Hybrid-sterility-mediated reproductive isolation is pivotal for speciation, yet the underlying molecular mechanisms and environmental responses remain unclear. Here, we report a temperature-sensitive pollen killer-protector system at a three-gene Sa locus for indica-japonica rice hybrid sterility. Genetic analyses identified SaFL+, a strong pollen protector from Sa-i (indica allele), and SaFL-, a weak japonica allele from Sa-j, which is exclusively functional under high temperatures. Protein interaction, ubiquitination, and degradation assays showed that SaF+ and SaM+ from Sa-i form a pollen-killer complex to bind and ubiquitinate the reactive oxygen species scavenger COX11 for degradation in mitochondria, causing male sterility of the Sa-j pollen. Protein affinity and competitive binding assays indicated that in the Sa-i pollen, SaFL+ binds SaM+ to disrupt the killer complex and restore fertility. However, the weak protector SaFL- can bind SaM+ under high temperatures, sparing the Sa-j pollen from sterility. Synteny comparisons and haplotype analyses showed that the Sa locus originated in ancient wild rice and underwent divergence within the Oryza genus during expansion from tropical to temperate environments, which might have driven latitudinal adaptation and reproductive isolation in rice populations. Thus, Sa represents a temperature-sensitive reproductive-isolation system associated with domestication and environmental adaptation in rice.

Plasma membrane intrinsic proteins (PIPs) are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress. To elucidate their roles in plant immunity to pathogen attack, we characterized the expression patterns, subcellular localizations, and H2O2-transport ability of 11 OsPIPs in rice (Oryza sativa), and identified OsPIP2;6 as necessary for rice disease resistance. OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H2O2. Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae, indicating a positive function in plant immunity. OsPIP2;6 interacts with OsPIP2;2, which has been reported to increase rice resistance to pathogens via H2O2 transport. Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Plant fertility regulation and reproductive development are not only essential for plant reproduction, but also the genetic basis of hybrid crop breeding. Post-translational protein modifications are the important regulation mechanism of different activities during plant development. In recent years, the molecular networks of fertility regulation and reproductive development in plants have been greatly advanced. However, very few reviews focus on how post-translational protein modifications involve in the plant fertility control. In this review, we summarize the function of phosphorylation, ubiquitination, SUMOylation and glycosylation on male fertility regulation, which provides some insights into further studies.

The base editing technology is developed from the CRISPR/Cas gene editing systems, which can perform accurate base or gene editing at the DNA level. Recent years, six types of novel DNA base editors have been developed for the editing of nuclear and organellar genomes, including the cytosine base editor (CBE), the adenine base editor (ABE), the glycosylase base editor (GBE), the adenine and cytosine dual base editor (DBE), the prime editor (PE) and the mitochondrial genome editor. In this review, we summarize the principles, optimization processes and current advances of the above six DNA editors and focus on their application in crop genetic improvement. Finally, the future development of base editing technology is prospected.

Background Avr-Pita was the first effector identified in the blast fungus ( Magnaporthe oryzae )–rice ( Oryza sativa ) pathosystem. However, the molecular mechanism underlying its effects on the host plant has remained a long-standing mystery. Results Here, we report that ectopically expressing Avr-Pita in rice enhances susceptibility to M. oryzae and suppresses pathogen-associated molecular pattern (PAMP)-triggered defense responses. Avr-Pita targets the host mitochondria and interacts with the cytochrome c oxidase (COX) assembly protein OsCOX11, a key regulator of mitochondrial reactive oxygen species (ROS) metabolism in rice. Overexpressing Avr-Pita or OsCOX11 increased COX activity and decreased ROS accumulation triggered by the fungal PAMP chitin. OsCOX11 -overexpressing plants showed increased susceptibility to M. oryzae , whereas OsCOX11 -knockdown plants showed resistance to M. oryzae . Conclusions Taken together, these findings suggest that the fungal pathogen M. oryzae delivers the effector Avr-Pita to the host plant, where it enhances COX activity thus decreasing ROS accumulation. Therefore, this effector suppresses host innate immunity by perturbing ROS metabolism in the mitochondria.