Large-scale genotyping plays an important role in genetic association studies. It has provided new opportunities for gene discovery, especially when combined with high-throughput sequencing technologies. Here, we report an efficient solution for large-scale genotyping. We call it specific-locus amplified fragment sequencing (SLAF-seq). SLAF-seq technology has several distinguishing characteristics: i) deep sequencing to ensure genotyping accuracy; ii) reduced representation strategy to reduce sequencing costs; iii) pre-designed reduced representation scheme to optimize marker efficiency; and iv) double barcode system for large populations. In this study, we tested the efficiency of SLAF-seq on rice and soybean data. Both sets of results showed strong consistency between predicted and practical SLAFs and considerable genotyping accuracy. We also report the highest density genetic map yet created for any organism without a reference genome sequence, common carp in this case, using SLAF-seq data. We detected 50,530 high-quality SLAFs with 13,291 SNPs genotyped in 211 individual carp. The genetic map contained 5,885 markers with 0.68 cM intervals on average. A comparative genomics study between common carp genetic map and zebrafish genome sequence map showed high-quality SLAF-seq genotyping results. SLAF-seq provides a high-resolution strategy for large-scale genotyping and can be generally applicable to various species and populations.

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1 Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage.

Linkage maps enable the study of important biological questions. The construction of high-density linkage maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. However, the marker number explosion and genotyping errors from NGS data challenge the computational efficiency and linkage map quality of linkage study methods. Here we report the HighMap method for constructing high-density linkage maps from NGS data. HighMap employs an iterative ordering and error correction strategy based on a k-nearest neighbor algorithm and a Monte Carlo multipoint maximum likelihood algorithm. Simulation study shows HighMap can create a linkage map with three times as many markers as ordering-only methods while offering more accurate marker orders and stable genetic distances. Using HighMap, we constructed a common carp linkage map with 10,004 markers. The singleton rate was less than one-ninth of that generated by JoinMap4.1. Its total map distance was 5,908 cM, consistent with reports on low-density maps. HighMap is an efficient method for constructing high-density, high-quality linkage maps from high-throughput population NGS data. It will facilitate genome assembling, comparative genomic analysis, and QTL studies. HighMap is available at http://highmap.biomarker.com.cn/.

BackgroundBacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application.Methodology/Principal FindingsIn this study, an approach, named “Step-by-Step” (SBS), has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD)>1.3×109 cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×103 cfu/mouse). Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD) of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×104 pfu, significantly smaller (p<0.01) than that of single monophage. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge.Conclusions/SignificanceOur data showed that the phage cocktail was more effective in reducing bacterial mutation frequency and in the rescue of murine bacteremia than monophage suggesting that phage cocktail established by SBS method has great therapeutic potential for multidrug-resistant bacteria infection.

Solid-phase microextraction (SPME), time-compressed chromatography (TCC), and time-of-flight mass spectrometry (TOFMS) were examined for their suitability and compatibility for rapid sampling, separation, and detection of apple flavor volatiles. Flavor-contributing volatile compounds were found to have relatively high partition coefficients on a 100 µm thick coating of polydimethylsiloxane (PDMS) on a SPME fiber. The time required to saturate the PDMS coating was highly volatiledependent, varying from less than 2 min to greater than 30 min. However, the response of this system was linear in the ppb to ppm range when the adsorption duration was standardized. The speed of the TOF mass spectrometer permitted identification and quantification of compounds having chromatographic peak widths of only a fraction of a second. The unskewed nature of fragmentation patterns obtained allowed individual component spectral characterization of unknown compounds even when not fully chromatographically separated. Thus, the time required for chromatography could be reduced by an order of magnitude without loss in analytical performance. Typical analysis times for complex mixtures were 2-5 min as compared with 20-60 min required for standard purgeand-trap analyses.

The apple (Malus × domestica Borkh.) is an economically and culturally important crop grown worldwide. Growers of this long-lived perennial must produce fruit of adequate quality while also combatting abiotic and biotic stress. Traditional apple breeding can take up to 20 yr from initial cross to commercial release, but genomics-assisted breeding can help accelerate this process. To advance genomics-assisted breeding in apple, we performed genome-wide association studies (GWAS) and genomic prediction in a collection of 172 apple accessions by linking over 55,000 single nucleotide polymorphisms (SNPs) with 10 phenotypes collected over 2 yr. Genomewide association studies revealed several known loci for skin color, harvest date and firmness at harvest. Several significant GWAS associations were detected for resistance to a major fungal pathogen, apple scab (Venturia ineaqualis [Cke.] Wint.), but we demonstrate that these hits likely represent a single ancestral source. Using genomic prediction, we show that most phenotypes are sufficiently predictable using genome-wide SNPs to be candidates for genomic selection. Finally, we detect a signal for firmness retention after storage on chromosome 10 and show that it may not stem from variation in PG1, a gene repeatedly identified in bi-parental mapping studies and widely believed to underlie a major QTL for firmness on chromosome 10. We provide evidence that this major QTL is more likely due to variation in a neighboring ethylene response factor (ERF) gene. The present study showcases the superior mapping resolution of GWAS compared to bi-parental linkage mapping by identifying a novel candidate gene underlying a well-studied, major QTL involved in apple firmness.

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