Genome shuffling for phenotypic improvement of industrial strains through recursive protoplast fusion technology.

Strains' improvement technology plays an essential role in enhancing the quality of industrial strains. Several traditional methods and modern techniques have been used to further improve strain engineering programs. The advances stated in strain engineering and the increasing demand for microbial metabolites leads to the invention of the genome shuffling technique, which ensures a specific phenotype improvement through inducing mutation and recursive protoplast fusion. In such technique, the selection of multi-parental strains with distinct phenotypic traits is crucial. In addition, as this evolutionary strain improvement technique involves combinative approaches, it does not require any gene sequence data for genome alteration and, therefore, strains developed by this elite technique will not be considered as genetically modified organisms. In this review, the different stages involved in the genome shuffling technique and its wide applications in various phenotype improvements will be addressed. Taken together, data discussed here highlight that the use of genome shuffling for strain improvement will be a plus for solving complex phenotypic traits and in promoting the rapid development of other industrially important strains.

Reshuffling yeast chromosomes with CRISPR/Cas9.

Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.

Quantitative Trait Locus Mapping of Salt Tolerance in Wild Rice Oryza longistaminata.

Salt stress is one of the most severe adverse environments in rice production; increasing salinization is seriously endangering rice production around the world. In this study, a rice backcross inbred line (BIL) population derived from the cross of 9311 and wild rice Oryza longistaminata was employed to identify the favorable genetic loci of O. longistaminata for salt tolerance. A total of 27 quantitative trait loci (QTLs) related to salt tolerance were identified in 140 rice BILs, and 17 QTLs formed seven QTL clusters on different chromosomes, of which 18 QTLs were derived from O. longistaminata, and a QTL for salt injury score (SIS), water content of seedlings (WCS) under salt treatment, and relative water content of seedlings (RWCS) was repeatedly detected and colocalized at the same site on chromosome 2, and a cytochrome P450 86B1 (MH02t0466900) was suggested as the potential candidate gene responsible for the salt tolerance based on sequence and expression analysis. These findings laid the foundation for further improving rice salt tolerance through molecular breeding in the future.

Maternal karyogene and cytoplasmic genotype affect the induction efficiency of doubled haploid inducer in Brassica napus.

BACKGROUND: Artificial synthesis of octoploid rapeseed double haploid (DH) induction lines Y3380 and Y3560 was made possible by interspecific hybridization and genome doubling techniques. Production of pure lines by DH induction provides a new way to achieve homozygosity earlier in B.napus. Previously, the mechanism of induction, and whether the induction has obvious maternal genotypic differences or not, are not known so far. RESULTS: In this study, different karyogene and cytoplasmic genotype of B.napus were pollinated with the previously reported DH inducers e.g. Y3380 and Y3560. Our study presents a fine comparison of different cytoplasmic genotypes hybridization to unravel the mechanism of DH induction. Ploidy identification, fertility and SSR marker analysis of induced F1 generation, revealed that ploidy and phenotype of the induced F1 plants were consistent with that type of maternal, rather than paternal parent. The SNP chip analysis revealed that induction efficiency of DH inducers were affected by the karyogene when the maternal cytoplasmic genotypes were the same. However, DH induction efficiency was also affected by cytoplasmic genotype when the karyogenes were same, and the offspring of the ogura cytoplasm showed high frequency inducer gene hybridization or low-frequency infiltration. CONCLUSION: The induction effect is influenced by the interaction between maternal karyogene and cytoplasmic genotype, and the results from the partial hybridization of progeny chromosomes indicate that the induction process may be attributed to the selective elimination of paternal chromosome. This study provides a basis for exploring the mechanism of DH inducer in B.napus, and provides new insights for utilization of inducers in molecular breeding.

Miniature inverted-repeat transposable elements (MITEs), derived insertional polymorphism as a tool of marker systems for molecular plant breeding.