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) systems produce double-strand breaks (DSBs) in targeted sites and generally cause insertion/deletions of one or several bases by non-homologous end joining (NHEJ)-mediated DNA repair. However, many edited cell lines with small insertions/deletions produce abnormal transcripts or proteins causing unexpected effects that complicate functional analysis (Tuladhar et al., 2019). Therefore, the ability to delete larger genomic fragments would have advantage for making true null alleles of coding and non-coding genes. The NHEJ pathway also produces fragment deletion with low efficiency (Pathak et al., 2019). Microhomology-mediated end joining (MMEJ) is another DNA repair mechanism based on recombination between microhomologous sequences (MHSs) of 4–25 bp located near DSBs (Decottignies, 2007). Studies in model organisms including plants showed that MMEJ usually results in deletions of sequences between pairs of MHSs near DSBs generated by genome editing (Pathak et al., 2019; Shen et al., 2017). The MMEJ approach has been explored for deletion or insertion of genomic fragments in animals and microorganisms (Bae et al., 2014; Nakade et al., 2014; Zhang et al., 2016), but this type of application as well as systematic investigation of the frequency of MMEJ events in plant genome editing is lacking. In addition, generation of fragment deletions could remove antibiotic or herbicide selectable marker genes from transgenic lines for minimizing public and regulatory concerns. Several strategies for obtaining marker-free transgenic plants have been reported (Perez and Angenon, 2013), but these methods require the use of a marker-free system for plant transformation. Therefore, a high-efficiency method for removing target DNA fragments in plants is needed. Here, we analysed the efficiency of CRISPR/Cas9-targeted deletion of genomic fragments mediated by MMEJ in rice (Oryza sativa L.) and demonstrated this approach as an effective tool for deleting marker genes from transgenic plants. Using the CRISPR/Cas9 multiplex genome editing system for plants (Ma et al., 2015), our laboratory produced hundreds of rice mutant lines. A proportion of these lines were edited at two or more sites within the target genes. Although we did not consider MHSs for MMEJ in our initial experimental design, we selected 48 edited populations (each representing a group of T0 plants generated with the same CRISPR/Cas9 construct) that had fragment deletions between the target sites, and determined whether some deletion events were mediated by MMEJ (two MHS sites recombined as one in the deletion junctions) or by NHEJ (no MHSs in the deletion junctions). Of these edited populations, 24 had MMEJ-mediated fragment deletions ranging from 28 to 20 667 bp with an average size of 1653 bp. The average fragment deletion efficiency was 18.9% (Figure 1a). Moreover, 43 edited populations had NHEJ-mediated deletions ranging from 25 to 21 000 bp with an average size of 948 bp. Thus, if the design of editing targets does not consider facilitating MMEJ, the frequencies of fragment deletions induced by MMEJ and NHEJ are similar, but the sizes of MMEJ-mediated deletions are somewhat larger. We next attempted to induce MMEJ to delete a marker gene from an existing transgenic line. We previously generated a transgenic line carrying a homozygous, single-copy T-DNA insertion of a transgene carrying MONOSACCHARIDE TRANSFERASE 3 (MST3). The MST3 transgene was in a pCAMBIA1300-based binary construct with the hygromycin-resistance (HPT) gene cassette (Figure 1b). To delete this HPT cassette using genome editing, we first determined the sequences flanking the T-DNA insertion by the modified high-efficiency thermal asymmetric interlaced PCR (mhiTAIL-PCR) (Tan et al., 2019). We then used our online microhomology-finder tool (http://skl.scau.edu.cn/repfinder/) to analyse the sequences flanking the HPT cassette in the T-DNA and the inserted genome to identify MHSs. This analysis identified four groups of MHSs (MHS1–4) of 5–7 bp in the flanking sequences (Figure 1b,c). To produce DSBs as close as