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.

BAC Library Development and Clone Characterization for Dormancy-Responsive DREB4A, DAM, and FT from Leafy Spurge (Euphorbia esula) Identifies Differential Splicing and Conserved Promoter Motifs

We developed two leafy spurge bacterial artificial chromosome (BAC) libraries that together represent approximately 5× coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the Arizona Genomics Institute. These libraries were used to clone full-length genomic copies of an AP2/ERF transcription factor of the A4 subfamily of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEINS (DREB) known to be differentially expressed in crown buds of leafy spurge during endodormancy, a DORMANCY ASSOCIATED MADS-BOX (DAM) gene, and several FLOWERING LOCUS T (FT) genes. Sequencing of these BAC clones revealed the presence of multiple FT genes in leafy spurge. Sequencing also provided evidence that two different DAM transcripts expressed in crown buds of leafy spurge during endo- and eco-dormancy result from alternate splicing of a single DAM gene. Sequence data from the FT promoters was used to identify several conserved elements previously recognized in Arabidopsis, as well as potential novel transcription factor binding sites that may regulate FT. These leafy spurge BAC libraries represent a new genomics-based tool that complements existing genomics resources for the study of plant growth and development in this model perennial weed. Furthermore, phylogenetic footprinting using genes identified with this resource demonstrate the usefulness of studying weedy species to further our general knowledge of agriculturally important genes.

Whole genome sequencing of sporadic Burkitt lymphoma in HIV-infected and uninfected patients.

8577 Background: Burkitt Lymphoma is defined by canonical translocations between MYC and immunoglobulin IgH, IgK or IgL (8:14, 8:2, 8:22, respectively), and is commonly associated with HIV. The identification of HIV from sequenced samples is critical to understanding HIV-associated Burkitt Lymphoma. While recent novel gene mutations (ID3 and TCF3) have been implicated in functional roles, concomitant genomic structural variants and the interaction of HIV with structural variation is less well defined. Methods: We sequenced the whole genomes of 15 patients with 100bp paired-end reads on Illumina Hi-Seq platform, resulting in an average insert size of 278 (+/- 63) and coverage of 60X tumor and 30X normal. We included 7 HIV-negative, and 8 HIV-positive subjects. Sequencing reads were mapped to the reference genome using BWA. Large-scale structural variation was detected by the BreakDancer and Crest programs. Functional annotation was used to prioritize structural variants for validation. Single nucleotide variants and small insertions and deletions were detected by CARNAC, a somatic variation discovery pipeline. The subset of WGS reads that failed to align to the human reference genome were tested for the presence of HIV sequences by comparing the unmapped reads to a database of viral DNA sequences which included the common subtypes of HIV defined by Los Alamos. Reads matching HIV or EBV with an expectation value of <10-4 were analyzed to determine virus coverage and viral integration sites. Results: Canonical MYC-IgH translocations were identified in 9/15 (60%) tumor samples, with 2 additional subjects harboring either a deletion or an inversion near exon1 of MYC; 4 had no MYC rearrangement. MYC translocations occurred equally in both groups. TP53 and SMARC4 point mutations were observed recurrently in the HIV uninfected group but not in the HIV infected patients. Variable levels of HIV DNA sequence were observed in normal tissue of all HIV infected patients. Conclusions: Whole genome sequencing has identified known somatic variants in HIV infected and uninfected patients. Two genes, TP53 and SMARC4, appear to be differentially mutated, but additional samples are needed to achieve statistical significance.

Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

Bacterial artificial chromosome (BAC) libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa), using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3 kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine.

Construction of a Llama Bacterial Artificial Chromosome Library with Approximately 9-Fold Genome Equivalent Coverage

The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be2.4×109 bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama.

Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

We describe the construction and characterization of a publicly available BAC library for the tea plant,Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers.

Construction of Papaya Male and Female BAC Libraries and Application in Physical Mapping of the Sex Chromosomes

Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzymeBstY I and consists of 36,864 clones with an average insert size of 104 kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzymeEcoR I and consists of 55,296 clones with an average insert size of 101 kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yhchromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes.