Plant molecular breeding is expected to give significant gains in cultivar development through development and utilization of suitable molecular marker systems for genetic diversity analysis, rapid DNA fingerprinting, identification of true hybrids, trait mapping and marker-assisted selection. Transposable elements (TEs) are the most abundant component in a genome and being used as genetic markers in the plant molecular breeding. Here, we review on the high copious transposable element belonging to class-II DNA TEs called "miniature inverted-repeat transposable elements" (MITEs). MITEs are ubiquitous, short and non-autonomous DNA transposable elements which have a tendency to insert into genes and genic regions have paved a way for the development of functional DNA marker systems in plant genomes. This review summarises the characteristics of MITEs, principles and methodologies for development of MITEs based DNA markers, bioinformatics tools and resources for plant MITE discovery and their utilization in crop improvement.

Genome shuffling based on different types of ribosome engineering mutants for enhanced production of 10-membered enediyne tiancimycin-A.

Tiancimycin-A (TNM-A) is an anthraquinone-fused ten-membered enediyne produced by Streptomyces sp. CB03234, which is very promising for the development of anticancer antibody-drug conjugates (ADCs). To improve the titer of TNM-A, we have generated high-producing mutants CB03234-S and CB03234-R through ribosome engineering, but still not sufficient for pilot production of TNM-A. As the follow-up work, gentamycin-induced ribosome engineering was further adopted here to generate the mutant CB03234-G, which produced similar level of TNM-A as in CB03234-S and CB03234-R. Benefiting from the distinct antibiotic resistances of three ribosome engineering mutants, genome shuffling between any two of them was respectively carried out, and finally obtained the recombinant CB03234-GS26. Under optimal conditions, CB03234-GS26 produced 40.6 ± 1.0 mg/L TNM-A in shaking flasks and 20.8 ± 0.4 mg/L in a scaled-up 30-L fermentor. Comparing with the parental high-producing mutants, the over 1.6-fold titer improvement of CB03234-GS26 in fermentor was more promising for pilot production of TNM-A. Besides the distinctive morphological features, genetic characterization revealed that CB03234-GS26 possessed 1.8 kb rsmG related deletion just the same as CB03234-S, but no mutation was found in rpsL. Subsequent knockouts proved that rsmG was unrelated to titer improvement of TNM-A, which implied other genomic variations and mechanisms rather than ribosome engineering to enhance the biosynthesis of TNM-A. Therefore, CB03234-GS26 provided a basis to locate potential novel genetic targets, and explore the interactions between complex metabolic network and TNM biosynthetic pathway, which shall promote future construction of high-yielding systems for TNM-A and other anthraquinone-fused enediynes.Key Points •United genome shuffling and ribosome engineering help further strain improvement. •CB03234-GS26 with improved titer is practical for the pilot production of TNM-A. •Enhanced TNM-A production should attribute to novel genetic features/mechanisms.

Applications and research advance of genome shuffling for industrial microbial strains improvement.

Genome shuffling, an efficient and practical strain improvement technology via recursive protoplasts fusion, can break through the limits of species even genus to accelerate the directed evolution of microbial strains, without requiring the comprehensively cognized genetic background and operable genetic system. Hence this technology has been widely used for many important strains to obtain the desirable industrial phenotypes. In this review, we introduce the procedure of genome shuffling, discuss the new aid strategies of genome shuffling, summarize the applications of genome shuffling for increasing metabolite yield, improving strain tolerance, enhancing substrate utilization, and put forward the outlook to the future development of this technology.

The genome and transcriptome of Lactococcus lactis ssp. lactis F44 and G423: Insights into adaptation to the acidic environment.

Nisin, as a common green (environmentally friendly), nontoxic antibacterial peptide secreted by Lactococcus lactis, is widely used to prevent the decomposition of meat and dairy products and maintains relatively high stability at low pH. However, the growth of Lc. lactis is frequently inhibited by high lactic acid concentrations produced during fermentation. This phenomenon has become a great challenge in enhancing the nisin yield for this strain. Here, the shuffled strain G423 that could survive on a solid plate at pH 3.7 was generated through protoplast fusion-mediated genome shuffling. The nisin titer of G423 peaked at 4,543 IU/mL, which was 59.9% higher than that of the same batch of the initial strain Lc. lactis F44. The whole genome comparisons between G423 and F44 indicated that 6 large fragments (86,725 bp) were inserted in G423 compared with that of Lc. lactis F44. Transcriptome data revealed that 4 novel noncoding transcripts, and the significantly upregulated genes were involved in multiple processes in G423. In particular, the expression of genes involved in cell wall and membrane biosynthesis was obviously perturbed under acidic stress. Quantitative real-time PCR analysis showed that the transcription of noncoding small RNA NC-1 increased by 2.35-fold at pH 3.0 compared with that of the control (pH 7.0). Overexpression assays indicated that small RNA NC-1 could significantly enhance the acid tolerance and nisin production of G423 and F44. Our work provided new insights into the sophisticated genetic mechanisms involved in Lc. lactis in an acidic environment, which might elucidate its potential application in food and dairy industries.

