First report of Passiflora latent virus(Passiflora latent virus;PLV) infecting persimmon in Korea

Passiflora latent virus (PLV), a member of the genus Carlavirus in the family Betaflexiviridae has been reported in Passiflora species in Australia, Germany, Israel, the United States, and New Zealand (Tang et al., 2008). In September 2019, leaves showing a virus-like disease with mosaic, curling and necrosis were collected from ten persimmon (Diospyros kaki Thunb.) orchards in Gyeongsang province, Korea. Total RNA from a pooled sample of leaves from 21 trees was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) and subjected to high throughput sequencing. After pre-processing and Ribo-Zero rRNA Removal, a cDNA library was prepared using an Illumina TruSeq Stranded Total RNA Kit and sequenced on an Illumina NovaSeq 6000 system (Macrogen Inc. Korea). De novo assembly of the 74,862,810 reads was performed using Trinity software (r20140717); the initially assembled 213,476 contigs were screened against the NCBI viral genome database using BLASTN. By these means, 12 contigs derived from PLV were identified. Contigs with lengths of 209 to 802 nt shared nt identities of 90.70 to 94.82% with PLV isolates, covering a total of 5,169 nt (~61.6% of the full PLV genome). Two additional viruses were also detected from the pooled sample: persimmon cryptic virus (PeCV) and persimmon virus A (PeVA). To confirm PLV infection, reverse transcription-polymerase chain reaction (RT-PCR) was performed using virus-specific primers, PLV-F (5’-ACACAAAACTGCGTGTTGGA-3’) and PLV-R (5’-CAAGACCCACCTACCTCAGTGTG-3’), designed based on a 633 nt contig sequence in the polymerase gene. RT-PCR products of the expected 571 bp were obtained from two of 21 individual original samples; no asymptomatic plants were tested. Amplicons were cloned into the pGEM-T Easy Vector, and two clones per sample Sanger sequenced bidirectionally (BIONEER, Korea). The identical Sequence (GenBank LC556232) showed 99.65% nt identity to the contig, and 93.87% identity with the corresponding polymerase sequence of PLV-Rehovot isolate from passion fruit in Israel (MH379331). The two PLV positive samples showing leaf necrosis were also co-infected with PeVA, identified by RT-PCR using previously reported primers PeVAfor/ PeVArev (Morell et al., 2014), but not with PeCV (mixed with PeVA in only 1/21 plants; PeVA was found in 19/21 plants). None of the tested viruses were detected in two trees, displaying mosaic, and leaf curling, respectively. The foliar symptoms of PLV infection on passionfruit have been reported to vary throughout the year (Spiegel et al., 2007). No such observations in persimmon was possible, as the infected persimmon trees were removed and destroyed because they might pose a threat to the cultivation of passion fruits in Korea. To our knowledge, this is the first report of persimmon as a host of PLV anywhere in the world, and the first report of PLV in Korea in any host. A further survey is needed to determine possible presence of PLV on persimmon and Passiflora species.

Occurrence of Soybean Yellow Common Mosaic Virus in Soybean in China Showing Yellow Common Mosaic Disease

