Sequence Analysis Of Coat Protein Gene Research Articles

Genotypic Analysis for Retrieval of MYMV Resistant Progenies from Certain Crosses of Mungbean [Vigna radiata (L.) Wilczek

The present investigation was conducted to study the nature of inheritance of MYMV among intra-specific crosses among three MYMV susceptible and five MYMV resistant genotypes of green gram. The molecular screening and hotspot evaluation is carried out in fifteen F1 progenies and 8 F2 families, respectively. The yellow mosaic-affected leaf samples gave positive results with MYMIV specific primer pairs YFP1/YRP1 and YFP2/YRP2 amplified fragments having size of 704bp and 648 bp, respectively. Coat protein gene sequence analysis revealed that the identified virus is homologous to MYMV. The inheritance study based on F2 data indicated the ratio of MYMV resistant plants and susceptible plants showed a good fit to expected 3:1 (Susceptible: Resistant) suggesting a typical monogenic recessive gene governing resistant reaction to MYMV besides resting the susceptibility from the susceptible parents.

First Report of Barley Yellow Dwarf Virus-PAV Infecting Oats (Avena sativa) in Taiwan

Barley yellow dwarf virus is a group of positive-sense single stranded RNA viruses with many species belonging to the family Luteoviridae and known to cause significant yield losses of cereal crops worldwide (Irwin and Thresh 1990). Oat (Avena sativa L.), a temperate cereal crop usually grown as forage in subtropical regions, has been recently reintroduced into Taiwan as a local winter forage source. In April 2017, yellowing, V-shape reddening, and purple symptoms were observed on 7.9% of the mature leaves of the oat plants grown in the experimental field of National Taiwan University (NTU) in Taipei city, Taiwan. We collected 37 leaf samples with suspected virus-infection symptoms in the experimental field for virus screening. The samples were expressing varying degrees of yellowing, browning, or typical barley yellow dwarf virus (BYDV)-associated V-shape reddening and purple symptoms. An additional five samples with no symptoms were also collected. These 37 samples belong to 22 oat lines, including two registered cultivars and 20 unreleased breeding lines. Some lines were replicated in the field and therefore were sampled more than once. A specific reverse transcription polymerase chain reaction assay (RT-PCR) for BYDV, covering the coat protein (CP) gene and partial RNA-dependent RNA polymerase regions, was performed with the primers Shu-F (5′-TACGGTAAGTGCCCAACTCC-3′) and Yan-R (5′-TGTTGAGGAGTCTACCTATTTG-3′) on the extracted total RNA of the samples (Malmstrom and Shu 2004). Among the examined samples, only the three expressing typical BYDV symptoms tested positive, whereas the other 34 samples with solely yellowing or browning symptoms and the five symptomless samples tested negative. The samples belonged to the cultivar Swan and two breeding lines, 15-39 and 16-47; the seeds of the three symptomatic oats were all imported from the United States. We further cross-examined the three positive samples with another RT-PCR assay, using specific primer pairs targeting the BYDV CP gene (for strains PAV, MAV, and GAV): Luteo1F (5′-TTCGGMSARTGGTTGTGGTCCA-3′) and YanR-new (5′-TGTTGAGGAGTCTACCTATTTNG-3′) (Mustafayev et al. 2013; Svanella-Dumas et al. 2013). The electrophoresis analysis of the RT-PCR amplicons revealed the expected 545-bp fragment from all. The amplicons were later cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) for sequencing analysis. All of the sequences obtained were identical (GenBank accession nos. MF618251, MK361042, and MK361043) and showed 99% similarity to the BYDV-PAV isolates from the United States (accession nos. EF521840 and DQ115532) in GenBank. Based on the phylogenetic analysis of CP gene sequences, the BYDV-PAV Taiwan isolates and these two U.S. isolates were in the same cluster. To further confirm the results, the BYDV-PAV specific enzyme-linked immunosorbent assay on the three symptomatic samples, using the commercialized kit (Agdia, Elkhart, IN), also aligned with the prior assay and showed positive reaction results. To our knowledge, this is the first report of BYDV-PAV in Taiwan. The phylogeny analysis of the virus suggests the BYDV-PAV might have been introduced through the seeds, which had gone through one cycle of multiplication at the border between Idaho and Oregon states prior to growing in Taiwan. Because the BYDV infection on oat presented a threat to cereal crop production in Taiwan, all oat plants in the NTU experimental field were eliminated immediately to prevent the spread of the disease. Agricultural agencies were informed of the incidence, and oat growers were educated to identify the BYDV symptoms on oat. Further field surveys of BYDV on the alleged vector insects will be carried to better understand the disease biology in Taiwan and to provide information on the disease system in the Southeast Asia region.

