A novel endogenous badnavirus exists in Alhagi sparsifolia#

A novel endogenous badnavirus exists in Alhagi sparsifolia # # 疏叶骆驼刺中包含一种新杆状DNA病毒 1 first-author 2 3 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:yaoqin@ujs.edu.cnliweimin01@caas.cn †E-mail:yaoqin@ujs.edu.cnliweimin01@caas.cn yaoqin@ujs.edu.cn 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:yaoqin@ujs.edu.cnliweimin01@caas.cn †E-mail:yaoqin@ujs.edu.cnliweimin01@caas.cn yaoqin@ujs.edu.cn Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China Inspection & Quarantine Technology Center, Fujian Entry-Exit Inspection and Quarantine Bureau, Fuzhou 350003, China Inspection & Quarantine Technology Center, Fujian Entry-Exit Inspection and Quarantine Bureau, Fuzhou 350003, China Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming 650201, China Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming 650201, China Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China We report the recovery of a 7068-nt viral sequence from the “viral fossils” embedded in the genome of Alhagi sparsifolia , a typical desert plant. Although the full viral genome remains to be completed, the putative genome structure, the deduced amino acids and phylogenetic analysis unambiguously demonstrate that this viral sequence represents a novel species of the genus Badnavirus . The putative virus is tentatively termed Alhagi bacilliform virus (ABV). Southern blotting and inverse polymerase chain reaction (PCR) data indicate that the ABV-related sequence is integrated into the A . sparsifolia genome, and probably does not give rise to functional episomal virus. Molecular evidence that the ABV sequence exists widely in A . sparsifolia is also presented. To our knowledge, this is the first endogenous badnavirus identified from plants in the Gobi desert, and may provide new clues on the evolution, geographical distribution as well as the host range of the badnaviruses. 目的 2 新杆状DNA病毒的分离与鉴定. 创新点 2 首次在高寒地区代表性植物--疏叶骆驼刺中发现杆状DNA病毒,为研究杆状DNA病毒的进化、地理分布及寄主范围提供了新证据. 方法 2 利用分段聚合酶链式反应(PCR)克隆疏叶骆驼刺杆状病毒( Alhagi bacilliform virus ,ABV)的基因组序列;通过基因组分析、序列比对和进化树分析阐明ABV的进化地位;用Southern印迹杂交和反向PCR分析ABV序列与宿主基因组的关系;并通过PCR检测确定ABV在我国西北地区疏叶骆驼刺中的分布. 结论 2 本研究获得了7068 nt 的ABV基因组序列,根据基因组结构、保守序列比对及进化树分析,推测ABV是一种新杆状DNA病毒.分子检测证据表明,ABV基因组序列已整合进入疏叶骆驼刺基因组中,但没有产生游离病毒.此外,对我国西北11个不同地区的疏叶骆驼刺进行PCR检测,结果显示其中9个地区的疏叶骆驼刺均含有ABV序列,由此表明ABV在我国西北地区的疏叶骆驼刺中广泛存在. Badnavirus Endogenous Alhagi bacilliform virus Nuclear integration 杆状DNA病毒 内生疏叶骆驼刺杆状病毒 基因组整合 Introduction 1 Badnaviruses, members of the plant virus family Caulimoviridae, possess an open-circular double-stranded DNA genome of 7–8 kb with non-enveloped bacilliform particles (Fauquet et al., 2005 2005 ). So far, forty species are assigned to the Badnavirus genus (Bhat et al., 2016 2016 ), which include Commelina yellow mottle virus (ComYMV) as the type species (Medberry et al., 1990 1990 ). The badnaviruses are known to be transmitted through vegetative propagation and via vector insects such as mealybug and aphid (Harper et al., 2004 2004 ; Geering et al., 2005a 2005a ; Laney et al., 2012 2012 ; Seal et al., 2014 2014 ; Bhat et al., 2016 2016 ). Their typical genome structure consists of three open reading frames (ORFs) solely on the plus strand (Xu et al., 2011 2011 ). ORF1 and ORF2 encode proteins of unclear function(s), but previous studies have suggested that the ComYMV ORF1-and ORF2-encoded proteins are associated with the cell-wall-enriched fraction and with virions (Cheng et al., 1996 1996 ), and that the Cacao swollen shoot virus (CSSV) ORF2 encodes a nucleic acid-binding protein (Jacquot et al., 1996 1996 ). ORF3, the largest ORF, encodes a about 200-kDa polyprotein which contains conserved domains of movement protein (MP), aspartic protease (AP), a cysteine-rich zinc finger-like RNA-binding region (RB), a second cysteine-rich region (2nd CR), reverse transcriptase (RT), and ribonuclease H (RNaseH) (Medberry et al., 1990 1990 ; Tzafrir et al., 1997 1997 ; Wang et al., 2014 2014 ; Kazmi et al., 2015 2015 ). The conserved domains, particularly the RT-RNaseH, are usually employed to discriminate between viruses within the Badnavirus genus (King et al., 2012 2012 ). The polyprotein is specifically hydrolyzed by AP, resulting in four to five mature proteins related to viral movement and replication as well as virion packaging (Medberry et al., 1990 1990 ; Jacquot et al., 1996 1996 ; Hohn et al., 1997 1997 ; Tzafrir et al., 1997 1997 ; Hany et al., 2014 2014 ). Like other members of the family Caulimoviridae, which are referred to as endogenous pararetroviruses (EPRVs), many badnaviruses also embed their genomes into host nuclear DNA (Geering et al., 2001 2001 , 2005a 2005a , 2005b 2005b ; Harper et al., 2002 2002 ; Philippe and Marie, 2009 2009 ; Chabannes et al., 2013 2013 ; Iskra-Caruana et al., 2014 2014 ; Seal et al., 2014 2014 ). It is proposed that this nuclear integration takes place by illegitimate recombination during DNA double strand break repair in the host genome (Staginnus and Richert-Pöggeler, 2006 