possible to the MHSs for efficient MMEJ-mediated fragment deletion, we used the web-based tool CRISPR-GE (http://skl.scau.edu.cn/) to design three target sites (T1–T3) that were located near any pair of the MHSs, including two target sites on the genomic flanking sequence and one target site on the T-DNA flanking sequence (Figure 1b,c). To induce DSBs at the target sites, we prepared a binary construct with three single-guide RNA (sgRNA) expression cassettes for the target sites based on the pYLCRISPR/Cas9Pubi-B vector with the glyphosate selectable marker gene Bar as described (Ma et al., 2015) and used it to retransform this transgenic line (Figure 1b,c). We obtained 21 Cas9-positive T0 plants as confirmed by PCR (Figure 1d). PCR with a Pf/Pr primer pair (Figure 1b,c) detected 3 plants (#1, #8 and #15) with homozygous deletion (ho△) and 11 plants (#3–#5, #9, #11, #14 and #17–#21) with heterozygous deletion (he△) (Figure 1d). To further determine whether these deletions were mediated by MMEJ or NHEJ, we examined the deletion-junction sequences. To this end, all the PCR-amplified fragments with reduced sizes and full length (Figure 1d) were recovered and sequenced. In the three ho△ plants, the HPT cassette was precisely excised with deletion-junction events between the pair of MHS1, MHS2 or MHS3 (Figure 1e,f). Of the 11 he△ plants, 8 had deletion-junction events between pair of MHS1, MHS 2 or MHS 4 (Figure 1f). The remaining 3 he△ plants (#3, #5 and #9) had fragment deletions generated by NHEJ, with junction sites between the targeted non-homologous DSB ends. In these he△ plants, sequencing of the full-length fragments showed the presence of the HPT cassette but with various base insertion/deletion mutations at the three targeted sites. In total, 3/21 (14.3%) of the edited plants had a homozygous marker gene deletion by MMEJ, and 11/21 (52.4%, including 8 plants by MMEJ and 3 plants by NHEJ) underwent mono-allelic (heterozygous) gene deletion (Figure 1f). Our results demonstrate that by designing suitable target sites near MHSs (see below), the efficiency of MMEJ-mediated fragment deletion is much higher than that of NHEJ-mediated deletions. In addition, we obtained marker-free T1 plants derived from the ho△ #1, #8 and #15 plants, which had no HPT and the T-DNA (containing the CRISPR/Cas9 components and Bar) removed by genetic segregation. Although studies have reported genomic fragment deletions by CRISPR/Cas systems, the efficiency is not high. The MMEJ pathway is active during cell cycle, which provides more chances for DSB repair than the other DNA repair pathways (Shen et al., 2017). Our results suggest that three factors may be related to the efficiency of genomic deletion using MMEJ-assisted CRISPR/Cas editing. First, the MHS1 and MHS4 sites, which are closer to the targeted sites (Figure 1c), had higher efficiency (in total 8/21) than that of the other two MHSs (in total 3/21, Figure 1f). It seems that the closer the MHSs are to the DSB positions, the higher the fragment deletion efficiency will be. Second, the length of MHSs may be less important than their positions relative to the DSBs; for example, MHS3 has the longest size (7 bp) but it produced only one he△ plant. Therefore, a MHS size of 4–5 bp (or even 3 bp, such as MHS4(a)) is sufficient for efficient MMEJ-mediated DNA repair. Third, production of multiple DSBs using multiplex sgRNAs may increase the frequency of genomic fragment deletions. In conclusion, the high-efficiency genomic fragment deletion strategy using MMEJ-assisted CRISPR/Cas9 editing is flexible in target design for many genomic regions to remove targeted genomic fragments. Moreover, this approach can facilitate functional genomics research and genetic improvement of organisms. We thank Zhe Zhao, Dongchang Zeng, Yaling Zhang and Jingluan Han in our laboratory for providing part of the genome editing data for analysis. This work was supported by grants from the Major Program of Guangdong Basic and Applied Research (2019B030302006), the National Natural Science Foundation of China (31971915; 31771740) and the Guangdong special support programme of young top-notch talent in science and technology innovation (2019TQ05N147). The authors declare no conflict of interest. Y.-G.L. and Q.Z. designed the studies. J.T., Y.Z., B.W., Y.H., Y.W., Y.L., W.L., W.Z., G.L., S.C., K.M. and X.X. performed the experiments. J.T., L.C., Q.Z. and Y.-G.L. analysed data. Q.Z., Y.-G.L. and J.T. wrote the paper.