Genome shuffling for improving the activity of alkaline pectinase in Bacillus subtilis FS105 and its molecular mechanism.

Genome shuffling for improving the activity of alkaline pectinase in Bacillus subtilis FS105 and its molecular mechanism were investigated. The fused strain B. subtilis FS105 with the highest activity of alkaline pectinase was obtained after two rounds of genome shuffling. The activity of alkaline pectinase in B. subtilis FS105 was 499 U/ml, which was improved by 1.6 times compared to that in original strain. To elucidate its molecular mechanism, rpsL gene sequences from original and fused strains were cloned and aligned, and the space structure of their coding proteins were also analyzed and compared. The alignment of the rpsL gene sequences indicated that three bases G, G and C were respectively replaced by A, A and G in the positions 52, 408 and 409 after genome shuffling. This resulted in the substitution of two amino acid residues in ribosomal protein S12: D18N and P137A, and therefore improving the biosynthesis of alkaline pectinase. This study lays a foundation for improving the activity of alkaline pectinase by genome shuffling and understanding its molecular mechanism.

Genome characterization of Sugarcane Yellow Leaf Virus with special reference to RNAi based molecular breeding.

Sugarcane is an essential crop for sugar and biofuel. Globally, its production is severely affected by sugarcane yellow leaf disease (SCYLD) caused by Sugarcane Yellow Leaf Virus (SCYLV). Many aphid vectors are involved in the spread of the disease which reduced the effectiveness of cultural and chemical management. Empirical methods of plant breeding such as introgression from wild and cultivated germplasm were not possible or at least challenging due to the absence of resistance in cultivated and wild germplasm of sugarcane. RNA interference (RNAi) transformation is an effective method to create virus-resistant varieties. Nevertheless, limited progress has been made due to lack of comprehensive research program on SCYLV based on RNAi technique. In order to show improvement and to propose future strategies for the feasibility of the RNAi technique to cope SCYLV, genome-wide consensus sequences of SCYLV were analyzed through GenBank. The coverage rates of every consensus sequence in SCYLV isolates were calculated to evaluate their practicability. Our analysis showed that single consensus sequence from SCYLV could not work well for RNAi based sugarcane breeding programs. This may be due to high mutation rate and continuous recombination within and between various viral strains. Alternative multi-target RNAi strategy is suggested to combat several strains of the viruses and to reduce the silencing escape. The multi-target small interfering RNA (siRNA) can be used together to construct RNAi plant expression plasmid, and to transform sugarcane tissues to develop new sugarcane varieties resistant to SCYLV.

Antibodies and Engineered Antibody Fragments against M13 Filamentous Phage to Facilitate Phage-Display-Based Molecular Breeding.

Antibodies are essential for characterizing various analytes. "Molecular-breeding" approaches enable rapid generation of antibody mutants with desirable antigen-binding abilities. Typically, prototype antibodies are converted to single-chain Fv fragments (scFvs), and random mutations are genetically introduced to construct molecular libraries with a vast diversity. Improved species therein are then isolated via phage display genotype-phenotype-connecting systems to separate them from a large excess of nonspecific scFvs. During these experiments, counting of phage particles is routinely performed. However, current methods depend on the time-consuming overnight cultivation of phage-infected bacteria on agar plates to estimate phage numbers as plaque-forming units (pfu) or colony-forming units, the results of which fluctuate considerably. Immunochemical systems capturing phage particles should be a more convenient and robust alternative. We therefore generated monoclonal antibodies against M13 filamentous phage, which is commonly used for phage display, by employing hybridoma technology. Combinatorial use of two such antibodies (Ab-M13#53 and #71; both specific to the major coat protein pVIII) enabled development of a sandwich enzyme-linked immunosorbent assay (ELISA) that could measure ca. 107-1010 phage pfu/mL. To construct a more convenient system, Ab-M13#71 was converted to the scFv form and further fused with an alkaline phosphatase variant. Using this fusion protein, the sandwich ELISA enabled rapid (within 90 min) and reliable phage counting without reducing the sensitivity, and the results were reasonably consistent with those of infection-based methods. The present anti-phage antibodies and scFvs might also enable visualization of individual phage particles by combining them with sensitive fluorescent staining.