2006 ; Laney et al., 2012 2012 ). The resulting integrants usually present diverse and complex structures, possibly because sequence rearrangement such as tandem repeat, inversion, duplication, and fragmentation occurs in the integrated viral genome (Hull et al., 2000 2000 ; Umber et al., 2014 2014 ). The integrants derived from most of the identified badnaviruses, including Fig badnavirus 1 (FBV-1), Kalanchoë top-spotting virus (KTSV), and Dracaena mottle virus (DrMV), have been considered to be non-infectious (Yang et al., 2005 2005 ; Su et al., 2007 2007 ; Laney et al., 2012 2012 ; Iskra-Caruana et al., 2014 2014 ). Only integrants of Banana streak GF virus (BSGFV), Banana streak OL virus (BSOLV), and Banana streak IM virus (BSIMV) that infect the Musa species have been found to be activated as episomal infectious viruses when crossing or on abiotic stresses (Gayral et al., 2008 2008 ; Chabannes et al., 2013 2013 ). The hosts of the known badnaviruses range from monocots to dicots, most of which are tropical and subtropical plants, including banana, potato, black pepper, citrus, cocoa, sugarcane, taro, and yam, as well as a few temperate crops such as red raspberry, gooseberry, and ornamental spiraea (Harper et al., 2002 2002 ; Hansen et al., 2005 2005 ; Staginnus and Richert-Pöggeler, 2006 2006 ; Bhat et al., 2016 2016 ). Therefore, the majority of the badnaviruses are distributed in tropical and subtropical climate zones, with a few in some temperate regions (Bhat et al., 2016 2016 ). Alhagi sparsifolia Shap. is a perennial subshrub belonging to the family Leguminosae, which grows in desert climates and is naturally distributed in the Gobi desert of Northwest China and adjacent countries in Middle Asia. Recent high-resolution RNA sequencing (RNA-Seq) of the primary roots of A . sparsifolia (Wu et al., 2015 2015 ) suggested the presence of a badnavirus. We initiated our study to determine the genome of this badnavirus, tentatively named Alhagi bacilliform virus (ABV), and have provided evidence that it is novel and is integrated in the A . sparsifolia genome. To our knowledge, ABV is the first badnavirus identified from a plant growing only in the Gobi desert, thus extending the geographical distribution of the badnaviruses. Materials and methods 1 Plant materials 2 A . sparsifolia seeds collected from Taklamakan of the Xinjiang Uygur Autonomous Region, Northwest China and previously subjected to RNA-seq analyses (Wu et al., 2015 2015 ) were used to clone the putative ABV sequence. To explore the distribution of the putative badnavirus in A . sparsifolia , more seeds were collected individually from 11 different places in Northwest China: ten from the Xinjiang Uygur Autonomous Region (Shihezi, Hutubi, Luntai, Manas Lake, Kuytun, Cele, Wensu, Yopurga, Wushi, and Alal) and one from Gansu Province (Minqin). As described by Wu et al. ( 2015 2015 ), the seeds were first treated with concentrated sulfuric acid, and were then cultured in sterilized dishes with a layer of fully wetted filter paper in the dark at 25 °C. After 7 d, the seedlings were harvested and stored at −80 °C until use. Total DNA extraction 2 By using the cetyltrimethylammonium bromide (CTAB) method (Gawel and Jarret, 1991 1991 ), the total DNA of A. sparsifolia was extracted from 100 mg of the 7-d old seedlings, and was subsequently treated with RNase A (1 mg/ml) at 65 °C for 60 min. Following two extractions with chloroform-isoamyl (24:1, v/v), the DNA was precipitated and resuspended in a final volume of 40 μl sterile distilled deionized H 2 O and stored at −20 °C until use. Genome cloning and assembly 2 Nine primer sets (Table S1 S1 ) were designed to generate polymerase chain reaction (PCR) products covering the entire putative genome of ABV following a genome walking strategy. PCR was performed using the total DNA prepared from A . sparsifolia seedlings from Taklamakan as a template. At least five independent clones were sequenced for each amplified product. A continuous nucleotide sequence (Fig. 1a 1a ; GenBank accession KY034642) was assembled with the overlapping clones (Figs. 1b and 1c 1b and 1c ) using DNAMAN Version 5.2.2 (Lynnon Biosoft, Montreal, QC, Canada). ORF finder ( http://www.ncbi.nlm.nih http://www.ncbi.nlm.nih . gov/projects/gorf) was used to identify the putative ORFs in the assembled viral genome. The theoretical molecular weights (MWs) of the deduced proteins were calculated using ExPASy ( http://web.expasy.org/ http://web.expasy.org/ compute_pi), and a CD-search ( http://www.ncbi.nlm http://www.ncbi.nlm . nih.gov/Structure/cdd/wrpsb.cgi) was employed to identify conserved domains within the protein sequences. (a) The putative ORFs are indicated by open boxes. The predicted domains, CP, RB, 2nd CR, AP, RT, and RNaseH, within ORF3 are shown. The dashed lines at the 5' and 3' ends of the nucleotide sequence represent the unidentified sequence of the ABV genome. (b) Relative positions of the three badnavirus sequences derived from RNA-Seq. (c) Relative positions of