The megabase-scale crossover landscape is independent of sequence divergence

Meiotic recombination frequency varies along chromosomes and strongly correlates with sequence divergence. However, the causality underlying this correlation is unclear. To untangle the relationship between recombination landscapes and polymorphisms, we characterized the genome-wide recombination landscape in the absence of polymorphisms, using Arabidopsis thaliana homozygous inbred lines in which a few hundred genetic markers were introduced through mutagenesis. We found that megabase-scale recombination landscapes in inbred lines are strikingly similar to the recombination landscapes in hybrids, with the sole exception of heterozygous large rearrangements where recombination is prevented locally. In addition, we found that the megabase-scale recombination landscape can be accurately predicted by chromatin features. Our results show that polymorphisms are not causal for the shape of the megabase-scale recombination landscape, rather, favor alternative models in which recombination and chromatin shape sequence divergence across the genome.

MG2C: a user-friendly online tool for drawing genetic maps

Genetic map is a linear arrangement of the relative positions of sites in the chromosome or genome based on the recombination frequency between genetic markers. It is the important basis for genetic analysis. Several kinds of software have been designed for genetic mapping, but all these tools require users to write or edit code, making it time-costing and difficult for researchers without programming skills to handle with. Here, MG2C, a new online tool was designed, based on PERL and SVG languages.Users can get a standard genetic map, only by providing the location of genes (or quantitative trait loci) and the length of the chromosome, without writing additional code. The operation interface of MG2C contains three sections: data input, data output and parameters. There are 33 attribute parameters in MG2C, which are further divided into 8 modules. Values of the parameters can be changed according to the users’ requirements. The information submitted by users will be transformed into the genetic map in SVG file, which can be further modified by other image processing tools.MG2C is a user-friendly and time-saving online tool for drawing genetic maps, especially for those without programming skills. The tool has been running smoothly since 2015, and updated to version 2.1. It significantly lowers the technical barriers for the users, and provides great convenience for the researchers.

Diagnostic value of Rf1 gene molecular markers in sunflower

Background. Modern production of sunflower seeds is currently based on the cultivation of high-yielding heterotic F1 hybrids from cross-breeding of lines with cytoplasmic male sterility (CMS) of PET1-type and fertility restorer lines. The paternal parent serves as a donor of the nuclear Rf1 gene functional allele, which is responsible for pollen fertility restoration in F1 plants. The detection of carriers of the Rf1 locus recessive and dominant alleles using diagnostic molecular markers accelerates breeding of female and male parental lines for creating hybrids. Materials and methods. The material for the study included 75 lines of various origins from the VIR sunflower genetic collection as well as hybrids from crosses of VIR 116A sterile line with fertile lines differing in the type of cytoplasm (fertile or sterile) and the presence of molecular markers, most of which were linked to the Rf1 locus. For marker validation, two different approaches were used: either by analyzing associations between the ability of a line to restore pollen fertility and the presence of molecular markers in its genotype, or by estimating recombination frequency between the Rf1 locus and marker loci in four segregating hybrid populations. Results. According to the obtained results, no markers demonstrated 100% efficiency in the analysis of the sample of genotypes. The ORS511 marker was most frequently observed among the lines presumably carrying the dominant allele Rf1. Pollen fertility of F1 hybrids from interline crossings was 89-99%. The segregation for fertility/sterility in F2 fitted the theoretical ratio of 3:1 expected in case of the monogenic control of the trait. The markers HRG01, HRG02 and ORS511 were linked to the fertility restoration trait, with recombination rates between Rf1 locus and markers varying in different cross combinations. The analysis of VIR 116А × VIR 740 and VIR 116А × RIL 130 hybrids showed that among the marker loci studied, the ORS511 was closest to the Rf1 locus Rf1 (recombination frequency of 2.2 and 3.3%, respectively). The recombination rate between the Rf1 and ORS511 loci equaled 7.5% in the cross VIR 116А × VIR 210 and 8.9% in VIR 116 × VIR 195. Conclusion. The markers ORS511, HRG01 and HRG02 are the most efficient for the identification of alleles of the Rf1 gene and for the marker assisted selection in hybrid populations produced involving sunflower lines from the VIR collection.

Concurrent Disruption of Genetic Interference and Increase of Genetic Recombination Frequency in Hybrid Rice Using CRISPR/Cas9

Manipulation of the distribution and frequency of meiotic recombination events to increase genetic diversity and disrupting genetic interference are long-standing goals in crop breeding. However, attenuation of genetic interference is usually accompanied by a reduction in recombination frequency and subsequent loss of plant fertility. In the present study, we generated null mutants of the ZEP1 gene, which encodes the central component of the meiotic synaptonemal complex (SC), in a hybrid rice using CRISPR/Cas9. The null mutants exhibited absolute male sterility but maintained nearly unaffected female fertility. By pollinating the zep1 null mutants with pollen from indica rice variety 93-11, we successfully conducted genetic analysis and found that genetic recombination frequency was greatly increased and genetic interference was completely eliminated in the absence of ZEP1. The findings provided direct evidence to support the controversial hypothesis that SC is involved in mediating interference. Additionally, the remained female fertility of the null mutants makes it possible to break linkage drag. Our study provides a potential approach to increase genetic diversity and fully eliminate genetic interference in rice breeding.