Soybean yellow common mosaic virus (SYCMV), a positive sense ssRNA virus classified in the genus Sobemovirus, was first reported and characterized in Korea (Nam et al., 2012). Currently, its only known host is soybean (Nam et al., 2012) on which it causes bright yellow mosaic and crinkling of the leaves (Lim et al., 2016). During a field survey in July 2019, bright yellow mosaic and mild crinkling symptoms were observed on soybean leaves (cv. Zhonghuang 13) in the Hubei province of China. To identify the possible pathogen(s) associated to the disease symptoms, leaves from five symptomatic plants were collected, pooled and total RNA was extracted using TRIzol® Reagent (Invitrogen, CA, USA). 10 μg of the total RNA was purified via magnetic beads (Thermo Fischer Scientific, USA) and a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) was then used to construct an RNA sequencing library. Transcriptome sequencing was performed on an Illumina HiSeq 4000 (LC Sciences, USA). The average insert size for the paired-end library was 300 ± 50 bp. After quality control, a total of 47.5 million clean reads were obtained and assembled using the Trinity software (version 2.8.5). The assembled contigs were searched against NCBI virus RefSeqs (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral) by the BLASTx algorithm with a cutoff E value of ≤10-5. 12 contigs sized from 3,421 to 4,093 bp were found to share a sequence identity of 77.5%-94.1% with SYCMV isolates from Japan (LC332541) and South Korea (JF495127.1). No other virus matches were identified. The largest contig (4,093 bp, MT816507) covers 99% of the expected complete genome of SYCMV (4,121 bp, KX096577). To verify the accuracy of the sequence assembled, RT-PCR-Sanger sequencing was performed on a single field plant sample using primers designed for SYCMV (Forward, 5′-GAACAAAGAGTCTGGATCTT-3′; Reverse, 5′-TCCTTCCAAAACCTCGCGGG-3′). The sequence of the amplicon (3854 bp, MT997092) exhibited an identity of 99.9% to the HTS-derived SYCMV contig sequence. Phylogenetic analysis of the amplicon sequence revealed that the SYCMV isolate from China formed a distinct branch in the tree (Fig. S1). Sap from symptomatic field plants was used to mechanically inoculate two soybean cultivars (Jiunong 9 and Kefeng 1, 10 plants per cultivar), and leaves inoculated with phosphate buffer saline (PBS, 0.01 M, pH 7.5) served as a control (3 plants per cultivar). All but the control plants developed systemic bright yellow mosaic symptoms 10 days after inoculation (Fig. S2A). The infection of the soybean plants with SYCMV was confirmed by RT-PCR with the newly designed primers for SYCMV (Forward, 5′- CCTACAGGCATTGGTTTCGT-3′; Reverse, 5′-CGTGAGGTTCTTGCTTCACA-3′, anticipated amplicon size: 2,210 bp) (Fig. S2B) and by amplicon sequencing (100% sequence identity with MT9979092). In addition, the infection was further confirmed by immuno-blotting using an antibody against SYCMV coat protein (synthesized by GenScript, USA) (Fig. S2C). Together, the results demonstrate that SYCMV is the causal agent of the bright yellow mosaic symptoms in soybean observed in the field. To the best of our knowledge, this is the first report of SYCMV on soybean in China. These findings shall not only alert local growers to a potential new threat to soybean production in their region, but also provide new insights on the transmission, epidemiology and pathological properties of SYCMV in China.

OMGS: Optical Map-based Genome Scaffolding

Due to the current limitations of sequencing technologies,de novogenome assembly is typically carried out in two stages, namely contig (sequence) assembly and scaffolding. While scaffolding is computationally easier than sequence assembly, the scaffolding problem can be challenging due to the high repetitive content of eukaryotic genomes, possible mis-joins in assembled contigs and inaccuracies in the linkage information. Genome scaffolding tools either use paired-end/mate-pair/linked/Hi-C reads or genome-wide maps (optical, physical or genetic) as linkage information. Optical maps (in particular Bionano Genomics maps) have been extensively used in many recent large-scale genome assembly projects (e.g., goat, apple, barley, maize, quinoa, sea bass, among others). However, the most commonly used scaffolding tools have a serious limitation: they can only deal with one optical map at a time, forcing users to alternate or iterate over multiple maps. In this paper, we introduce a novel scaffolding algorithm called OMGS that for the first time can take advantages of multiple optical maps. OMGS solves several optimization problems to generate scaffolds with optimal contiguity and correctness. Extensive experimental results demonstrate that our tool outperforms existing methods when multiple optical maps are available, and produces comparable scaffolds using a single optical map. OMGS can be obtained fromhttps://github.com/ucrbioinfo/OMGS

CENP-B box, a nucleotide motif involved in centromere formation, occurs in a New World monkey

Centromere protein B (CENP-B) is one of the major proteins involved in centromere formation, binding to centromeric repetitive DNA by recognizing a 17 bp motif called the CENP-B box. Hominids (humans and great apes) carry large numbers of CENP-B boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have been reported regarding the presence of the CENP-B box in other primate taxa. Consequently, it is widely believed that the CENP-B box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an abundance of CENP-B boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B box may depend largely on the sequencing methods used, and that the CENP-B box in centromeric repetitive DNA may be more common than researchers previously thought.

Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.Key words: HMW glutenin, evolution, retrotransposon, comparative genomics.