cDNA clones used to assemble the ABV sequence Sequence analysis 2 Multiple sequence alignments were performed on nucleotide (nt) or deduced amino acid (aa) sequences of the putative ABV and those of the representative members in the genus Badnavirus using DNAMAN Version 5.2.2 (Lynnon Biosoft, Montreal, QC, Canada). Phylogenetic analysis was inferred on the 579-nt RT-RNaseH-coding region, the 456-nt AP-coding region, and the 816-nt coat protein (CP)-coding region of ABV, and 40 known badnaviruses, whose names and sequence accession numbers are listed in Table S2 S2 . Strawberry vein banding virus (SVBV) of the genus Caulimovirus was used as an outgroup since it is one of the most closely related non-badnavirus members of the family Caulimoviridae. The phylogenetic tree was built with MEGA6 using the neighbor joining method (Tamura et al., 2011 2011 ). The significance of branching order was assessed by bootstrap resampling of 1000 replicates, and the cut-off value was 50%. Southern blotting analysis 2 Total DNA (30 μg) prepared from the 7-d old seedlings was fully digested overnight with 100 U of Xba I or Hin dIII, which does not cut the sequence of the RT-RNaseH domain within ABV. The digested DNA samples, along with the equivalent amount of untreated total DNA, were separated in a 0.8% (0.008 g/ml) agarose gel, and immediately transferred to the Hybond N + membrane (GE Healthcare, Life Sciences, Indianapolis, USA). Following prehybridization, the membrane was hybridized overnight at 65 °C with the 32 P-labeled probes prepared from the 579-nt RT-RNaseH-coding region of ABV using the Prime-a-Gene ® kit (Promega, Madison, WI, USA). The membrane was washed twice in 0.5×saline-sodium citrate (SSC) and 0.1% (1 g/L) sodium dodecyl sulfide (SDS) at 65 °C for 30 min, and exposed to X-ray film at −80 °C. Developing and fixing were performed after 2–3 d. Inverse PCR 2 Total DNA (2 μg) prepared from the 7-d-old seedlings was completely digested overnight with 20 U of Xba I. After chloroform extraction and ethanol precipitation, 1 μg of the Xba I-digested DNA was ligated at 16 °C in a volume of 500 μl containing 10 μl T 4 DNA ligase overnight. The resulting ligation product was further precipitated with ethanol, and was resuspended in 10 μl of sterile distilled deionized H 2 O. One microliter of the purified ligation product was immediately used as a template to perform the 1st round of PCR in a 50-μl reaction volume with the primer pair IR1-F/IR1-R. The 2nd round of PCR was conducted with 1 μl of a 200-fold dilution of the primary PCR product with the nested primer pair IR2-F/IR2-R (Table S1 S1 ). The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 3 min, and final extension at 72 °C for 10 min. PCR products were analyzed by electrophoresis in 1% (0.01 g/ml) agarose gel, and a band with the expected size of approximately 3 kb was purified and ligated into the pMD18-T Vector (TaKaRa, Dalian, China) for sequencing. Amplification of the nucleotide sequence of the RT-RNaseH domain 2 With a degenerate primer pair Badna-FP/Badna-RP, as show in Table S1 S1 (Yang et al., 2003 2003 ), the PCR amplification was performed in a 50-μl reaction volume containing LA Taq DNA polymerase (TaKaRa) as well as a 50-ng DNA template prepared from A . sparsifolia seedlings from Shihezi, Hutubi, Luntai, Manas Lake, Kuytun, Cele, Wensu, Yopurga, Wushi, Alal, and Minqin. The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 40 s, and final extension at 72 °C for 10 min. PCR products were analyzed by 1% agarose gel electrophoresis, and a band with the expected size of approximately 580 bp was purified and ligated into the pMD18-T Vector (TaKaRa). After transformation, the positive clones were screened for sequencing. Results and discussion 1 A putative badnavirus found in A . sparsifolia 2 Using RNA-Seq, we recently constructed a transcriptome database containing 33 255 unigenes with the polyadenylated RNAs purified from the primary roots of A . sparsifolia from Taklamakan (Wu et al., 2015 2015 ). By BlastX searching against the NCBI nr database, two unigenes, comp6965_c1 with a 246-bp length and comp22399_c0 with a size of 10 144 bp, were identified to have homology with the genome of Pagoda yellow mosaic associated virus (PYMAV; GenBank accession KJ013302.1), a known badnavirus. The comp6965_c1 matches with nt 3526 to 3771 of PYMAV with an identity of approximately 63%. For comp22399_c0, the fragment ranging from nt 1 to 1136 matches with nt 847 to 2004 of PYMAV genome with an identity of approximately 50%, and the complementary sequence of nt 1950 to 4547 matches with nt 4224 to 6903 of PYMAV genome with an identity of 55%. The three badnavirus-like sequences were successfully cloned through PCR amplification with the corresponding primer pairs (Table S1 S1 ), and were individually termed as B1, B2, and B3 (Fig. 1b 1b ). It is worth emphasizing that total DNA of A . sparsifolia from Taklamakan was used as the PCR template. These data collectively implied that the A . sparsifolia plant