Development of microsatellite markers for two endangered grassland butterflies, Melitaea ambigua and M. protomedia (Nymphalidae), using Ion Torrent next-generation sequencing

2020

Ecological Research

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Museum specimens include genetic information from when they were collected. This historical information, which is very difficult to ascertain from samples collected currently, could be a valuable material for use in conservation genetics. However, the genetic analysis of museum specimens is technically difficult because of DNA fragmentation and the deamination of cytosine to uracil. In recent years, various methods have been developed for the genetic analysis of museum specimens, such as data analysis techniques including next-generation sequencing. The development of approaches that extract historical genetic information from museum specimens is expected to provide a new perspective on conservation genetics. This review focuses on the availability of museum specimens as genetic resources for conservation genetics. Some case studies are introduced, and perspectives on the future utility of conservation genetic studies using museum specimens are discussed. Moreover, recommended genetic analysis methods and important points for the usage of museum specimens are presented. This review provides a strong case for increasing the usage of museum specimens in conservation genetics studies in the future.

Section: Genetic Analysis Methods Using Museum Specimensmentioning

confidence: 99%

Museum specimens: An overlooked and valuable material for conservation genetics

2020

Ecological Research

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“…The genotypes of archival and contemporary samples were determined at eight microsatellite loci characterised by Nakahama et al . (): Mpr002 , Mpr006 , Mpr007 , Mpr015 , Mpr019 , Mpr021 , Mpr024 and Mpr026 . PCR amplifications were performed in 5 μl total volume, which included 1 ng template DNA, 2.5 μl of 2× Multiplex PCR Master Mix (QIAGEN), 0.01 μM forward primer with the tag‐sequence (5′‐CACGACGTTGTAAAACGAC‐3′, 5′‐CTATAGGGCACGCGTGGT‐3′ and 5′‐TGTGGAATTGTGAGCGG‐3′; Boutin‐Ganache et al ., ), 0.2 μM reverse primer and 0.1 μM M13 (fluorescently labelled) primer.…”

Section: Methodsmentioning

confidence: 99%

Recent transitions in genetic diversity and structure in the endangered semi‐natural grassland butterfly, Melitaea protomedia, in Japan

Insect Conserv Diversity

Isagi

2017

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The monitoring of genetic characters and definition of effective conservation units are important for conservation of endangered semi‐natural grassland species. Using molecular markers, we elucidated the recent transitions in genetic diversity for the endangered grassland butterfly Melitaea protomedia (Lepidoptera; Nymphalidae) in Japan. First, we examined changes in genetic diversity and structure from the 1980s to the 2010s from archival and contemporary DNA samples using eight microsatellite markers. Second, we estimated the genetic structure based on 1374 bp of mitochondrial COI gene from contemporary samples. We also defined the conservation units of M. protomedia based on the above analysis. The genetic diversity of M. protomedia has significantly declined from the 1980s to the 2010s. Although genetic differentiation was very strong among populations in c. 2010, there was only weak genetic structure in c. 1990. In addition, the number of haplotypes based on mitochondrial DNA is now very low due to recent declines. These findings suggest that effective conservation units for critically endangered species should be determined based on historic (i.e. prior to population declines) as well as contemporary genetic diversity and differentiation, because genetic structure may have changed over time. Genetic analysis of archival DNA is useful to obtain historic genetic information.

“…The genotypes of specimen samples were clarified at nine microsatellite loci characterized by Nakahama et al. (): Mam011 , Mam013 , Mam016 , Mam017 , Mam020 , Mam023 , Mam026 , Mam028 and Mam031 . Polymerase chain reaction (PCR) amplifications were performed in 5 μL total volume, which included 1 ng template DNA, 2.5 μL of 2× Multiplex PCR Master Mix (Qiagen Japan, Tokyo, Japan), 0.01 μM forward primer with the M13 sequence (5′‐CACGACGTTGTAAAACGAC‐3′, 5′‐CTATAGGGCACGCGTGGT‐3′ and 5′‐TGTGGAATTGTGAGCGG‐3′; Boutin–Ganache et al.…”

Section: Numbers Of Melitaea Ambigua Butterfly Specimens Per Period Fmentioning

confidence: 99%

“…The fluorescence intensity ratio of the DNA > 150 bp to the entire DNA was calculated for collections made in the 1960s-1970s, 1980s-1990s and 2000s-2010s. The genotypes of specimen samples were clarified at nine microsatellite loci characterized by Nakahama et al (2015): Mam011, Mam013, Mam016, Mam017, Mam020, Mam023, Mam026, Mam028 and Mam031.…”

mentioning

confidence: 99%

Availability of short microsatellite markers from butterfly museums and private specimens

Isagi

2017

Entomological Science

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Knowledge of the temporal changes in genetic diversity and structure is important for identifying factors causing a decline in threatened insect species, and for establishing conservation programs for these species. Thus, there is recently an increasing interest in the restoration of genetic diversity in conservation programs using DNA data from historical museum specimens. For butterfly specimens, we measured the yields and fragment sizes of the extracted DNA and investigated the genotyping success probability of nine short microsatellite markers (allele size 73–191 bp). We used leg samples of specimens of a medium‐sized butterfly species, Melitaea ambigua (Lepidoptera; Nymphalidae), collected from the 1960s to the 2010s. The yields of specimen‐extracted DNA longer than 150 bp decreased with increasing specimen age. There were negative correlations between the genotyping success probability and specimen age for each of all microsatellite markers. A negative correlation was also observed between the genotyping success probability and allele size of each microsatellite marker. We conclude that short microsatellite markers and analysis of recently obtained specimens are particularly suitable for microsatellite analysis of butterfly specimens.