Analisis Gen Selubung Protein Chilli Veinal Mottle Potyvirus dari Beberapa Daerah di Indonesia

<p>Variation on symptoms and virulence was<br />observed on different isolates of ChiVMV collected from<br />West Java, Central Java, East Java, South Kalimantan, West<br />Sumatera and Central Aceh. Research was conducted to<br />study genetic variation of six ChiVMV isolates based on<br />sequence analysis of coat protein (CP) gene and amino<br />acid. Sequence analysis of CP gene showed 87% to 99%<br />homology among the six isolates with level of variation<br />ranging from 0.02% to 1.48%. Sequence analysis of amino<br />acid derived from CP gene showed 85% to 99% homology.<br />Further analysis on amino acid motives of CP gene indicated<br />mutation of octapeptide motif, i.e<br />LSGQVQPQSRQSEMETEVPQVR on ChiVMV CKB and<br />RMETFGLDGRVGTQEEDTERHT on other ChiVMV isolates.<br />Other differences was observed on amino acid number 61<br />and 84 i.e. deletion of MET and mutation of GG to KV on<br />ChiVMV BL and KR. Phyllogenetic analysis based on<br />nucleotide and amino acid sequence showed that six<br />isolates of ChiVMV can be differentiated into three groups.<br />ChiVMV KR and BL were in the same group with ChiVMV<br />Pataruman (GeneBank No. access DQ854961), ChiVMV CKB<br />was in the same group with ChiVMV Cikabayan 2<br />(GeneBank No. access DQ854960), and ChiVMV TD, ChiVMV<br />NI and GB ChiVMV were in the same group with ChiVMV<br />Taiwan (GeneBank No. access DQ854948). Analysis of CP<br />gene confirmed the occurrence of genetic variation among<br />ChiVMV isolates although symptom variation is weak.</p>

Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

Wheat streak mosaic virus detected in winter wheat in Lithuania

Wheat streak mosaic virus (WSMV) causing viral diseases in graminaceous plants worldwide has been isolated in Lithuania from natural infected winter wheat (Triticum aestivum L.) plants. The diseased wheat plants with pronounced symptoms (chlorotic mottling, streaks of leaves and stunting) were found in various localities of Vilnius and Kaunas regions. Virus isolates were investigated by serology, electron microscopy (EM) and reverse transcription-polymerase chain reaction (RT-PCR) methods. The identification of the virus was based on the results of symptomology on host-plants, positive DAS-ELISA (double-antibody sandwich enzyme-linked immunosorbent assay) test, particle morphology (presence of flexuous, filamentous virus particles ~15 nm in diameter and 700 nm in length) and a specific amplification fragment size (750 bp) of virus RNA (ribonucleic acid) in RT-PCR. Isolate (WSMV-1313) of WSMV from wheat plants was characterized by a coat protein gene sequence analysis. Comparison with other WSMV isolates from the GenBank database showed that the Lithuanian WSMV isolate shares the highest sequence similarity to WSMV isolates from Russia (98.3%) and Italy (98.1%). This is the first report of wheat as a natural host for WSMV in Lithuania.

