

Sequence polymorphisms in <Emphasis Type="Italic">Zmisa2</Emphasis> gene are significantly associated with starch pasting and gelatinization properties in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Researches on the genetic basis of starch pasting and gelatinization properties will provide foundation of maize improvement for quality, feed and industrial applications. Maize gene Zmisa2, encoding an isoamylase-type starch debranching enzyme, plays important roles in starch biosynthesis. In this study, the genomic sequences of the gene Zmisa2 in 72 elite maize inbred lines were obtained, and the nucleotide polymorphisms and haplotype diversity were detected. In addition, seven pasting and four gelatinization properties of maize were measured for the tested inbred lines using rapid visco analyzer and differential scanning calorimeter, respectively. A total of 99 sequence variants, including 91 SNPs and 8 indels, were identified at the promoter and coding regions of this gene. Although the frequency of polymorphism in promoter region is much higher than that of coding region, the SNPs in the coding region of maize gene Zmisa2 classified this gene into 21 haplotypes, which encode 11 different ISA2 proteins. Furthermore, the association of the variants of Zmisa2 gene with maize starch pasting and gelatinization properties was estimated, and the results revealed that seven SNPs in coding region, including four nonsynonymous sites, were significantly associated with phenotypic variations of pasting time and enthalpy of transition (ΔH). These results suggested that the polymorphism in maize Zmisa2 locus could be used in molecular marker-assisted selection for improvement of quality in maize breeding programs.     

Fine mapping of the novel male-sterile mutant gene <Emphasis Type="Italic">ms39</Emphasis> in maize originated from outer space flight

A novel male-sterile maize mutant male sterility 39 (ms39) was obtained from offspring of the commercial hybrid Chuandan No. 9 that had been carried into outer space. A previous study demonstrated that ms39 is controlled by a single recessive nuclear gene, located on the long arm of chromosome 3. Here, we used 1073 mutant individuals derived from the (ms39?×?Mo17) F₂ population and sequentially developed new primers to identify markers supporting the fine mapping of ms39. A 365-kb region on chromosome 3 flanked by markers L8 and M30 at a genetic distance of 0.18 and 0.47 cM, respectively, was identified. According to the reference sequence of ZmB73_Ref-Gen_v4, 12 candidate genes were identified within the 365-kb mapping region. Based on cloning and sequence BLAST analysis of the 12 candidate genes, a four-base-pair deletion was found within the exon of Zm00001d043909, which encoded callose synthase12. This four-base-pair deletion resulted in a frameshift mutation in ms39, leading to the earlier termination of the coding protein, and ultimately caused abnormal performance of the callose synthase. Additionally, cytological observation was conducted on a sister cross population (ms39/ms39?×?ms39/Ms39). These observations showed that the tapetum cells of the ms39 mutant appeared abnormal from the dyad stage, and aborted microspores were observed during pollen development. These results lay the foundation for the cloning of ms39 and exploration of the molecular mechanism underlying aborted pollen development in ms39 maize.     

Nucleotide Diversity of Maize ZmBT1 Gene and Association with Starch Physicochemical Properties

Cereal Brittle1 protein has been demonstrated to be involved in the ADP-Glc transport into endosperm plastids, and plays vital roles in the biosynthesis of starch. In this study, the genomic sequences of the ZmBT1 gene in 80 elite maize inbred lines were obtained, and the nucleotide polymorphisms and haplotype diversity were detected. A total of 30 variants, including 22 SNPs and 8 indels, were detected from the full sequences of this gene. Among these polymorphic sites, 9 SNPs and 2 indels were found to be located in the coding region. The polymorphisms of CDS sequences classified the maize ZmBT1 gene into 6 haplotypes, which encode 6 different ZmBT1 proteins. Neutrality tests revealed a decrease in population size and/or balancing selection on the maize ZmBT1 locus. To detect the association between sequence variations of this gene and the starch physicochemical properties, 7 pasting and 4 gelatinization traits of starch were measured for the tested inbred lines using rapid visco analyzer (RVA) and differential scanning calorimeter (DSC), respectively. The result of association analysis revealed that an indel in the coding region was significantly associated with the phenotypic variation of starch gelatinization enthalpy.     

An enhanced U6 promoter for synthesis of short hairpin RNA

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1^G93A) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.