might harbor a badnavirus, which was tentatively named as ABV. In order to fill gaps between the three known sequences of ABV, five sets of primer pairs (Table S1 S1 ) were further designed and used to perform PCR amplification. Due to the circular genome of the badnaviruses, the primer pair of A1-F/A1-R was used to amplify the viral sequence harboring the intergenic region (IR). The obtained overlapping PCR products, along with nucleotide sequences B1, B2, and B3, were assembled, finally resulting in a continuous nucleotide sequence (7068 nt) with a G+C content of 46.9% (Fig. 1 1 ). The IR regions of the badnaviruses are known to have a putative plant initiator methionine transfer RNA (tRNA met )-binding site, the first nucleotide of which is usually used to start numbering the viral genome (Geijskes et al., 2002 2002 ; Yang et al., 2003 2003 ; Seal and Muller, 2007 2007 ; Su et al., 2007 2007 ; Kazmi et al., 2015 2015 ). Because the initial 12 nucleotides (5'-TGGTATCA GAGC-3') of the putative tRNA met -binding site are conserved in plant pararetroviruses, we analyzed the 7068-nt sequence by first searching for the 12 conserved nucleotides from the IR region of the assembled ABV sequence. Unfortunately, no nucleotide representing the 12-nt conserved sequence was detected, indicating that the 7068-nt ABV sequence may represent an incomplete viral genome. We, therefore, tentatively numbered the start of the ABV sequence by comparing it with the putative IR of ABV and the known badnaviruses. This needs to be refined. Typically, the badnavirus genome encompasses three ORFs (ORF1, ORF2, and ORF3) on the plus strand (Bhat et al., 2016 2016 ). Further analysis of the coding capacity of the ABV sequence identified no ORF1, probably due to the incomplete genome (Fig. 1a 1a ). However, the fragment ranging from nt 508 to 936 should be of the ABV ORF2 (Fig. 1a 1a ). The deduced protein comprises 142 aa with an MW of 16.2 kDa, and has the highest similarity at 34.9% with the putative protein encoded by the PYMAV ORF2. The fragment from nt 936 to 6573 was thought to represent the ABV ORF3, although its coding capacity was interrupted by multiple internal stop codons. The amino acids deduced from this fragment contain domains of CP, AP, RT, RNaseH as well as two “Cys” motifs, RB and 2nd CR (Figs. 1a 1a and 2 2 ), which are conserved in the polyproteins encoded by the ORF3 of the known badnaviruses (Medberry et al., 1990 1990 ; Tzafrir et al., 1997 1997 ; Wang et al., 2014 2014 ; Kazmi et al., 2015 2015 ). In addition, the nt 6138–6620 of ABV potentially encoded for a 160-aa protein with an MW of 17.8 kDa (Fig. 1a 1a ), and was assumed as the counterpart of the PYMAV ORF4 (Wang et al., 2014 2014 ), because BLASTP analysis showed that this putative protein only produces significant alignment with the PYMAV ORF4-encoded protein. Virus names are showed before each sequence. Identical (*) and conserved (:) amino acids are indicated Despite the absence of the putative tRNA met binding site and ORF1, the remaining genetic properties of this incomplete ABV genome demonstrated conclusively that ABV is indeed a badnavirus. According to the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV) (King et al., 2012 2012 ), badnaviruses with less than 80% nucleotide sequence identity or 89% amino acid sequence identity in the RT-RNaseH-coding region are regarded as species. In the current study, comparison of the putative RT-RNaseH of ABV with the same domain of the known badnaviruses exhibited amino acid sequence identities ranging from 57% to 72% and nucleotide sequence identities from 58% to 65% (Table 1 1 ), suggesting that ABV is a novel member of the genus Badnavirus . a Viruses used are summarized in Table S2 S2 2 Virus 11 Amino acid sequence identity (%)/nucleotide sequence identity (%) ABV PYMAV DrMV SCBIMV ComYMV HBV DBV TaBV KTSV BSGFV CSSV ABV − PYMAV 72/65 − DrMV 62/62 60/63 − SCBIMV 59/58 60/60 63/59 − ComYMV 57/58 61/60 61/60 61/64 − HBV 63/63 62/64 64/60 60/59 64/64 − DBV 58/62 66/65 63/63 64/63 65/63 69/71 − TaBV 61/62 63/63 64/61 62/62 64/60 63/62 66/61 − KTSV 62/61 61/63 64/61 60/60 59/63 63/64 65/66 64/59 − BSGFV 60/60 64/59 63/62 60/59 59/64 65/65 63/64 61/61 67/64 − CSSV 60/61 60/62 60/60 60/61 60/62 67/63 62/64 64/59 57/62 55/63 − Phylogenetic analysis of the conserved domains within the putative polyprotein of ABV 2 The relationship between the putative ABV with the other members of the genus Badnavirus and SVBV, a member of the genus Caulimovirus , was further estimated using nucleotide sequences of RT-RNaseH, CP, and AP for phylogenetic analyses. The inferred phylogenetic trees disclosed that ABV was always clustered with the badnaviruses, supporting placement of this virus within the genus Badnavirus . The RT-RNaseH domains of ABV and PYMAV cluster together as part of a larger cluster including the RT-RNaseH domains of nine distinct badnaviruses, BbVF, PVBV, DrMV, YNMoV, BsCVBV, GVCV, RYNV, TaBV, and GVBaV, as do the CP of ABV and PYMAV (Figs. 3a and 3b 3a