Cooled propylene glycol as a pragmatic choice for preservation of DNA from remote field-collected Diptera for next-generation sequence analysis

Next-generation sequencing (NGS)-based methods can now be applied to large population-scale studies, but this demands very high-quality DNA. For specimens collected from remote field locations, DNA degradation can be a problem, requiring logistically challenging preservation techniques. Simpler preservation techniques are therefore required. Prior to collection of exotic fruit fly (Tephritidae) species, a number of readily available preservatives with storage at either 4°C or room temperature were trialed here to determine the DNA quality for three locally available Diptera species,Fannia canicularis(L.),Musca domesticaL., and Lucilia sericataMeigen. Considerable variation was observed between the different preservatives, species, and temperatures, but several preservatives at 4°C were favored. Chilled propylene glycol was subsequently used for the storage and carriage of Australian field-collectedBactrocera fruit fly specimens to New Zealand. When processed up to 20 d later, DNA fragments of ∼10-20 kb were obtained for successful genotyping by sequencing analysis. This protocol is therefore recommended as a logistically simple and safe approach for distant collection of dipteran samples for NGS population genomic studies

Cooled propylene glycol as a pragmatic choice for preservation of DNA from remote field-collected Diptera for next-generation sequence analysis

Next-generation sequencing (NGS)-based methods can now be applied to large population-scale studies, but this demands very high-quality DNA. For specimens collected from remote field locations, DNA degradation can be a problem, requiring logistically challenging preservation techniques. Simpler preservation techniques are therefore required. Prior to collection of exotic fruit fly (Tephritidae) species, a number of readily available preservatives with storage at either 4°C or room temperature were trialed here to determine the DNA quality for three locally available Diptera species,Fannia canicularis(L.),Musca domesticaL., and Lucilia sericataMeigen. Considerable variation was observed between the different preservatives, species, and temperatures, but several preservatives at 4°C were favored. Chilled propylene glycol was subsequently used for the storage and carriage of Australian field-collectedBactrocera fruit fly specimens to New Zealand. When processed up to 20 d later, DNA fragments of ∼10-20 kb were obtained for successful genotyping by sequencing analysis. This protocol is therefore recommended as a logistically simple and safe approach for distant collection of dipteran samples for NGS population genomic studies

Colour break in reverse bicolour daffodils is associated with the presence of Narcissus mosaic virus

<p>Abstract</p> <p>Background</p> <p>Daffodils (<it>Narcissus pseudonarcissus</it>) are one of the world's most popular ornamentals. They also provide a scientific model for studying the carotenoid pigments responsible for their yellow and orange flower colours. In reverse bicolour daffodils, the yellow flower trumpet fades to white with age. The flowers of this type of daffodil are particularly prone to colour break whereby, upon opening, the yellow colour of the perianth is observed to be 'broken' into patches of white. This colour break symptom is characteristic of potyviral infections in other ornamentals such as tulips whose colour break is due to alterations in the presence of anthocyanins. However, reverse bicolour flowers displaying colour break show no other virus-like symptoms such as leaf mottling or plant stunting, leading some to argue that the carotenoid-based colour breaking in reverse bicolour flowers may not be caused by virus infection.</p> <p>Results</p> <p>Although potyviruses have been reported to cause colour break in other flower species, enzyme-linked-immunoassays with an antibody specific to the potyviral family showed that potyviruses were not responsible for the occurrence of colour break in reverse bicolour daffodils. Colour break in this type of daffodil was clearly associated with the presence of large quantities of rod-shaped viral particles of lengths 502-580 nm in tepals. Sap from flowers displaying colour break caused red necrotic lesions on <it>Gomphrena globosa</it>, suggesting the presence of potexvirus. Red necrotic lesions were not observed in this indicator plant when sap from reverse bicolour flowers not showing colour break was used. The reverse transcriptase polymerase reactions using degenerate primers to carla-, potex- and poty-viruses linked viral RNA with colour break and sequencing of the amplified products indicated that the potexvirus <it>Narcissisus mosaic virus </it>was the predominant virus associated with the occurrence of the colour break.</p> <p>Conclusions</p> <p>High viral counts were associated with the reverse bicolour daffodil flowers that were displaying colour break but otherwise showed no other symptoms of infection. <it>Narcissus mosaic virus </it>was the virus that was clearly linked to the carotenoid-based colour break.</p

Cooled Propylene Glycol as a Pragmatic Choice for Preservation of DNA From Remote Field-Collected Diptera for Next-Generation Sequence Analysis

Next-generation sequencing (NGS)-based methods can now be applied to large population-scale studies, but this demands very high-quality DNA. For specimens collected from remote field locations, DNA degradation can be a problem, requiring logistically challenging preservation techniques. Simpler preservation techniques are therefore required. Prior to collection of exotic fruit fly (Tephritidae) species, a number of readily available preservatives with storage at either 4°C or room temperature were trialed here to determine the DNA quality for three locally available Diptera species,Fannia canicularis(L.),Musca domesticaL., and Lucilia sericataMeigen. Considerable variation was observed between the different preservatives, species, and temperatures, but several preservatives at 4°C were favored. Chilled propylene glycol was subsequently used for the storage and carriage of Australian field-collectedBactrocera fruit fly specimens to New Zealand. When processed up to 20 d later, DNA fragments of ∼10-20 kb were obtained for successful genotyping by sequencing analysis. This protocol is therefore recommended as a logistically simple and safe approach for distant collection of dipteran samples for NGS population genomic studies

A generic RT-PCR assay for the detection of Luteoviridae

This study, using RT-PCR, is the first comprehensive assessment since 1991 of a generic detection method for the Luteoviridae. Thirteen Luteoviridae species were detected using three separate sets of low-degeneracy generic primers with RT-PCR to amplify 68-, 75- and 129⁄156-bp regions of the Luteoviridae coat-protein gene. Species detected include all members of the genus Luteovirus [Barley yellow dwarf virus (BYDV)-PAV, BYDV-PAS, BYDV-MAV (129 and⁄or 156 bp amplicons), Soybean dwarf virus, Bean leafroll virus (68 bpamplicon)] and eight of nine species from the genus Polerovirus [Beet western yellows virus, Beet chlorosis virus, Beet mild yellowing virus, Turnip yellows virus, Potato leafroll virus, Cucurbit aphid-borne yellows virus, Cereal yellow dwarf virus-RPV (68-bp amplicon) and Sugarcane yellow leaf virus (75-bp amplicon)]. These primers were not able to detect Carrot red leaf virus, Sweet potato leaf speckling virus (both belong to unassigned Luteoviridae) and Pea enation mosaic virus-1 (genus Enamovirus). A synthetic positive control containing all primer sequence priming sites was designed to facilitate this method as a generic tool for use with a variety of host plants. The Luteoviridae primers described in this study present a simple infection-detection tool of benefit to biosecurity authorities in nursery-stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as-yet undiscovered species within the Luteovirus and Polerovirus genera