Phylogenetic analysis of American hop latent virus in Humulus lupulus surveyed in Tasmania and Victoria, Australia

During July to August 2013 hop (Humulus lupulus) buds growing in Tasmania (n = 280) and Victoria (n = 200) from nine varieties were sampled and tested for the presence of American hop latent virus (AHLV) by enzyme linked immunosorbent assay and reverse transcription polymerase chain reaction using coat protein gene specific primers. Immunological results showed 153/480 bud samples were positive. RT-PCR resulted in an additional 19 positive samples. Coat protein gene sequence analysis showed 98–99 % identity with AHLV isolates from the USA.

First Report of Bidens mottle virus Infecting Calendula in Taiwan

In March of 2010, calendula (Calendula officinalis L.), a perennial herb known as the pot marigold, showing chlorotic spots on leaves, chlorosis, and stunting were collected from Puli Township, Nantou County, Taiwan. The disorder occurred in more than 50% of the calendula plants in the field. A virus culture isolated from one of the symptomatic calendulas was established in Chenopodium quinoa through triple single-lesion isolation and designated as TwCa1. With transmission electron microscopy (TEM), negatively stained flexuous filamentous virions approximately 12 × 720 nm were observed in the crude sap of TwCa1-infected C. quinoa leaves and pinwheel inclusion bodies were found in the infected cells. On the basis of the sizes of the viral particles and inclusion bodies, isolate TwCa1 was a suspected potyvirus. By reverse transcription (RT)-PCR and potyvirus degenerate primers (Hrp5/Pot1) (1,2), a 0.65-kb DNA fragment, which included the 3'-end of the NIb gene and the 5'-end of coat protein (CP) gene of the virus, was amplified from total RNA isolated from TwCa1-infected plants. The amplified DNA fragment was cloned and sequenced. A homology search indicated that the new calendula-infecting virus in Taiwan might belong to Bidens mottle virus (BiMoV) because its partial genomic sequence shared 94.9 to 97.3% nucleotide and 96.6 to 98.1% amino acid identity with 11 BiMoV isolates available in NCBI GenBank. Primer pairs Hrp5/oligo d(T) were used to amplify the 3'-end genome of BioMV TwCa1 including the 3'-end of the NIb gene, the full-length CP gene, and the 3'-nontranslatable region of the virus. The 807-nt CP gene of TwCa1 (Accession No. HQ117871) shared 97.3 to 98.6% nucleotide and 98.5 to 98.9% amino acid identity with those of 11 BiMoV isolates available in GenBank. Results from TEM observations and CP gene sequence analysis indicated that TwCa1 is an isolate of BiMoV. BiMoV was later detected by RT-PCR in eight symptomatic calendulas collected from the same field. To our knowledge, this is the first report of BiMoV infecting calendula in Taiwan. This newly identified calendula-infecting BiMoV could have a direct impact on the economically important vegetable and floral industry in Taiwan.

Coat Protein Gene Sequence Analysis of Potato virus X and Potato virus Y: Conserved Regions to Design Gene Silencing Cassette

Potato virus X(PVX) and Potato virus Y(PVY) are two of the three most prevalent viruses that cause significant yield declines in potato. Twenty-seven PVX and thirty-seven PVY accessions were analyzed for nucleotide sequence variation of the coat protein gene. The average and variance of genetic distance for PVX were estimated at 0.118 and 0.004 and 0.118 and 0.005 for PVY using the neighbour joining method. Results of phylogenetic trees and their certification via stepwise discriminant analysis led us to classify of PVX sequences in four groups and PVY sequences in three groups. One purpose of this project was to determine suitable conserved regions to make of gene silencing constructs. Length of identified conserved regions were enough to silence of the virus coat protein genes on infected plants, many of which were located consequently with short gap spacers. In this term, some of groups were divided into subgroups to obtain conserved regions under minimum length of25 nt, enough length to design specific diagnostic-primers.

Identification ofTomato aspermy virusas the cause of yellow mosaic and flower deformation of chrysanthemums in India

Using indirect ELISA, RT-PCR and coat protein gene sequence analysis, Tomato aspermy virus is identified as the cause of yellow mosaic and flower deformation of Chrysanthemum morifolium cultivars in India.