and 3b ). A large cluster was also viewed in the AP-inferred phylogenetic tree, the only difference being that the AP of CyNLV, rather than TaBV, was included in this cluster (Fig. 3c 3c ). These data suggest the close relationship of ABV with the badnaviruses, in particular, PYMAV, the host of which also belongs to the family Leguminosae (Wang et al., 2014 2014 ). The phylogenetic trees were inferred with MEGA6 using the neighbor joining method with Strawberry vein banding virus (SVBV) as an outgroup. Branch lengths are proportional to genetic distances. Significance of branching order was assessed by bootstrap resampling of 1000 replicates and bootstrap values are given when ≥50%. The clusters including ABV are shaded. The viruses used (listed with abbreviation and GenBank accession number for nucleotide sequence) are presented in Table S2 S2 . These are unrooted trees Integration of the ABV sequence into the A. sparsifolia genome 2 Many, if not all, badnaviruses are known to have integrants in their respective host plant genomes (Geering et al., 2001 2001 , 2005a 2005a , 2005b 2005b ; Harper et al., 2002 2002 ; Philippe and Marie, 2009 2009 ; Chabannes et al., 2013 2013 ; Iskra-Caruana et al., 2014 2014 ; Seal et al., 2014 2014 ). To test whether ABV is endogenous to the A . sparsifolia genome, Southern blotting was performed to analyze the untreated total DNA from Taklamakan A . sparsifolia with the 32 P-labeled probes corresponding to the RT-RNaseH-coding region of ABV. As shown in Fig. 4a 4a , the undigested total DNA sample yielded a unique hybridized band at the location of the A . sparsifolia genomic DNA rather than at the location (about 8 kb) expected for the ABV genome, suggesting nuclear integration of the ABV sequence. One might argue that no hybridized band corresponding to the unit length of the ABV genome was detected because there is too little, if any, episomal virus in the plant to be detected. However, considering the seriously interrupted coding capacity of the ABV ORF3 (Fig. 1 1 ), the endogenous ABV sequences should be incapable of giving rise to a functional episomal virus, but may present as long-lasting imprints within the A . sparsifolia genome, known as “viral fossils” (Feschotte and Gilbert, 2012 2012 ). In accordance with this hypothesis, no product was generated with rolling circle amplification (RCA) to test the total DNA prepared from A . sparsifolia of Taklamakan (data not shown). Therefore, the recovered 7068-nt ABV sequence may reflect an ancient badnavirus that was once active but now silenced in A . sparsifolia . (a) Southern blotting analysis of the A . sparsifolia genomic DNA with radiolabelled probes corresponding to the RT-RNaseH-coding region of ABV. M: λ- Hin dIII digest DNA marker; Lane 1: undigested DNA; Lane 2: DNA digested with Hin dIII; Lane 3: DNA digested with Xba I. (b) A schematic diagram of inverse PCR employed to clone an endogenous ABV element. The primer pairs IR1-F/IR1-R and IR2-F/IR2-R (Table S1 S1 ) were designed based on nt 5203–5781 (the putative RT-RNaseH-coding region) of ABV. A 2925-bp endogenous ABV element was cloned, its position in the putative ABV genome is shown, and the grey bars “a” and “b” represent the right and left flanking plant genome sequences, respectively In addition, the same probes were also used to analyze the restriction endonuclease-digested total DNA of A . sparsifolia . The resulting data disclosed that, for the Hin dIII-digested DNA, only a fragment of about 4.5 kb was detected (Fig. 4a 4a , Lane 2). However, the Xba I-digested DNA gave rise to two discrete bands at approximately 6.5 and 3.0 kb (Fig. 4a 4a , Lane 3), indicating at least two copies of the ABV elements in the A . sparsifolia genome. To further verify the endogenous nature of the ABV-related elements, we further designed two nested primer pairs according to the 579-nt RT-RNaseH-coding region of ABV, which was used to prepare the labeled probes for Southern blotting as described above, and employed the approach of inverse PCR to clone the DNA fragments representing the lower hybridization band from the Xba I-digested DNA. As expected, a 2925-bp DNA fragment (GenBank accession KY677913) was obtained, and sequence analysis (Fig. 4b 4b ) showed that this DNA fragment comprises a viral element corresponding to nt 5052 to 5946 of the putative ABV, which is flanked by the plant genome sequences “a” and “b” sharing homology with nt 3220 to 3480 and nt 2295 to 3173 of Medicago truncatula chromosome 8 clone mth2-6f11 (GenBank accession AC151460), respectively. This DNA fragment was further confirmed by a PCR analysis across the virus-plant junctions with a primer pair VPJ-F/VPJ-R (Table S1 S1 ) anchored to the plant genome sequences “a” and “b”. Taken together, these data provide additional evidence for the integration of the ABV sequence into the A . sparsifolia genome, and indicate that the integrated ABV sequence underwent complex sequence rearrangement, like integrants of other badnaviruses (Geering et al., 2001 