LociScan,a tool for screening genetic marker combinations for plant variety discrimination

To reduce the cost and increase the efficiency of plant genetic marker fingerprinting for variety discrimination,it is desirable to identify the optimal marker combinations.We describe a marker combination screening model based on the genetic algorithm(GA)and implemented in a software tool,Loci Scan.Ratio-based variety discrimination power provided the largest optimization space among multiple fitness functions.Among GA parameters,an increase in population size and generation number enlarged optimization depth but also calculation workload.Exhaustive algorithm afforded the same optimization depth as GA but vastly increased calculation time.In comparison with two other software tools,Loci Scan accommodated missing data,reduced calculation time,and offered more fitness functions.In large datasets,the sample size of training data exerted the strongest influence on calculation time,whereas the marker size of training data showed no effect,and target marker number had limited effect on analysis speed.

Identification of a locus associated with genic male sterility in maize via EMS mutagenesis and bulked-segregant RNA-seq

Genic male sterility(GMS) is one of the most important resources for exploiting heterosis in crop breeding, so that identifying genomic loci regulating GMS is desirable. However, many regulatory genes controlling GMS have not yet been characterized in maize, owing partly to a lack of genetic materials. We generated a recessive male-sterile maize mutant in the Jing 724 genetic background via ethyl methanesulfonate treatment, and found the male sterility to be due to a single gene mutation. Bulk-segregant RNA sequencing of three replicates indicated that one genomic region located at the end of chromosome 4 was associated with the observed mutant phenotype. Among genes with nonsynonymous mutations,Zm00001 d053895(bHLH51) showed abolished expression in the sterile bulks and was annotated as a bHLH transcription factor orthologous to Arabidopsis AMS, suggesting an association with the male sterility of the mutant. Kompetitive Allele-Specific PCR assays further validated the exclusive correlation of male sterility with the single C-to-T mutation in the fifth exon. The new maize mutant and the potential SNP locus provide novel genetic material for investigating the molecular mechanism underlying tapetal development and may facilitate the improvement of hybrid production systems.

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

Efficient and precise genomic deletion in rice using enhanced prime editing

Efficient and precise genomic deletion shows promise for investigating the function of proteins in plant research and enhancing agricultural traits.In this study,we tested the PRIME-Del(PDel)strategy using a pair of prime editing guide RNAs(pegRNAs)that targeted opposite DNA strands and achieved an average deletion efficiency of 55.8%for 60 bp fragment deletions at six endogenous targets.Moreover,as high as 84.2%precise deletion efficiency was obtained for a 2000 bp deletion at the OsGS1 site in transgenic rice plants.To add the bases that were unintentionally deleted between the two nicking sequences,we used the PDel/Syn strategy,which introduced multiple synonymous base mutations in the region that had to be patched in the RT template.The PDel/Syn strategy achieved an average of 58.1%deletion efficiency at six endogenous targets,which was higher than the PDel strategy.The strategies presented in this study contribute to achieving more accurate and flexible deletions in transgenic rice plants.

Trash to treasure:lactate and protein lactylation in maize root impacts response to drought

Lactate,protein lactylation(Kla),and specifically histone lactylation have recently been shown to regulate antipathogenic immune responses in mammals.Herein,after we confirmed the presence and accumulation of lactate in maize roots under drought conditions,a lactylome profiling analysis revealed that Kla modifications were invariably present in maize roots,that there were obvious differences in the lactylomes of drought-sensitive(B73)vs.drought-tolerant(Jing2416)lines,and that growing Jing2416 under drought conditions caused significant decreases in the lactylation of multiple enzymes responsible for fatty acid degradation.Inspired by findings of histone-Kla based epigenetic regulation of immune functions in animals,we initially discovered 37 Kla sites on 16 histones in the maize genome,and again detected obvious differential histone Klamediated trends between two lines by Ch IP-Seq.Notably,only 2.7%of genes with differential histone Kla peaks detected during drought stress were commonly present in both lines,a finding demonstrating that abiotic stress triggers distinct epigenetic activities in diverse germplasm while also strongly supporting that a histone Kla layer of regulation is associated with physiological responses to drought stress.Interestingly,exogenous application of spermidine improved the drought tolerance of B73and substantially altered the levels of lactate,protein lactylation,and histone Kla modification.Thus,beyond extending the known domain of Kla-based biochemical and epigenetic regulation from animal immunity to plant stress physiology,our study suggests the physiological,biochemical,and genetic function of“the best-known metabolic waste”,lactate.

