A DNA barcode based on 648 bp of cytochrome c oxidase I (COI) gene aims to build species-specific libraries for animal groups. However, it is hard to recover full-length (648 bp) barcode gene from environmental fecal samples due to DNA degradation. In this study, we designed a new primer set (K_Bird...
A DNA barcode based on 648 bp of cytochrome c oxidase I (COI) gene aims to build species-specific libraries for animal groups. However, it is hard to recover full-length (648 bp) barcode gene from environmental fecal samples due to DNA degradation. In this study, we designed a new primer set (K_Bird), which amplifies a 226 bp fragment targeted an inner position of full-length COI barcode based on 102 species of Korean birds to improve amplification success, and we attempted to identify bird species from 39 avian fecal samples collected during 4 months from Jinan, South Korea. Simultaneously, we conducted a dietary analysis using a universal DNA mini-barcode (Uni_Minibar) from same fecal samples. In silico analysis on newly designed mini-barcode represented that genetic distances were 0.5% in species and 9.1% in genera. Intraspecific variations of 149 species out of 174 species (86%) between Korea and North America were within the threshold (5.3% threshold in this study). From environmental fecal samples collected in Jinan, we identified seven avian species, which have high similarity (99-100%) with registered COI sequences in GenBank. Eight kinds of prey species, such as moth, spider, fly, and dragonfly, were identified in dietary analysis. We suppose that our strategy applying mini-barcode for environmental fecal samples, might be a useful and convenient tool for species identification and dietary analysis for birds.
A DNA barcode based on 648 bp of cytochrome c oxidase I (COI) gene aims to build species-specific libraries for animal groups. However, it is hard to recover full-length (648 bp) barcode gene from environmental fecal samples due to DNA degradation. In this study, we designed a new primer set (K_Bird), which amplifies a 226 bp fragment targeted an inner position of full-length COI barcode based on 102 species of Korean birds to improve amplification success, and we attempted to identify bird species from 39 avian fecal samples collected during 4 months from Jinan, South Korea. Simultaneously, we conducted a dietary analysis using a universal DNA mini-barcode (Uni_Minibar) from same fecal samples. In silico analysis on newly designed mini-barcode represented that genetic distances were 0.5% in species and 9.1% in genera. Intraspecific variations of 149 species out of 174 species (86%) between Korea and North America were within the threshold (5.3% threshold in this study). From environmental fecal samples collected in Jinan, we identified seven avian species, which have high similarity (99-100%) with registered COI sequences in GenBank. Eight kinds of prey species, such as moth, spider, fly, and dragonfly, were identified in dietary analysis. We suppose that our strategy applying mini-barcode for environmental fecal samples, might be a useful and convenient tool for species identification and dietary analysis for birds.
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제안 방법
In this reason, it is essential to construct local DNA barcode database related to target dietary species such as insect, spider, and so on. Construction of local database can be helpful to identify each species exactly and increase the resolutions for dietary analysis. In addition, after determining the groups at the order level using universal primer set, the use of group-specific primer for accurate identification of dietary species can be compensated low resolution problem
However, it is not always feasible to follow their suggestion in the field. In this study, to improve DNA amplification rate, we collected samples on fallen leaves or rocks under trees, which were known as resting and excreting place of birds. In addition, all of samples were collected in early morning to reduce effect of UV and weather condition.
org). In this study, we added IUPAC code for polymorphic nucleic acids inside forward primer to amplify COI sequences of the Korean birds for greater efficiency. However, we should consider the possibility that some of bird species can not be amplified by our mini-barcode primer set.
In this study, we devised avian-specific mini-barcode (K_Bird), which is effective for bird identification, and applied universal mini-barcode (Uni-Minibar) designed by Meusnier et al. (2008) for dietary analysis from fecal samples. And, we tested the performances of two mini-barcode from unknown fecal samples collected from maeulsoop, simultaneously.
We analyzed 39 unidentified avian fecal samples from village groves and forests of the Korean agricultural landscape located in Jinan during experimental periods using two different primer set both K_Bird_F1 & R1 for bird identification and Uni_Minibar F1 & R1 for prey detection (Table 2).
대상 데이터
Mai in Jinan, in the middle of South Korea (E 127°825´49´´, N 35°47´30´´). Avian fecal samples were collected at the selected study sites bimonthly from May to September 2011. We collected primarily fecal samples on leaves and rocks to prevent contamination from soil materials.
데이터처리
minibarcode by calculating P for each mini-barcode in comparison to the full-length barcode. Paired t-test was conducted using R software packages (stats-package), version 2.15 (R Core Team 2012) and P values were calculated in paired t-test. Genetic distances were calculated by using MEGA5 software (Tamura et al.
이론/모형
org) (Ratnasingham and Hebert 2007). Sequence divergences were calculated using the Kimura-2-parameter (K2P) model (Kimura 1980). Genetic distance was represented with each taxonomic level based on sequence divergences results.
성능/효과
Seven avian species, Cyanopica cyanus, Garrulus glandarius, Passer montanus, Pica pica, Oriolus chinensis, Sturnus cineraceus, and Streptopelia orientalis, were identified from amplified 24 fecal samples. All of identified seven avian species showed high similarity (99-100%) with registered COI sequences in GenBank from the BLASTN results. Two avian species of Corvidae, P.
Despite these benefits, however, some of taxonomy groups (Lepidoptera, in case of our results) could not be resolved in species level. For example, some unidentified COI sequences were matched with nine different species, Acronicta mansueta, A. superans, A. quadrata, A. innotata, A. rumicis, Agriopodes fallax, Concana mundissima DHJ01, Capis curvata, and Pharetra rumicis, which had same similarity values (100%) in BOLD-IDS. In addition, the identification results both BLASTN and BOLD-IDS represented just order level in case of Diptera, Hemiptera, and so on.
Regarding success rates of amplification, amplicon size of our mini-barcode (226 bp) is appropriate for successful amplification from avian fecal samples. Our results showed that PCR success rates using two primer pair, K_Bird and Uni_Minibar, were about 6065%. We assumed that 6065% of success were not low rates considering the negative effect of environmental factors such as UV and rain.
More than one-third of fecal samples (16 specimen) were identified for both birds and prey species. Seven avian species, Cyanopica cyanus, Garrulus glandarius, Passer montanus, Pica pica, Oriolus chinensis, Sturnus cineraceus, and Streptopelia orientalis, were identified from amplified 24 fecal samples. All of identified seven avian species showed high similarity (99-100%) with registered COI sequences in GenBank from the BLASTN results.
In dietary analysis, most of COI sequences amplified from avian fecal samples belonged to one of four Order (Araneae, Diptera, Hemiptera, and Megaloptera). Three species, Acronicta rumicis, Polygonia caureum, and Stomoxys calcitrans, had 100% similarity from BLASTN. Seven out of 25 amplified fecal samples showed description as birds or fungi.
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