Construction of a Coix BAC library and isolation of the 22 kDa α-coixin gene cluster

Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa α-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa α-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome.

Integrative Physical and Genetic Mapping of the Chickpea Genome for Fine Mapping and Analysis of Agronomic Traits

Chickpea is the third most important pulse crop in the world and ranks first in the Middle East; however, it has been subjected to only limited research in modern genomics. In the first period of this project (US-3034-98R) we constructed two large-insert BAC and BIBAC libraries, developed 325 SSR markers and mapped QTLs controlling ascochyta blight resistance (ABR) and days to first flower (DTF). Nevertheless, the utilities of these tools and results in gene discovery and marker-assisted breeding are limited due to the absence of an essential platform. The goals of this period of the project were to use the resources and tools developed in the first period of the project to develop a BAC/BIBAC physical map for chickpea and using it to identify BAC/BIBACcontigs containing agronomic genes of interest, with an emphasis on ABR and DTF, and develop DNA markers suitable for marker-assisted breeding. Toward these goals, we proposed: 1) Fingerprint ~50,000 (10x) BACs from the BAC and BIBAC libraries, assemble the clones into a genome-wide BAC/BIBAC physical map, and integrate the BAC/BIBAC map with the existing chickpea genetic maps (Zhang, USA); 2) fine-map ABR and DTFQTLs and enhance molecular tools for chickpea genetics and breeding (Shahal, Sherman and DaniShtienberg, Israel; Chen and Muehlbauer; USA); and 3) integrate the BAC/BIBAC map with the existing chickpea genetic maps (Sherman, Israel; Zhang and Chen, USA). For these objectives, a total of $460,000 was requested originally, but a total of $300,000 was awarded to the project. We first developed two new BAC and BIBAC libraries, Chickpea-CME and Chickpea- CHV. The chickpea-CMEBAC library contains 22,272 clones, with an average insert size of 130 kb and equivalent to 4.0 fold of the chickpea genome. The chickpea-CHVBIBAC library contains 38,400 clones, with an average insert size of 140 kb and equivalent to 7.5 fold of the chickpea genome. The two new libraries (11.5 x), along with the two BAC (Chickpea-CHI) and BIBAC (Chickpea-CBV) libraries (7.1 x) constructed in the first period of the project, provide libraries essential for chickpea genome physical mapping and many other genomics researches. Using these four libraries we then developed the proposed BAC/BIBAC physical map of chickpea. A total of 67,584 clones were fingerprinted, and 64,211 (~11.6 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBACcontigs, with each containing an average of 39.2 clones and having an average physical length of 559 kb. The contigs collectively span ~1,088 Mb, being 1.49 fold of the 740- Mb chickpea genome. Third, we integrated the physical map with the two existing chickpea genetic maps using a total of 172 (124 + 48) SSR markers. Fourth, we identified tightly linked markers for ABR-QTL1, increased marker density at ABR-QTL2 and studied the genetic basis of resistance to pod abortion, a major problem in the east Mediterranean, caused by heat stress. Finally, we, using the integrated map, isolated the BAC/BIBACcontigs containing or closely linked to QTL4.1, QTL4.2 and QTL8 for ABR and QTL8 for DTF. The integrated BAC/BIBAC map resulted from the project will provide a powerful platform and tools essential for many aspects of advanced genomics and genetics research of this crop and related species. These includes, but are not limited to, targeted development of SNP, InDel and SSR markers, high-resolution mapping of the chickpea genome and its agronomic genes and QTLs, sequencing and decoding of all genes of the genome using the next-generation sequencing technology, and comparative genome analysis of chickpea versus other legumes. The DNA markers and BAC/BIBACcontigs containing or closely linked to ABR and DTF provide essential tools to develop SSR and SNP markers well-suited for marker-assisted breeding of the traits and clone their corresponding genes. The development of the tools and knowledge will thus promote enhanced and substantial genetic improvement of the crop and related legumes.

Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene

Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck ‘Cara Cara’ navel orange and Citrus reticulata (L.) Blanco ‘Egan No. 1’ Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 × 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 × 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.