ORF355 confers enhanced salinity stress adaptability to S-type cytoplasmic male sterility maize by modulating the mitochondrial metabolic homeostasis

Moderate stimuli in mitochondria improve wideranging stress adaptability in animals, but whether mitochondria play similar roles in plants is largely unknown. Here, we report the enhanced stress adaptability of S-type cytoplasmic male sterility(CMS-S) maize and its association with mild expression of sterilizing gene ORF355. A CMS-S maize line exhibited superior growth potential and higher yield than those of the near-isogenic N-type line in saline fields. Moderate expression of ORF355 induced the accumulation of reactive oxygen species and activated the cellular antioxidative defense system. This adaptive response was mediated by elevation of the nicotinamide adenine dinucleotide concentration and associated metabolic homeostasis. Metabolome analysis revealed broad metabolic changes in CMS-S lines, even in the absence of salinity stress. Metabolic products associated with amino acid metabolism and galactose metabolism were substantially changed, which underpinned the alteration of the antioxidative defense system in CMS-S plants. The results reveal the ORF355-mediated superior stress adaptability in CMS-S maize and might provide an important route to developing salt-tolerant maize varieties.

The HuangZaoSi Maize Genome Provides Insights into Genomic Variation and Improvement History of Maize

Maize is a globally important crop that was a classic model plant for genetic studies. Here, we report a 2.2 Gb draft genome sequence of an elite maize line, HuangZaoSi (HZS). Hybrids bred from HZS-improved lines (HILs) are planted in more than 60% of maize fields in China. Proteome clustering of six completed sequeneed maize genomes show that 638 proteins fall into 264 HZS-specific gene families with the majority of contributions from tandem duplication events. Resequencing and comparative analysis of 40 HZSrelated lines reveals the breeding history of HILs. More than 60% of identified selective sweeps were clustered in identity.by.descent conserved regions, and yield-related genes/QTLs were enriched in HZS characteristic selected regions. Furthermore, we dem on strated that HZS-specific family genes were not uniformly distributed in the genome but enriched in improvement/function.related genomic regions. This study provides an important and novel resource for maize genome research and expands our knowledge on the breadth of genomic variation and improvement history of maize.

Discriminated sgRNAs-Based SurroGate System Greatly Enhances the Screening Efficiency of Plant Base-Edited Cells

The development of CRISPR/Cas9-mediated base editing has made genomic modification more efficient. However, selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. In this study, an efficient surrogate reporter system based on a defective hygromycin resistance gene was established in rice to enrich base-edited cells. After step-by-step optimization, the Discriminated sgRNAs-based SurroGate system (DisSUGs) was established by artificially differentiating the editing abilities of a wild-type single guide RNA (sgRNA) targeting the surrogate reporter gene and an enhanced sgRNA targeting endogenous sites. The DisSUGs enhanced the efficiency of screening base-edited cells by 3- to 5-fold for a PmCDA1-based cytosine-to-tyrosine base editor (PCBE), and 2.5- to 6.5-fold for an adenine base editor (ABE) at endogenous targets. These targets showed editing efficiencies of <25% in the conventional systems. The DisSUGs greatly enhanced the frequency of homozygous substitutions and expanded the activity window slightly for both a PCBE and an ABE. Analyses of the total number of single-nucleotide variants from whole-genome sequencing revealed that, compared with the no-enrichment PCBE strategy, the DisSUGs did not alter the frequency of genome-wide sgRNA-independent off-target mutations, but slightly increased the frequency of target-dependent off-target mutations. Collectively, the DisSUGs developed in this study greatly enhances the efficiency of screening plant base-edited cells and will be a useful system in future applications.

Activation of Mitochondrial orf355 Gene Expression by a Nuclear-Encoded DREB Transcription Factor Causes Cytoplasmic Male Sterility in Maize

Coordination between mitochondria and the nucleus is crucial for fertility determination in plants with cytoplasmic male sterility(CMS).Using yeast one-hybrid screening,we identified a transcription factor,ZmDREB1.7,that is highly expressed in sterile microspores at the large vacuole stage and activates the expression of mitochondria-encoded CMS gene orf355.Δpro,a weak allele of ZmDREB1.7 with the loss of a key unfolded protein response(UPR)motif in the promoter,partially restores male fertility of CMS-S maize.ZmDREB1.7 expression increases rapidly in response to antimycin A treatment,but this response is attenuated in theΔpro allele.Furthermore,we found that expression of orf355 in mitochondria activates mitochondrial retrograde signaling,which in turn induces ZmDREB1.7 expression.Taken together,these findings demonstrate that positive-feedback transcriptional regulation between a nuclear regulator and a mitochondrial CMS gene determines male sterility in maize,providing new insights into nucleus-mitochondria communication in plants.