2001 ; Gregor et al., 2004 2004 ; Umber et al., 2014 2014 ). In accordance with this hypothesis, one DNA fragment encompassing nt 1–79, 712–795, 904–2283, and 6653–7055 of the 7059-nt ABV sequence was cloned using the primer pair of A1-F/A1-R (data not shown). Widespread presence of the ABV sequence in A . sparsifolia 2 The presence of ABV in A . sparsifolia from Taklamakan prompted us to explore the distribution of the endogenous virus in this plant species. We subjected A . sparsifolia seedlings from 11 different places in Northwest China, Shihezi, Hutubi, Luntai, Manas Lake, Kuytun, Cele, Wensu, Yopurga, Wushi, Alal, and Minqin, to PCR detection using the degenerate primer pair Badna-FP and Badna-RP (Yang et al., 2003 2003 ). Except for seedlings from three adjacent places (Wensu, Wushi, and Alal), the RT-RNaseH-coding region of ABV was amplified from all tested samples, showing widespread presence of this badnavirus in A . sparsifolia (Fig. 5 5 ). We could not rule out the possibility that the ABV-related sequence is embedded in the genome from plants in Wensu, Wushi, and Alal, but that we were unable to detect it because of sequence rearrangement within the entire RT-RNaseH-coding region. The RT-RNaseH-coding region of ABV was detected in A . sparsifolia seedlings from nine different places of the Xinjiang Uygur Autonomous Region and Gansu Province, China, except for those from Wensu, Wushi, and Alal, the three adjacent places which are circled with dashed lines Conclusions 2 This study reports evidence for an ancient endogenous badnavirus, tentatively termed ABV, in A . sparsifolia , a typical desert plant which differs from the known badnavirus hosts which are tropical, subtropical, and temperate plants. While the full genome sequence of ABV remains to be completed, the 7068-nt viral sequence recovered from the endogenous badnavirus elements in A . sparsifolia may shed fresh light on the evolution, geographical distribution as well as the host range of the badnaviruses. Dating of the endogenization event of ABV may not be possible. However, it seems to have occurred after the speciation of A . sparsifolia because no ABV sequence has so far been identified from 15 Leguminosae species with complete genomes available in GenBank. The endogenous badnavirus sequences in A . sparsifolia imply a potential contribution to the complexity and evolution of the host genome, and merit further investigation. References AI Bhat , T Hohn , R Selvarajan . Badnaviruses: the current global scenario . Viruses , 2016 . 8 ( 6 ): 177 DOI: 10.3390/v8060177 http://doi.org/10.3390/v8060177 . Badnaviruses: the current global scenario M Chabannes , FC Baurens , PO Duroy , 等 . Three infectious viral species lying in wait in the banana genome . J Virol , 2013 . 87 ( 15 ): 8624 - 8637 . 23720724 DOI: 10.1128/JVI.00899-13 http://doi.org/10.1128/JVI.00899-13 . Three infectious viral species lying in wait in the banana genome CP Cheng , BEL Lockhart , NE Olszewski . The ORF I and II proteins of Commelina yellow mottle virus are virion-associated . Virology , 1996 . 223 ( 2 ): 263 - 271 . 8806562 DOI: 10.1006/viro.1996.0478 http://doi.org/10.1006/viro.1996.0478 . The ORF I and II proteins of Commelina yellow mottle virus are virion-associated CM Fauquet , MA Mayo , J Maniloff , 等 . Virus taxonomy, classification and nomenclature of viruses , 2005 . Eighth Report of the International Committee on the Taxonomy of Viruses. Elsevier Academic Press, San Diego , . Virus taxonomy, classification and nomenclature of viruses C Feschotte , C Gilbert . Endogenous viruses: insights into viral evolution and impact on host biology . Nat Rev Genet , 2012 . 13 ( 4 ): 283 - 296 . 22421730 DOI: 10.1038/nrg3199 http://doi.org/10.1038/nrg3199 . Endogenous viruses: insights into viral evolution and impact on host biology NJ Gawel , RL Jarret . A modified CTAB DNA extraction procedure for Musa and Ipomoea . Plant Mol Biol Rep , 1991 . 9 ( 3 ): 262 - 266 . DOI: 10.1007/BF02672076 http://doi.org/10.1007/BF02672076 . A modified CTAB DNA extraction procedure for Musa and Ipomoea P Gayral , JC Noa-Carrazana , M Lescot , 等 . A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus . J Virol , 2008 . 82 ( 13 ): 6697 - 6710 . 18417582 DOI: 10.1128/JVI.00212-08 http://doi.org/10.1128/JVI.00212-08 . A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus ADW Geering , NE Olszewski , G Dahal , 等 . Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars . Mol Plant Pathol , 2001 . 2 ( 4 ): 207 - 213 . 20573008 DOI: 10.1046/j.1464-6722.2001.00071.x http://doi.org/10.1046/j.1464-6722.2001.00071.x . Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars ADW Geering , NE Olszewski , G Harper , 等 . Banana contains a diverse array of endogenous badnaviruses . J Gen Virol , 2005 . 86 ( 2 ): 511 - 520 . 15659772 DOI: 10.1099/vir.0.80261-0 http://doi.org/10.1099/vir.0.80261-0 . Banana contains a diverse array of endogenous badnaviruses ADW Geering , MM Pooggin , NE Olszewski , 等 . Characterisation of Banana streak Mysore virus and evidence that its DNA is integrated in the B genome of cultivated Musa . Arch Virol , 2005 . 