Development of genome editing technologies provides efficient tools for functional genomics and crop molecular breeding. Owing to its simplicity and high efficiency, CRISPR/Cas systems, including CRISPR/Cas9 and CRISPR/Cas12a, have been widely used for genome editing in many organisms. In this review, we summarize the recent advances on improvements and applications of CRISPR/Cas systems in plants, as well as the methods for analyzing targeted mutations in edited plants. Finally, we discuss current problems of CRISPR/Cas systems and give a prospect of genome editing technologies.

>Genome editing is a new technology for manipulating genomic DNA sequences at specific sites,and researchers worldwide are rapidly developing new variants of genomeediting applications that target mRNAs,viruses,chromatin structure,and other cellular processes.Genome editing uses sequence-specific nucleases such as zinc-finger nucleases,transcription-activator-like effector nucleases,or clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) endonucleases,such as Cas9 (Ran et al.,2017).These nucleases,of which Cas proteins are guided

Plant height has a major effect on grain yield in crops such as rice (Oryza sativa), and the hormone gibberellic acid (GA) regulates many developmental processes that feed into plant height. Rice ELONGATED UPPERMOST INTERNODE1 (Eui1) encodes a GA-deactivating enzyme governing elongation of the uppermost internode. The expression of Eui1 is finely tuned, thereby maintaining homeostasis of endogenous bioactive GA and producing plants of normal plant height. Here, we identified a dominant dwarf mutant, dEui1, caused by the deletion of an RY motif-containing cis-silencing element (SE1) in the intron of Eui1. Detailed genetic and molecular analysis of SE1 revealed that this intronic cis element recruits at least one trans-acting repressor complex, containing the B3 repressors OsVAL2 and OsGD1, the SAP18 corepressor, and the histone deacetylase OsHDA710, to negatively regulate the expression of Eui1. This complex generates closed chromatin at Eui1, suppressing Eui1 expression and modulating GA homeostasis. Loss of SE1 or dysfunction of the complex components impairs histone deacetylation and H3K27me3 methylation of Eui1 chromatin, thereby increasing Eui1 transcription and decreasing bioactive GA, producing dwarfism in rice. Together, our results reveal a novel silencing mechanism in which the intronic cis element SE1 negatively regulates Eui1 expression via repressor complexes that modulate histone deacetylation and/or methylation.

Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. (c) 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

Use of the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) and Cpf1 systems in plants (Ma et al., 2016Ma X. Zhu Q. Chen Y. Liu Y.-G. CRISPR/Cas9 platforms for genome editing in plants: developments and applications.Mol. Plant. 2016; 9: 961-974Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, Wang et al., 2017Wang M. Mao Y. Lu Y. Tao X. Zhu J. Multiplex gene editing in rice using the CRISPR-Cpf1 system.Mol. Plant. 2017; https://doi.org/10.1016/j.molp.2017.03.001Abstract Full Text Full Text PDF Scopus (198) Google Scholar) involves many steps, including the selection of appropriate specific target site(s) that should have no highly homologous sequences as the potential off-target sites in the genome, the design and synthesis of oligonucleotides involving the target sequences, the preparation of expression cassette(s) for the target single guide RNAs (sgRNAs) that provide target sequence specificity, the construction of plant-transformation/expression vector(s), and the transformation of plants, followed by the detection and determination of the targeted mutations in the transgenic plants. Several software tools have been developed for designing target sites or evaluating the outcome of genome/gene editing; however, to date, the toolkit that could aid a genome editing experiment simultaneously at all the steps is not available. Here we present a web-based software package, CRISPR-GE (Genome Editing) (http://skl.scau.edu.cn/), a convenient, integrated toolkit to expedite all experimental designs and analyses of mutations for CRISPR/Cas9/Cpf1-based genome editing in plants and other organisms. CRISPR-GE provides a set of powerful tools for the design of target sgRNAs (targetDesign), prediction of off-target sites (offTarget), design of primers for construction of the sgRNA expression cassettes and amplification of target site-containing genomic fragments (primerDesign), determination of mutant sequences from sequencing chromatograms of genomic PCR amplicons containing target sites (DSDecodeM), and download of genomic sequences of certain regions from reference genomes (seqDownload). Using our previously reported CRISPR/Cas9 vector system (Ma et al., 2015aMa X. Zhang Q. Zhu Q. Liu W. Chen Y. Qiu R. Wang B. Yang Z. Li H. Lin Y. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar) or other CRISPR/Cas9/Cpf1 vector systems, CRISPR-GE offers an efficient and complete solution for genome editing in plants. Figure 1A shows the overall workflow of CRISPR-GE. The first tool in CRISPR-GE, targetDesign, aids in the key step of choosing appropriate target site(s) for the Cas9 or Cpf1 nucleases. Moreover, the associated offTarget program facilitates the selection of specific target site(s) and prevents the cleavage at non-target sequences in the target genome. Using the targetDesign program, researchers can rapidly find all possible target sites in a given sequence of interest and make a prediction of potential off-target sites and estimated scores in the assigned genome by invoking the offTarget algorithm. Meanwhile, researchers can also select target sites with different types of protospacer-adjacent motif (PAM, including NGG for SpCas9, TTN for FnCpf1, TTTN for AsCpf1, or a specific PAM defined by the user) for the CRISPR systems. First, the user selects or defines the PAM type and a target/reference genome, and inputs a genomic sequence (up to 10 000 bp) of the target plant variety/line, or a gene locus name, whereby the program will design the target site(s) (Figure 1B). If using a gene locus, the target gene sequence is derived from the selected target/reference genome. Currently, 27 plant genomes and genomes of other non-plant organisms, including human, mouse, zebrafish and Caenorhabditis elegans (Supplemental Table 1), are provided as the targets/references. After submitting the task, targetDesign aligns the query sequence to the target/reference genome using BLASTN to locate its chromosomal positions. Then, the program (1) searches all possible target sites with the defined PAM within the forward and reverse strands, and (2) analyzes the secondary structure of the candidate target sgRNAs and indicates the target site(s) that have eight or more contiguous nucleotides to pair with the sgRNA scaffold sequence, which may affect the normal folding of the target sgRNA, thus reducing the editing efficiency (Ma et al., 2015aMa X. Zhang Q. Zhu Q. Liu W. Chen Y. Qiu R. Wang B. Yang Z. Li H. Lin Y. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). Also, (3) targetDesign invokes the offTarget program to predict potential off-target sites in the target/reference genome and assign an estimated score to each potential off-target site of the CRISPR/Cas9 system according to the previous analysis of the off-target effects of each base in the target sgRNA in mammalian cells (Doench et al., 2016Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1863) Google Scholar). Potential off-target sites with higher scores may have higher chances of being targeted by the sgRNA/Cas9 complex. After identifying potential off-target sequences, (4) targetDesign will query the annotation of each target site and off-target site. To quickly perform the procedure, we built and included a database containing annotations of the reference genomes in CRISPR-GE. Moreover, (5) targetDesign can output the results in an interactive table (Figure 1C). The table lists all candidate target sites and their positions in the input genomic sequence, GC contents of the target sites, genomic locations, and corresponding potential off-target sites and their scores. All mismatched base(s) of the potential off-target sites to the target sgRNA are marked in red. Furthermore, restriction enzyme sites that are present in the candidate targets can be displayed by clicking the “Show Restriction Enzyme Sites” button. Since four or more contiguous “T” bases in target sgRNAs may serve as a transcription termination signal for RNA polymerase III, which produces transcripts from the U3/U6 promoters, targetDesign marks such poly-T sites in candidate target sequences in yellow for attention. The detailed off-target information can be displayed by clicking the “see detail” link. The editing efficiency of target sites by CRISPR/Cas9/Cpf1 systems is related to the target sequence compositions, but also may be largely affected by other unknown factors, for example, the different chromatin states (e.g., euchromatin or heterochromatin, transcriptionally active or silencing) of the target site regions. Therefore, it may be difficult or impossible to precisely determine target sites of “high efficiency” for editing by the program based on the nucleotide compositions of the target sites. However, it is possible to anticipate the “low-efficiency” sites based on the common features that negatively affect the editing as described above. In summary, candidate sites with very low or very high GC content (≤25% or ≥80%), poly-T site(s), contiguous base-pairing with the sgRNA sequence, which are indicated by a “Bad site warning” mark (!, !!, or !!!), and those having potential off-target site(s) of high score values (0.7 or higher), if this issue is concerned, should not be used for the genome editing. Finally, (6) users can select appropriate target site(s) and clicks “Primer design” to enter primerDesign-V (Vector), a subprogram of primerDesign, to generate primers for the subsequent vector construction using our CRISPR/Cas9 vector system (Ma et al., 2015aMa X. Zhang Q. Zhu Q. Liu W. Chen Y. Qiu R. Wang B. Yang Z. Li H. Lin Y. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar), which has gained wide usage, or other CRISPR/Cas9 vectors. Users also can directly enter the offTarget program to evaluate potential off-target sites of defined target site(s) in a target/reference genome. To perform this task, the user should select (or define) the PAM type and the target/reference genome, and input the target site sequence(s) and their corresponding PAM(s) (Figure 1D). The users can input multiple target sites by clicking the “Insert” button. After submitting the job, the query target site sequence(s) are aligned to the target/reference genome with mismatches of up to five nucleotides using BatMis algorithms (Tennakoon et al., 2012Tennakoon C. Purbojati R.W. Sung W.K. BatMis: a fast algorithm for k-mismatch mapping.Bioinformatics. 2012; 28: 2122-2128Crossref PubMed Scopus (28) Google Scholar), and the potential off-target sites for the CRISPR/Cas9 system are scored. Since no experimental analysis or reliable models for off-target effects of CRISPR editing systems using Cpf1 nucleases or other Cas9 variants are available, the program will skip the scoring step for off-target effects of these systems. The resulting table of the off-target predictor lists potential off-target sites with their scores, including the target sequence itself (score = 1). The mismatched bases between the target sgRNAs and off-target sites are highlighted in red (Figure 1C). Compared with other target-sgRNA design tools such as CRISPR-P (Liu et al., 2017Liu H. Ding Y. Zhou Y. Jin W. Xie K. Chen L. CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants.Mol. Plant. 2017; 10: 530-532Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), E-CRISPR (Heigwer et al., 2014Heigwer F. Kerr G. Boutros M. E-CRISP: fast CRISPR target site identification.Nat. Methods. 2014; 11: 122-123Crossref PubMed Scopus (532) Google Scholar), and Breaking-Cas (Oliveros et al., 2016Oliveros J.C. Franch M. Tabas-Madrid D. San-León D. Montoliu L. Cubas P. Pazos F. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.Nucleic Acids Res. 2016; 44: W267-W271Crossref PubMed Scopus (73) Google Scholar), the targetDesign/offTarget programs have the following advantages. First, targetDesign/offTarget allows users to design target sites (and predict potential off-target sites) in the inputted genomic sequences of the target varieties/lines that may actually have nucleotide variations with the selected reference genome (or the genome of the used close relative species), or in the gene sequences that are directly derived from the reference genome by the program (with the gene locus name). Second, if the reference genome sequence or the genome of a close relative species is not available, targetDesign also can be used to design target site(s) in the input genomic sequence with evaluations under the factors described above except for the prediction of potential off-target sites in the target/reference genome. To prepare CRISPR/Cas9/sgRNA constructs, the first step is to generate target-sgRNA expression cassette(s). The target site(s) selected through targetDesign and/or offTarget are transferred to primerDesign-V, which automatically outputs the primers for preparation of the sgRNA expression cassettes. Users also can directly enter primerDesign/primerDesign-V for the primer generation. Our CRISPR/Cas9/sgRNA vector system provides four U3 and U6 small nuclear RNA promoters from rice and four U3 and U6 promoters from Arabidopsis to express the target-sgRNAs in plants. Two methods can be used to prepare the target-sgRNA expression cassettes: Adapter-ligation/PCR (Method 1), or Overlapping PCR (Method 2) (Ma et al., 2015aMa X. Zhang Q. Zhu Q. Liu W. Chen Y. Qiu R. Wang B. Yang Z. Li H. Lin Y. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, Ma and Liu, 2016Ma X. Liu Y.-G. CRISPR/Cas9-based multiplex genome editing in monocot and dicot plants.Curr. Protoc. Mol. Biol. 2016; 115: 31.6.1-31.6.21Crossref Scopus (76) Google Scholar). Depending on the selected promoter(s) and method, different primers are generated to introduce the target sequences into the target-sgRNA expression cassettes (Figure 1E). The primerDesign-V tool also can be used to generate the adapter primers for other sgRNA vector systems that use the Adapter-ligation method. To facilitate PCR-amplification of genomic sequences that contain one or multiple target sites for analysis of the targeted mutations, we included another subprogram, primerDesign-A (Amplification), in primerDesign. With this tool, highly specific primers for the genomic PCR, and those for consequent sequencing, are designed by analyzing the reference genome sequence using the Primer3 (Untergasser et al., 2012Untergasser A. Cutcutache I. Koressaar T. Ye J. Faircloth B.C. Remm M. Rozen S.G. Primer3—new capabilities and interfaces.Nucleic Acids Res. 2012; 40: e115Crossref PubMed Scopus (5830) Google Scholar) and BatMis algorithms (Tennakoon et al., 2012Tennakoon C. Purbojati R.W. Sung W.K. BatMis: a fast algorithm for k-mismatch mapping.Bioinformatics. 