Gene-lndexed Mutations in Maize

The availability of the B73 inbred reference genome sets the stage for high-throughput functional charac- terization of maize genes on a whole-genome scale. Among the 39 324 protein-coding genes predicted, the vast majority are untapped due to the lack of suitable high-throughput reverse genetic resources. We have generated a gene-indexed maize mutant collection through ethyl methanesulfonate mutagenesis and de- tected the mutations by combining exome capture and next-generation sequencing. A total of 1086 mutated MI plants were sequenced, and 195 268 CG〉TA-type point mutations, including stop gain/loss, missplice, start gain/loss, and various non-synonymous protein mutations as well as 4610 InDel mutations, were identified. These mutations were distributed on 32 069 genes, representing 82% of the predicted protein-coding genes in the maize genome. We detected an average of 180 mutations per mutant line and 6.1 mutations per gene. As many as 27 214 mutations of start codons, stop codons, or missplice sites were identified in 14 101 genes, among which 6232 individual genes harbored more than two such muta- tions. Application of this mutant collection is exemplified by the identification of the ent-kaurene synthase gene, which encodes a key enzyme in the gibberellin biosynthesis pathway. This gene-indexed genome- wide mutation collection provides an important resource for functional analysis of maize genes and may bring desirable allelic variants for genetic breeding in maize.

Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants

Dear Editor,Base editors(BEs),including cytosine base editor(CBE)and adenine base editor(ABE),have been widely used to generate irreversible nucleotide substitution in plants and animals.However,their wide applications are largely hindered by the strict NG protospacer adjacent motif(PAM)sequences recognized by Streptococcus pyogenes Cas9(SpCas9)and its engineered variants,such as SpCas9-NG and xCas9(Hua et al.,2019;Ren et al.,2019;Wu et al.,2019;Zhong et al.,2019;Zhang et al.,2020).Most recently,it was reported that three new SpCas9 variants,SpCas9-NRRH,SpCas9-NRTH,and SpCas9-NRCH,could recognize non-G PAMs(NRNH,where R is A or G and H is A,C,or T)in human cells(Miller et al.,2020).Meanwhile,SPRY,another new SpCas9 variant,was developed to greatly expand the editing scope of BEs to nearly PAMless(Walton et al.,2020).In this study,we generated a series of efficient BE toolkits and almost achieved C-to-T mutation without PAM restriction except for NTG PAM,and largely expanded A-to-G mutation scope in stable transformed rice,providing a reference for application in other plants.

HTPdb and HTPtools:Exploiting maize haplotypetag polymorphisms for germplasm resource analyses and genomics-informed breeding

Along with rapid advances in high-throughput-sequencing technology,the development and application of molecular markers has been critical for the progress that has been made in crop breeding and genetic research.Desirable molecular markers should be able to rapidly genotype tens of thousands of breeding accessions with tens to hundreds of markers.In this study,we developed a multiplex molecular marker,the haplotype-tag polymorphism(HTP),that integrates Maize6H-60K array data from 3,587 maize inbred lines with 6,375 blocks from the recombination block map.After applying strict filtering criteria,we obtained 6,163 highly polymorphic HTPs,which were evenly distributed in the genome.Furthermore,we developed a genome-wide HTP analysis toolkit,HTPtools,which we used to establish an HTP database(HTPdb)covering the whole genomes of 3,587 maize inbred lines commonly used in breeding.A total of 172,921 non-redundant HTP allelic variations were obtained.Three major HTPtools modules combine seven algorithms(e.g.,chain Bayes probability and the heterotic-pattern prediction algorithm)and a new plotting engine named“BCplot”that enables rapid visualization of the background information of multiple backcross groups.HTPtools was designed for big-data analyses such as complex pedigree reconstruction and maize heterotic-pattern prediction.The HTP-based analytical strategy and the toolkit developed in this study are applicable for high-throughput genotyping and for genetic mapping,germplasm resource analyses,and genomics-informed breeding in maize.