150 ( 4 ): 787 - 796 . 15785970 DOI: 10.1007/s00705-004-0471-z http://doi.org/10.1007/s00705-004-0471-z . Characterisation of Banana streak Mysore virus and evidence that its DNA is integrated in the B genome of cultivated Musa RJ Geijskes , KS Braithwaite , JL Dale , 等 . Sequence analysis of an Australian isolate of Sugarcane bacilliform badnavirus . Arch Virol , 2002 . 147 ( 12 ): 2393 - 2404 . 12491105 DOI: 10.1007/s00705-002-0879-2 http://doi.org/10.1007/s00705-002-0879-2 . Sequence analysis of an Australian isolate of Sugarcane bacilliform badnavirus W Gregor , MF Mette , C Staginnus , 等 . A distinct endogenous pararetrovirus family in Nicotiana tomentosiformis , a diploid progenitor of polyploid tobacco . Plant Physiol , 2004 . 134 ( 3 ): 1191 - 1199 . 14988473 DOI: 10.1104/pp.103.031112 http://doi.org/10.1104/pp.103.031112 . A distinct endogenous pararetrovirus family in Nicotiana tomentosiformis, a diploid progenitor of polyploid tobacco CN Hansen , G Harper , JS Heslop-Harrison . Characterisation of pararetrovirus-like sequences in the genome of potato ( Solanum tuberosum ) . Cytogenet Genome Res , 2005 . 110 ( 1-4 ): 559 - 565 . 16093709 DOI: 10.1159/000084989 http://doi.org/10.1159/000084989 . Characterisation of pararetrovirus-like sequences in the genome of potato (Solanum tuberosum) U Hany , IP Adams , R Glover , 等 . The complete genome sequence of Piper yellow mottle virus (PYMoV) . Arch Virol , 2014 . 159 ( 2 ): 385 - 388 . 24005374 DOI: 10.1007/s00705-013-1824-2 http://doi.org/10.1007/s00705-013-1824-2 . The complete genome sequence of Piper yellow mottle virus (PYMoV) G Harper , R Hull , B Lockhart , 等 . Viral sequences integrated into plant genomes . Ann Rev Phytopathol , 2002 . 40 ( 1 ): 119 - 136 . DOI: 10.1146/annurev.phyto.40.120301.105642 http://doi.org/10.1146/annurev.phyto.40.120301.105642 . Viral sequences integrated into plant genomes G Harper , D Hart , S Moult , 等 . Banana streak virus is very diverse in Uganda . Virus Res , 2004 . 100 ( 1 ): 51 - 56 . 15036835 DOI: 10.1016/j.virusres.2003.12.024 http://doi.org/10.1016/j.virusres.2003.12.024 . Banana streak virus is very diverse in Uganda T Hohn , J Fütterer , R Hull . The Proteins and functions of plant pararetroviruses: knowns and unknowns . Crit Rev Plant Sci , 1997 . 16 ( 1 ): 133 - 161 . DOI: 10.1080/713608145 http://doi.org/10.1080/713608145 . The Proteins and functions of plant pararetroviruses: knowns and unknowns R Hull , G Harper , B Lockhart . Viral sequences integrated into plant genomes . Trends Plant Sci , 2000 . 5 ( 9 ): 362 - 365 . 11203277 DOI: 10.1016/S1360-1385(00)01723-4 http://doi.org/10.1016/S1360-1385(00)01723-4 . Viral sequences integrated into plant genomes ML Iskra-Caruana , PO Duroy , M Chabannes , 等 . The common evolutionary history of badnaviruses and banana . Infect Genet Evol , 2014 . 21 83 - 89 . 24184704 DOI: 10.1016/j.meegid.2013.10.013 http://doi.org/10.1016/j.meegid.2013.10.013 . The common evolutionary history of badnaviruses and banana E Jacquot , LS Hagen , M Jacquemond , 等 . The open reading frame 2 product of cacao swollen shoot badnavirus is a nucleic acid-binding protein . Virology , 1996 . 225 ( 1 ): 191 - 195 . 8918546 DOI: 10.1006/viro.1996.0587 http://doi.org/10.1006/viro.1996.0587 . The open reading frame 2 product of cacao swollen shoot badnavirus is a nucleic acid-binding protein SA Kazmi , Z Yang , N Hong , 等 . Characterization by small RNA sequencing of Taro bacilliform CH virus (TaBCHV), a novel badnavirus . PLoS ONE , 2015 . 10 ( 7 ): e0134147 26207896 DOI: 10.1371/journal.pone.0134147 http://doi.org/10.1371/journal.pone.0134147 . Characterization by small RNA sequencing of Taro bacilliform CH virus (TaBCHV), a novel badnavirus AMQ King , MJ Adams , EJ Lefkowitz , 等 . Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses , 2012 . Academic Press, San Diego, CA, USA , . Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses AG Laney , M Hassan , IE Tzanetakis . An integrated badnavirus is prevalent in fig germplasm . Phytopathology , 2012 . 102 ( 12 ): 1182 - 1189 . 22992110 DOI: 10.1094/PHYTO-12-11-0351 http://doi.org/10.1094/PHYTO-12-11-0351 . An integrated badnavirus is prevalent in fig germplasm SL Medberry , BE Lockhart , NE Olszewski . Properties of Commelina yellow mottle virus’s complete DNA sequence, genomic discontinuities and transcript suggest that it is a pararetrovirus . Nucleic Acids Res , 1990 . 18 ( 18 ): 5505 - 5513 . 1699203 DOI: 10.1093/nar/18.18.5505 http://doi.org/10.1093/nar/18.18.5505 . Properties of Commelina yellow mottle virus’s complete DNA sequence, genomic discontinuities and transcript suggest that it is a pararetrovirus G Philippe , I Marie . Phylogeny of Banana streak virus reveals recent and repetitive endogenization in the genome of its banana host ( Musa sp.) . J Mol Evol , 2009 . 69 ( 1 ): 65 - 80 . 