2012; 28: 2122-2128Crossref PubMed Scopus (28) Google Scholar). The locations of the target site(s), the selected primers, and the sequencing primer(s) are displayed directly in the genomic sequence. The amplicons using these primers can be used for direct sequencing followed by decoding using the tool described below, for cloning in plasmid vectors or for polymorphism detection by other methods. For analysis of transgenic plants obtained with the editing constructs, CRISPR-GE provides the DSDecodeM program, an updated version of DSDecode (Liu et al., 2015Liu W. Xie X. Ma X. Li J. Chen J. Liu Y.-G. DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations.Mol. Plant. 2015; 8: 1431-1433Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), to determine the allelic mutant sequences of targeted sites. In diploid organisms, CRISPR-mediated genome editing can often cause uniform (biallelic, heterozygous, and homozygous) mutations. Direct sequencing of PCR amplicons containing such biallelic and heterozygous mutant sites produces superimposed sequencing chromatograms. Previously we developed the DSD (Degenerate Sequence Decoding) method and its web-based software tool (DSDecode) for decoding allelic mutant sequences of various uniform mutations from sequencing chromatograms with superimposed peaks (Liu et al., 2015Liu W. Xie X. Ma X. Li J. Chen J. Liu Y.-G. DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations.Mol. Plant. 2015; 8: 1431-1433Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, Ma et al., 2015bMa X. Chen L. Zhu Q. Liu Y.-G. Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products.Mol. Plant. 2015; 8: 1285-1287Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). However, DSDecode can only analyze one sequencing chromatogram file (ab1 format) at a time. Here, the updated DSDecodeM can analyze up to 20 sequencing chromatograms at the same time. In addition, DSDecodeM has several other improvements compared with DSDecode. (1) If higher noise signals are produced in the region before the target sites of sequencing chromatograms, the noise may interfere with the analysis of the program, leading to failure of the decoding. In DSDecodeM, the target sequence can be provided, as an optional input, which may allow the program to exclude these noise signals, thus enabling correct decoding. (2) To make the program more flexible in separating sequencing signal from noise, DSDecodeM has an optional parameter setting to adjust the cutoff of the noise-peak/base-peak signal ratio (0.1–0.8) if using the default ratio (0.3) fails to decode sequencing chromatograms of lower qualities. (3) DSDecode reads the sequencing digital information from ab1 files using the sangerseqR package, but this consumes more computational resources and thus requires a relatively long time for the decoding. In DSDecodeM we developed a Python-based program that can rapidly read the digital information from ab1 files. Therefore, the decoding speed of DSDecodeM is much faster (∼2 s per ab1 file if multiple files are decoded at the same time) than that of DSDecode (∼13 s per ab1 file). (4) The result page of DSDecodeM displays all decoding results of multiple sequencing files, which can be downloaded in a txt-format file (Figure 1F). The tool seqDownload is convenient for downloading genomic sequences of various lengths (up to ca. 200 kb) of certain regions from the selected reference genomes (Supplemental Table 1), for various analyses. For this sequence download, a user needs to input a gene locus name, a short marker sequence, or a pair of marker primer sequences located on the target region, and to define the lengths of the upstream and downstream flanking sequences to the gene locus/marker sequence. In summary, the overarching purpose of CRISPR-GE is to establish an intuitive and powerful toolkit for researchers to perform genome editing. This toolkit will greatly facilitate the entire design and analysis process, enabling high-efficiency CRISPR-based genome editing in plants and other organisms. This work was supported by grants from Guangdong Province Public Interest Research and Capacity Building Special Fund (2015B020201002), the Ministry of Agriculture of the People's Republic of China (2016ZX08010-001, 2016ZX08009-002), and the Postdoctoral Science Foundation of China (2016M602480).