Genome shuffling and ribosome engineering of Streptomyces virginiae for improved virginiamycin production.

The production of virginiamycin (VGM) from Streptomyces virginiae was improved by genome shuffling and ribosome engineering companied with a high-throughput screening method integrating deep-well cultivation and the cylinder-plate detecting. First, a novel high-throughput method was developed to rapidly screen large numbers of VGM-producing mutants. Then, the starting population of genome shuffling was obtained through ultraviolet (UV) and microwave mutagenesis, and four mutants with higher productivity of VGM were selected for genome shuffling. Next, the parent protoplasts were inactivated by UV and heat when a fusant probability was about 98%. Streptomycin resistance was used as an evolutionary pressure to extend positive effects on VGM synthesis. Finally, after five rounds of genome shuffling, a genetically stable strain G5-103 was obtained and characterized to be able to yield 251 mg/L VGM, which was 3.1- and 11.6-fold higher than that of the mutant strain UV 1150 and the wild-type strain, respectively.

Development of an efficient host-vector system of Ruminiclostridium josui.

Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host-vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5'-Gmet ATC-3', where the A nucleotide is Dam-methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam- /dcm- ) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 103 transformants/µg of DNA was obtained using a square-wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin-encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui.

Genome shuffling and high-throughput screening of Brevibacterium flavum MDV1 for enhanced L-valine production.

L-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for L-valine grows rapidly, there is an increasing interest to develop an efficient L-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield L-valine strains to replace the traditional L-valine assay. L-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six L-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an L-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of L-valine (0.598 mol L-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m3 fermentor, this strain produced > 66.8 g/L L-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of L-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.

Enhanced production of tanshinone IIA in endophytic fungi Emericella foeniculicola by genome shuffling.

CONTEXT: Tanshinone IIA, commercially produced from Salvia miltiorrhiza Bunge (C.Y.Wu) (Labiatae), has various biological benefits. Currently, this compound is mainly extracted from plants. However, because of the long growth cycle and the unstable quality of plants, the market demands can barely be satisfied. OBJECTIVE: The genomic shuffling technology is applied to screen the high-yield tanshinone IIA strain, which could be used to replace the plant S. miltiorrhiza for the production of tanshinone IIA. The change in the production of tanshinone IIA is clarified by comparing it with the original strain. MATERIALS AND METHODS: Tanshinone IIA was extracted from Strains cells, which was prepared through 0.5 mL protoplast samples by using hypertonic solution I from two different strains. Then, it was analyzed by high-performance liquid chromatography at 30 °C and UV 270 nm. Total DNA from the strains was extracted for RAPD amplification and electrophoresis to isolate the product. RESULTS: In this study, a high-yield tanshinone IIA strain F-3.4 was screened and the yield of tanshinone IIA was increased by 387.56 ± 0.02 mg/g, 11.07 times higher than that of the original strain TR21. DISCUSSION: This study shows that the genetic basis of high-yield strains is achieved through genome shuffling, which proves that genome shuffling can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining the strains with good traits and it is a useful method for the molecular breeding of industrial strains.

Enhanced production of fructosyltransferase in Aspergillus oryzae by genome shuffling.

OBJECTIVE: To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling. RESULTS: A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180 U g-1, approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 U g-1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201. CONCLUSIONS: Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

Improving fruit and wine: what does genomics have to offer?