19517051 DOI: 10.1007/s00239-009-9253-2 http://doi.org/10.1007/s00239-009-9253-2 . Phylogeny of Banana streak virus reveals recent and repetitive endogenization in the genome of its banana host (Musa sp.) S Seal , E Muller . Molecular analysis of a full-length sequence of a new yam badnavirus from Dioscorea sansibarensis . Arch Virol , 2007 . 152 ( 4 ): 819 - 825 . 17195956 DOI: 10.1007/s00705-006-0888-7 http://doi.org/10.1007/s00705-006-0888-7 . Molecular analysis of a full-length sequence of a new yam badnavirus from Dioscorea sansibarensis S Seal , A Turaki , E Muller , 等 . The prevalence of badnaviruses in West African yams ( Dioscorea cayenensis-rotundata ) and evidence of endogenous pararetrovirus sequences in their genomes . Virus Res , 2014 . 186 144 - 154 . 24457074 DOI: 10.1016/j.virusres.2014.01.007 http://doi.org/10.1016/j.virusres.2014.01.007 . The prevalence of badnaviruses in West African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous pararetrovirus sequences in their genomes C Staginnus , KR Richert-Pöggeler . Endogenous pararetroviruses: two-faced travelers in the plant genome . Trends Plant Sci , 2006 . 11 ( 10 ): 485 - 491 . 16949329 DOI: 10.1016/j.tplants.2006.08.008 http://doi.org/10.1016/j.tplants.2006.08.008 . Endogenous pararetroviruses: two-faced travelers in the plant genome L Su , S Gao , Y Huang , 等 . Complete genomic sequence of Dracaena mottle virus, a distinct badnavirus . Virus Genes , 2007 . 35 ( 2 ): 423 - 429 . 17497213 DOI: 10.1007/s11262-007-0102-3 http://doi.org/10.1007/s11262-007-0102-3 . Complete genomic sequence of Dracaena mottle virus, a distinct badnavirus K Tamura , D Peterson , N Peterson , 等 . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Mol Biol Evol , 2011 . 28 ( 10 ): 2731 - 2739 . 21546353 DOI: 10.1093/molbev/msr121 http://doi.org/10.1093/molbev/msr121 . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods I Tzafrir , L Ayala-Navarrete , BEL Lockhart , 等 . The N-terminal portion of the 216-kDa polyprotein of Commelina yellow mottle badnavirus is required for virus movement but not for replication . Virology , 1997 . 232 ( 2 ): 359 - 368 . 9191850 DOI: 10.1006/viro.1997.8569 http://doi.org/10.1006/viro.1997.8569 . The N-terminal portion of the 216-kDa polyprotein of Commelina yellow mottle badnavirus is required for virus movement but not for replication M Umber , D Filloux , E Muller , 等 . The genome of African yam ( Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct badnavirus species . Mol Plant Pathol , 2014 . 15 ( 8 ): 790 - 801 . 24605894 DOI: 10.1111/mpp.12137 http://doi.org/10.1111/mpp.12137 . The genome of African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct badnavirus species Y Wang , X Cheng , X Wu , 等 . Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China . Virus Res , 2014 . 188 103 - 108 . 24751798 DOI: 10.1016/j.virusres.2014.04.006 http://doi.org/10.1016/j.virusres.2014.04.006 . Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China H Wu , Y Zhang , W Zhang , 等 . Transcriptomic analysis of the primary roots of Alhagi sparsifolia in response to water stress . PLoS ONE , 2015 . 10 ( 3 ): e0120791 25822368 DOI: 10.1371/journal.pone.0120791 http://doi.org/10.1371/journal.pone.0120791 . Transcriptomic analysis of the primary roots of Alhagi sparsifolia in response to water stress D Xu , R Mock , G Kinard , 等 . Molecular analysis of the complete genomic sequences of four isolates of Gooseberry vein banding associated virus . Virus Genes , 2011 . 43 ( 1 ): 130 - 137 . 21533750 DOI: 10.1007/s11262-011-0614-8 http://doi.org/10.1007/s11262-011-0614-8 . Molecular analysis of the complete genomic sequences of four isolates of Gooseberry vein banding associated virus IC Yang , GJ Hafner , JL Dale , 等 . Genomic characterisation of Taro bacilliform virus . Arch Virol , 2003 . 148 ( 5 ): 937 - 949 . 12721801 DOI: 10.1007/s00705-002-0969-1 http://doi.org/10.1007/s00705-002-0969-1 . Genomic characterisation of Taro bacilliform virus Z Yang , M Nicolaisen , NE Olszewski , 等 . Sequencing, improved detection, and a novel form of Kalanchoë top-spotting virus . Plant Dis , 2005 . 89 ( 3 ): 298 - 302 . DOI: 10.1094/PD-89-0298 http://doi.org/10.1094/PD-89-0298 . Sequencing, improved detection, and a novel form of Kalanchoë top-spotting virus We thank Dr. Wang-bin ZHANG (College of Plant Science, Tarim University, the Xinjiang Uygur Autonomous Region, China) and Dr. Xin-wu PEI (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China) for helping collect the seeds of A . sparsifolia , and Dr. Hong-yan SHAN (Institute of Botany, Chinese Academy of Sciences, Beijing, China) for her helpful suggestion to construct the phylogenetic trees. Compliance with ethics guidelines Yong-chao LI, Jian-guo SHEN, Guo-huan ZHAO, Qin YAO, and Wei-min LI declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1700171 Project supported by the National Natural Science Foundation of China (Nos. 31370181 and 31570146) and the Fujian Natural Science Funds for Distinguished Young Scholar (No. 2014J06008), China 2017-03-28 2017-05-11 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 19