Effective RNAi-Mediated Silencing of the Mismatch Repair MSH2 Gene Induces Sterility of Tomato Plants but Not an Increase in Meiotic Recombination

In plant breeding, the ability to manipulate meiotic recombination aids in the efficient construction of new allelic compositions of chromosomes and facilitates gene transfer from wild relatives of crop plants. The DNA mismatch repair system antagonizes meiotic recombination. In this research, a trial was conducted to evaluate transgenic tomato plants carrying an RNA interference (RNAi) construct designed to inhibit the expression of the mismatch repair MSH2 gene. To drive the RNAi construct, we used either a pro-SmAMP2 promoter from Stellaria media ANTIMICROBIAL PEPTIDE2 or a Cauliflower mosaic virus 35S promoter (CaMV35S). The results of real-time PCR showed that, with a 16 h light/8 h dark photoperiod, MSH2-RNAi tomato transgenic plants exhibited MSH2 gene transcript contents ranging from 0% to 3% in the leaves, relative to untransformed controls. However, with this lighting mode, the MSH2-RNAi transgenic plants grew slowly, flowered poorly, and did not form seed sets. During cultivation with a 12 h light/12 h dark photoperiod, MSH2-RNAi transgenic plants exhibited MSH2 gene transcript contents ranging from 3% to 42%, relative to untransformed controls. Under these conditions, F1 hybrid seed sets formed for most of the MSH2-RNAi transgenic plants with the RNAi construct driven by the CaMV35S promoter, and for one transformant with the RNAi construct driven by the pro-SmAMP2 promoter. Under conditions of a 12 h light/12 h dark photoperiod, most of the F1 transgenic hybrids showed MSH2 gene transcript contents ranging from 3% to 34% and formed F2 offspring sets, which made it possible to assess the meiotic recombination frequency. We showed that the effective inhibition of MSH2 in MSH2-RNAi tomato transgenic plants is not associated with an increase in meiotic recombination compared to the control, but it stimulates the sterility of plants. It was established that the expression of the MSH2 gene in tomato plants is about 50 times higher with a 12 h light/12 h dark than with a 16 h light/8 h dark photoperiod. It is discussed that, in Solanum lycopersicum tomato plants, which are not sensitive to the day length for flowering, changing the lighting time may be a means of controlling the meiotic recombination frequency within certain limits.

Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

Direct fast estimator for recombination frequency in the F2 cross

In this short note, a new unbiased maximum-likelihood estimator is proposed for the recombination frequency in the F2 cross. The estimator is much faster to calculate than its EM algorithm equivalent, yet as efficient. Simulation studies are carried to illustrate the gain over another simple estimate proposed by Benito & Gallego (2004).

Interplay between Pds5 and Rec8 in regulating chromosome axis length and crossover frequency

Meiotic chromosomes have a loop/axis architecture, with axis length determining crossover frequency. Meiosis-specific Pds5 depletion mutants have shorter chromosome axes and lower homologous chromosome pairing and recombination frequency. However, it is poorly understood how Pds5 coordinately regulates these processes. In this study, we show that only ~20% of wild-type level of Pds5 is required for homolog pairing and that higher levels of Pds5 dosage-dependently regulate axis length and crossover frequency. Moderate changes in Pds5 protein levels do not explicitly impair the basic recombination process. Further investigations show that Pds5 does not regulate chromosome axes by altering Rec8 abundance. Conversely, Rec8 regulates chromosome axis length by modulating Pds5. These findings highlight the important role of Pds5 in regulating meiosis and its relationship with Rec8 to regulate chromosome axis length and crossover frequency with implications for evolutionary adaptation.

Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive

Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Drive mechanisms that are self-limiting are attractive because they allow control over the duration and extent of trait spread in time and space, and are reversible through natural selection as drive wanes. Self-sustainingCleave and Rescue(ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (theRescue), and a Cargo.ClvRspreads by creating loss-of-function (LOF) conditions in which those withoutClvRdie because they lack functional copies of the essential gene. We use modeling to show that when theRescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (splitClvR), drive ofRescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner—which is still self-limiting—by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking withRescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement splitClvRinDrosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make upClvRelements—Cas9 and gRNAs and aRescue/Cargo.

A Genetic Map and Linkage Panel for the Large-fruited Fresh-market Tomato

Genetic maps saturated with genetic markers are useful for genetic research and crop breeding; however, the genetic map for the large-fruited fresh-market tomato (Solanum lycopersicum) has never been constructed, and the recombination frequency between DNA fragments is only partly understood for fresh-market tomato. We constructed a novel fresh-market tomato genetic map by using 3614 single nucleotide polymorphism (SNP) markers and a 93 F2 segregating progeny derived from a cross between two United States large-fruited fresh-market tomato lines. The average distance between markers was less than 1 cM, and substantial recombination densities between markers were observed across the approximate centromere locations. A linkage panel for large-fruited fresh-market tomato was also established using the combined dataset of the genetic map and 58 SNP-genotyped core tomato lines. The allelic information in the linkage panel will be a significant resource for both tomato genetics and future breeding approaches.