Will we still be drinking wines made from Pinot Noir and eating McIntosh apples in the 23rd century? Elite grape and apple cultivars, vegetatively propagated for centuries, are highly susceptible to evolving pathogens. In response, growers continually expand their agrochemical weaponry at enormous environmental costs. By contrast, breeders are seeking disease-resistant, tastier alternatives to the handful of dominant cultivars by exploring genetic diversity in these fruits. However, this is a formidable task because consumers cling to ancient cultivars, and breeding long-lived woody perennials is laborious and expensive. Although genomics tools may not solve the former sociocultural dilemma, they can help overcome the latter practical obstacles. Screening seedlings for desirable genetic profiles using molecular techniques reduces the time and high costs associated with growing plants to maturity and evaluating fruit. Such screening is currently in its infancy in apples and grapes, but the adoption of modern DNA sequencing technologies and statistical approaches promises to accelerate cultivar improvement significantly. Here, I describe standard approaches for molecular breeding in apples and grapes, and some of the challenges associated with the collection and analysis of next-generation DNA sequence data. In addition, I urge breeders to establish populations specifically designed for a future of inexpensive genome sequencing.

A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops.

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical ß-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops.

Optimization of genome shuffling for high-yield production of the antitumor deacetylmycoepoxydiene in an endophytic fungus of mangrove plants.

As an accelerated evolutionary tool, genome shuffling is largely dependent on the high fusion frequency of different parental protoplasts. However, it was unclear how many types of parental protoplasts would afford the highest fusion frequency. Here, we applied the Monte Carlo method to simulate the simplified processes of protoplast fusion, to achieve maximal useful fusions in genome shuffling. The basic principle of this simulation is that valid fusions would take place when the minimum distance between two different types of parent protoplasts is smaller than that between two of the same types. Accordingly, simulations indicated that the highest fusion frequency would be achieved from eight to 12 different parental protoplasts. Based on the simulation results, eight parental protoplasts of the fungal endophyte Phomopsis sp. A123 were subjected to genome shuffling for yield improvement of deacetylmycoepoxydiene (DAM), an antitumor natural product with a novel chemical structure. After only two rounds of genome shuffling, four high-yield DAM-producing strains, namely G2-119, G2-448, G2-866, and G2-919, were obtained with the aid of activity screening and HPLC analysis. The results showed that the DAM yield in these four strains were 243-, 241-, 225-, and 275-fold, respectively, higher than that of the starting strain A123. This is the first time Monte Carlo simulation is introduced into the field of cell fusion and is also the first report on the optimization of genome shuffling focusing on the number of parental types in protoplast fusions.

A new approach for breeding low-temperature-resistant Volvariella volvacea strains: Genome shuffling in edible fungi.

Volvariella volvacea is difficult to store fresh because of the lack of low-temperature resistance. Many traditional mutagenic strategies have been applied in order to select out strains resistant to low temperature, but few commercially efficient strains have been produced. In order to break through the bottleneck of traditional breeding and significantly improve low-temperature resistance of the edible fungus V. volvacea, strains resistant to low temperature were constructed by genome shuffling. The optimum conditions of V. volvacea strain mutation, protoplast regeneration, and fusion were determined. After protoplasts were treated with 1% (v/v) ethylmethylsulfonate (EMS), 40 Sec of ultraviolet (UV) irradiation, 600 Gy electron beam implantation, and 750 Gy60 Co-γ irradiation, separately, the lethality was within 70%-80%, which favored generating protoplasts being used in following forward mutation. Under these conditions, 16 strains of V. volvacea mutated by EMS, electron beam, UV irradiation, and 60 Co-γ irradiation were obtained. The 16 mutated protoplasts were selected to serve as the shuffling pool based on their excellent low-temperature resistance. After four rounds of genome shuffling and low-temperature resistance testing, three strains (VF1 , VF2 , and VF3 ) with high genetic stability were screened. VF1 , VF2 , and VF3 significantly enhanced fruit body shelf life to 20, 28, and 28 H at 10 °C, respectively, which exceeded 25%, 75%, and 75%, respectively, compared with the storage time of V23, the most low-temperature-resistant strain. Genome shuffling greatly improved the low-temperature resistance of V. volvacea, and shortened the course of screening required to generate desirable strains. To our knowledge, this is the first paper to apply genome shuffling to breeding new varieties of mushroom, and offers a new approach for breeding edible fungi with optimized phenotype.