Chromosome-scale genome assembly of Prunus pusilliflora provides novel insights into genome evolution, disease resistance, and dormancy release in Cerasus L.

Prunus pusilliflora is a wild cherry germplasm resource distributed mainly in Southwest China.Despite its ornamental and economic value,a high-quality assembled P.pusilliflora genome is unavailable,hindering our understanding of its genetic background,population diversity,and evolutionary processes.Here,we de novo assembled a chromosome-scale P.pusilliflora genome using Oxford Nanopore,Illumina,and chromosome conformation capture sequencing.The assembled genome size was 309.62 Mb,with 76 scaffolds anchored to eight pseudochromosomes.We predicted 33035 protein-coding genes,functionally annotated 98.27%of them,and identified repetitive sequences covering 49.08%of the genome.We found that P.pusilliflora is closely related to Prunus serrulata and Prunus yedoensis,having diverged from them∼41.8 million years ago.A comparative genomic analysis revealed that P.pusilliflora has 643 expanded and 1128 contracted gene families.Furthermore,we found that P.pusilliflora is more resistant to Colletotrichum viniferum,Phytophthora capsici,and Pseudomonas syringae pv.tomato(Pst)DC3000 infections than cultivated Prunus avium.P.pusilliflora also has considerably more nucleotide-binding site-type resistance gene analogs than P.avium,which explains its stronger disease resistance.The cytochrome P450 and WRKY families of 263 and 61 proteins were divided into 42 and 8 subfamilies respectively in P.pusilliflora.Furthermore,81 MADS-box genes were identified in P.pusilliflora,accompanying expansions of the SVP and AGL15 subfamilies and loss of the TM3 subfamily.Our assembly of a high-quality P.pusilliflora genome will be valuable for further research on cherries and molecular breeding.

Using MiddRAD-seq data to develop polymorphic microsatellite markers for an endangered yew species

Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies.In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite loci, but compared with the more common shotgun genomic sequencing or transcriptome data, the potential utility of RAD-seq data for microsatellite ascertainment is comparatively under-used.In this study, we employed MiddRAD-seq data to develop polymorphic microsatellite loci for the endangered yew species Taxus florinii. Of 8,823,053 clean reads generated for ten individuals of a population, 94,851(~1%) contained microsatellite motifs. These corresponded to 2993 unique loci, of which 526(~18%) exhibited polymorphism. Of which, 237 were suitable for designing microsatellite primer pairs, and 128 loci were randomly selected for PCR validation and microsatellite screening. Out of the 128 primer pairs, 16 loci gave clear, reproducible patterns, and were then screened and characterized in 24 individuals from two populations. The total number of alleles per locus ranged from two to ten(mean=4.875), and within-population expected heterozygosity from zero to 0.789(mean = 0.530),indicating that these microsatellite loci will be useful for population genetics and speciation studies of T. florinii. This study represents one of few examples to mine polymorphic microsatellite loci from ddRAD data.

A comparison of different methods for preserving plant molecular materials and the effect of degraded DNA on ddRAD sequencing

Obtaining high-quality plant materials for experiments is challenging for many research projects.Therefore, it is of special importance to determine the best method for preserving biological macromolecules like DNA, which degrade over time. Although some research has demonstrated that DNA degradation has little effect on traditional molecular markers, the effects of DNA degradation on dd RADseq, a popular reduced-representation sequencing technology, have not been adequately investigated. In this study, we first chose six woody bamboo species(Bambusoideae, Poaceae) to explore appropriate methods for preserving molecular materials with two DNA extraction approaches. Then we sequenced twenty-one bamboos and examined the effects of DNA quality on data generation using the dd RAD-seq technique(Midd RAD-seq). Finally, we reconstructed phylogenies of twenty woody bamboo species. We found that the integrity of dry-powdered DNA was preserved longer than that of TE-dissolved DNA,regardless of whether the DNA was extracted by a modified CTAB protocol or DNAsecure plant kit. The dd RAD-seq data were robust, except when DNA was severely degraded. In addition, we resolved the phylogenetic positions of the sampled Phyllostachys spp. Our results suggest that dry-powdered DNA is the most appropriate preservation method for plant molecular materials. Furthermore, a moderate level of DNA degradation has little effect on reduced representation sequencing techniques represented by dd RAD-seq.

The C-terminal 17 amino acids of the photoreceptor UVR8 is involved in the fine-tuning of UV-B signaling^FA

Plant UV-B responses are mediated by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). In re-sponse to UV-B irradiation, UVR8 homodimers dissociate into monomers that bind to the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). The in-teraction of the C27 domain in the C-terminal tail of UVR8 with the WD40 domain of COP1 is critical for UV-B sig-naling. However, the function of the last 17 amino acids (C17) of the C-terminus of UVR8, which are adjacent to C27, is unknown, although they are largely conserved in land plants. In this study, we established that Arabidopsis thaliana UVR8 C17 binds to full-length UVR8, but not to COP1, and reduces COP1 binding to the remaining portion of UVR8, including C27. We hypothesized that overexpression of C17 in a wild-type background would have a dominant negative effect on UVR8 activity;however, C17 overexpression caused strong silencing of endogenous UVR8, precluding a detailed analysis. We therefore generated YFP-UVR8N423 transgenic lines, in which C17 was deleted, to examine C17 function in-directly. YFP-UVR8N423 was more active than YFP-UVR8, suggesting that C17 inhibits UV-B signaling by attenuating binding between C27 and COP1. Our study reveals an inhibitory role for UVR8 C17 in fine-tuning UVR8–COP1 interactions during UV-B signaling.