Rice blast is one of the most destructive rice diseases in the world.Growing resistant cultivars is the most effective approach to control the disease.The objective of this study is to obtain resistant mutants against rice blast from a japonica rice landrace LiJiangXinTuanHeiGu (LTH) which is susceptible to nearly 2 800 races of rice blast in the world.The seeds of LTH were irradiated with 60Co-γrays,and the resultant seeds (M0) were planted independently to produce individual M1 generation.The resistant mutants were screened from the M2 and M3 populations by natural inoculation in rice blast nursery or by spore spaying inoculation in the lab.During the screening,we found that the background of mutant is easily to be influenced by undesired out-crossing and this phenomenon is often be ignored in mutant selecting from some certain resistant varieties.Therefore,to improve the accuracy of mutant screening,the candidate resistant mutants were subjected to a molecular marker-assisted selection using 12 In/Del markers which show polymorphic between japonica rice and indica rice from 12 chromosomes.Then one and four resistant mutants with pure LTH background were obtained by above two screening strategies,respectively.The resistant LTH mutants will help to understand the mechanism of acquired immunity in the susceptible rice.One of the LTH resistant mutants was crossed with wild type LTH and indica rice susceptible variety CO39,respectively,and the F2 generations were inoculated with rice blast by spore spaying.Genetic analysis results suggested that the phenotype of the mutant is controlled by a single dominant gene.This study also indicated that undesired crossing rate with other rice varieties will cause artifact in mutant screening,even if the bagging is applied in the breeding of rice mutants.Therefore,the molecular markers assisted selection is a practical solution to minimize the risk of undesired out-crossing to improve the screening accuracy of resistant mutants from highly susceptible rice.

Research on plant molecular biology, genetic engineering, and crop molecular breeding involves various manipulations of plant genomic DNAs (gDNAs). These studies require preparing gDNAs from plant materials using suitable methods according to the yield, intactness, and purity of the resultant gDNA samples. Here we describe protocols for preparation of rice plant gDNAs for various applications including: (1) maxipreps and (2) minipreps of gDNAs for restriction analysis, gene cloning, PCR amplification, genomic sequencing, and genotyping; (3) preparation of megabase-sized nuclear DNA for construction of large-insert genomic libraries and long-range physical mapping; and (4) 96-well-format high-throughput gDNA preparation for PCR-based genotyping. The methods are also suitable for other plant species including dicots. © 2016 by John Wiley & Sons, Inc.

稻瘟病和白叶枯病是由稻温病菌(Magnaporthe oryzae)和白叶枯病菌(Xanthomonas oryzae pv.oryzae)引起的两种主要水稻病害,也是制约中国水稻生产的主要病害.为了从DNA水平探索造成水稻感病品种‘丽江新团黑谷’(LTH)和高抗品种‘特特普’(Tetep,TTP)间抗病性差异的分子基础,该研究对其已知的3个抗稻瘟病基因和3个抗白叶枯基因所在DNA区段分别进行PCR扩增,将等量混合的PCR产物再与基因组重测序样品按Ct值差值(ΔCt)～10的比例混合,采用二代测序技术进行一次性测序和比较分析,并对有差异的基因区域进行常规传统测序验证,以确定这2个品种中抗性基因(R基因)的数目和结构与品种抗病或感病表型的关联性.实验结果表明,二代测序能够快速并准确地寻找到2个不同水稻品种中多个特定基因的序列差异,且差异位点与常规测序结果相符.从LTH和TTP这2种抗性不同水稻品种在多个抗性基因的DNA水平差异来看,有差异的抗性基因位点在高抗品种TTP中大都与原始抗性基因序列相同,而对应的普感品种LTH的抗性基因往往多表现为氨基酸突变,这些序列差异很可能就是导致TTP与LTH抗性差异的分子基础.