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Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

Perera OP, Allen KC, Jain D, Purcell M, Little NS, Luttrell RG - J. Insect Sci. (2015)

Bottom Line: Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species.In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified.Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS Southern Insect Management Research Unit, Stoneville, MS 38776 op.perera@ars.usda.gov.

No MeSH data available.


Related in: MedlinePlus

Alignments of nucleotide sequences. (A) Alignment of nucleotide sequences of the ITS1 region used for species-specific primer design. H. armigera, H. assulta, Helio. subflexa, and Helio. virescens ITS1 nucleotide sequences were aligned with consensus H. zea ITS 1 nucleotide sequences. Identical nucleotides are given in plain text, and mismatched nucleotides are shown in white text with black background. Alignment gaps are indicated by a hyphen (-). Common forward primer sequence for both species is underlined. Reverse primer sequences specific to H. armigera and H. zea are marked by dashed-line and solid line boxes, respectively. (B) Alignment of 18S rRNA subunit region used for developing control amplicon. Primer binding sequences for forward and reverse primers are marked by forward and reverse arrows, respectively. ITS1 sequence entries are as follows; H. armigera: KT343376.1 (F1 hybrid), KT343377.1 (China), and KT343378.1 (Australia); ITS1 sequences of H. zea: KT343375.1 (F1 hybrid), KT343380.1 (laboratory colony), KT343381.1 (Mississippi); Helio. virescens: KT343379.1 (laboratory colony); H. assulta: KT343382.1 (Thailand); Helio. subflexa: KT62150.1 (laboratory colony); the ITS1 sequences AB620127.1, AF401740.1, and AJ577253.1 of H. armigera and the 18S rRNA subunit sequences Papilio xuthus (L) (AB674749.1) and H. assulta (EU051777.1), respectively, were obtained from GenBank.
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iev137-F1: Alignments of nucleotide sequences. (A) Alignment of nucleotide sequences of the ITS1 region used for species-specific primer design. H. armigera, H. assulta, Helio. subflexa, and Helio. virescens ITS1 nucleotide sequences were aligned with consensus H. zea ITS 1 nucleotide sequences. Identical nucleotides are given in plain text, and mismatched nucleotides are shown in white text with black background. Alignment gaps are indicated by a hyphen (-). Common forward primer sequence for both species is underlined. Reverse primer sequences specific to H. armigera and H. zea are marked by dashed-line and solid line boxes, respectively. (B) Alignment of 18S rRNA subunit region used for developing control amplicon. Primer binding sequences for forward and reverse primers are marked by forward and reverse arrows, respectively. ITS1 sequence entries are as follows; H. armigera: KT343376.1 (F1 hybrid), KT343377.1 (China), and KT343378.1 (Australia); ITS1 sequences of H. zea: KT343375.1 (F1 hybrid), KT343380.1 (laboratory colony), KT343381.1 (Mississippi); Helio. virescens: KT343379.1 (laboratory colony); H. assulta: KT343382.1 (Thailand); Helio. subflexa: KT62150.1 (laboratory colony); the ITS1 sequences AB620127.1, AF401740.1, and AJ577253.1 of H. armigera and the 18S rRNA subunit sequences Papilio xuthus (L) (AB674749.1) and H. assulta (EU051777.1), respectively, were obtained from GenBank.

Mentions: Complete 18S rRNA and ITS1 sequences of H. armigera, H. zea, and Helio. virescens, and ITS1 sequences of H. assulta, the F1 hybrid male (H. zea ♀X H. armigera ♂), and Helio.subflexa were deposited in GenBank (accession numbers KT343375.1 through KT343382.1, and KT762150.1). Nucleotide sequences of ITS1 amplicons from the F1 hybrid male identified two ITS1 sequences, one matching H. zea (accession no. KT343375.1) and the other matching H. armigera (accession no. KT343376.1). Nucleotide sequences of ITS1 of H. armigera from Australia, China, India, and Kenya were 97, 98, 98, and 98% identical, respectively, to the previously reported nucleotide sequences (Ji et al. 2003 and GenBank accession AB620127.1). Minor differences were observed in the repeat number of a dinucleotide (CA) microsatellite found in the ITS1 sequences of H. armigera. Nucleotide sequences of ITS1 of H. armigera and H. zea had significant differences and shared only 86% nucleotide identities (Fig. 1). ITS1 nucleotide sequence of H. assulta (accession no. KT343382.1) was 86% and 93% identical to H. armigera and H. zea ITS1, respectively. Nucleotide sequence regions with conserved differences (polymorphisms and insertion or deletions) unique to H. armigera and H. zea were used to develop oligonucleotide primer pairs. A species-specific primer combination, designed to amplify ITS1 fragments that differ in size, consistently differentiated the two Helicoverpa species. This primer set consisted of a forward primer common to both species and two species-specific reverse primers (Table 1). The primer pairs 3373 and 3374 and 3373 and 3377 produced species-specific amplicons of 147 and 334 bp from genomic DNA of H. armigera and H. zea, respectively (Fig. 2a). The F1 hybrid male (Fig. 2a, Lane 4) and the mixture of DNA from both species (Fig. 2a, Lane 6) produced two bands each corresponding to H. armigera- and H. zea-specific fragments. Amplification efficiency of the 314 bp H. zea ITS1 fragment from the F1 hybrid was low compared with the artificial hybrid. DNA degradation in the preserved F1 hybrid was the most likely reason for inefficient amplification of the larger ITS1 fragment of H. zea compared with the smaller (147 bp) ITS1 fragment from H. armigera.Fig. 1.


Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

Perera OP, Allen KC, Jain D, Purcell M, Little NS, Luttrell RG - J. Insect Sci. (2015)

Alignments of nucleotide sequences. (A) Alignment of nucleotide sequences of the ITS1 region used for species-specific primer design. H. armigera, H. assulta, Helio. subflexa, and Helio. virescens ITS1 nucleotide sequences were aligned with consensus H. zea ITS 1 nucleotide sequences. Identical nucleotides are given in plain text, and mismatched nucleotides are shown in white text with black background. Alignment gaps are indicated by a hyphen (-). Common forward primer sequence for both species is underlined. Reverse primer sequences specific to H. armigera and H. zea are marked by dashed-line and solid line boxes, respectively. (B) Alignment of 18S rRNA subunit region used for developing control amplicon. Primer binding sequences for forward and reverse primers are marked by forward and reverse arrows, respectively. ITS1 sequence entries are as follows; H. armigera: KT343376.1 (F1 hybrid), KT343377.1 (China), and KT343378.1 (Australia); ITS1 sequences of H. zea: KT343375.1 (F1 hybrid), KT343380.1 (laboratory colony), KT343381.1 (Mississippi); Helio. virescens: KT343379.1 (laboratory colony); H. assulta: KT343382.1 (Thailand); Helio. subflexa: KT62150.1 (laboratory colony); the ITS1 sequences AB620127.1, AF401740.1, and AJ577253.1 of H. armigera and the 18S rRNA subunit sequences Papilio xuthus (L) (AB674749.1) and H. assulta (EU051777.1), respectively, were obtained from GenBank.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4625950&req=5

iev137-F1: Alignments of nucleotide sequences. (A) Alignment of nucleotide sequences of the ITS1 region used for species-specific primer design. H. armigera, H. assulta, Helio. subflexa, and Helio. virescens ITS1 nucleotide sequences were aligned with consensus H. zea ITS 1 nucleotide sequences. Identical nucleotides are given in plain text, and mismatched nucleotides are shown in white text with black background. Alignment gaps are indicated by a hyphen (-). Common forward primer sequence for both species is underlined. Reverse primer sequences specific to H. armigera and H. zea are marked by dashed-line and solid line boxes, respectively. (B) Alignment of 18S rRNA subunit region used for developing control amplicon. Primer binding sequences for forward and reverse primers are marked by forward and reverse arrows, respectively. ITS1 sequence entries are as follows; H. armigera: KT343376.1 (F1 hybrid), KT343377.1 (China), and KT343378.1 (Australia); ITS1 sequences of H. zea: KT343375.1 (F1 hybrid), KT343380.1 (laboratory colony), KT343381.1 (Mississippi); Helio. virescens: KT343379.1 (laboratory colony); H. assulta: KT343382.1 (Thailand); Helio. subflexa: KT62150.1 (laboratory colony); the ITS1 sequences AB620127.1, AF401740.1, and AJ577253.1 of H. armigera and the 18S rRNA subunit sequences Papilio xuthus (L) (AB674749.1) and H. assulta (EU051777.1), respectively, were obtained from GenBank.
Mentions: Complete 18S rRNA and ITS1 sequences of H. armigera, H. zea, and Helio. virescens, and ITS1 sequences of H. assulta, the F1 hybrid male (H. zea ♀X H. armigera ♂), and Helio.subflexa were deposited in GenBank (accession numbers KT343375.1 through KT343382.1, and KT762150.1). Nucleotide sequences of ITS1 amplicons from the F1 hybrid male identified two ITS1 sequences, one matching H. zea (accession no. KT343375.1) and the other matching H. armigera (accession no. KT343376.1). Nucleotide sequences of ITS1 of H. armigera from Australia, China, India, and Kenya were 97, 98, 98, and 98% identical, respectively, to the previously reported nucleotide sequences (Ji et al. 2003 and GenBank accession AB620127.1). Minor differences were observed in the repeat number of a dinucleotide (CA) microsatellite found in the ITS1 sequences of H. armigera. Nucleotide sequences of ITS1 of H. armigera and H. zea had significant differences and shared only 86% nucleotide identities (Fig. 1). ITS1 nucleotide sequence of H. assulta (accession no. KT343382.1) was 86% and 93% identical to H. armigera and H. zea ITS1, respectively. Nucleotide sequence regions with conserved differences (polymorphisms and insertion or deletions) unique to H. armigera and H. zea were used to develop oligonucleotide primer pairs. A species-specific primer combination, designed to amplify ITS1 fragments that differ in size, consistently differentiated the two Helicoverpa species. This primer set consisted of a forward primer common to both species and two species-specific reverse primers (Table 1). The primer pairs 3373 and 3374 and 3373 and 3377 produced species-specific amplicons of 147 and 334 bp from genomic DNA of H. armigera and H. zea, respectively (Fig. 2a). The F1 hybrid male (Fig. 2a, Lane 4) and the mixture of DNA from both species (Fig. 2a, Lane 6) produced two bands each corresponding to H. armigera- and H. zea-specific fragments. Amplification efficiency of the 314 bp H. zea ITS1 fragment from the F1 hybrid was low compared with the artificial hybrid. DNA degradation in the preserved F1 hybrid was the most likely reason for inefficient amplification of the larger ITS1 fragment of H. zea compared with the smaller (147 bp) ITS1 fragment from H. armigera.Fig. 1.

Bottom Line: Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species.In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified.Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS Southern Insect Management Research Unit, Stoneville, MS 38776 op.perera@ars.usda.gov.

No MeSH data available.


Related in: MedlinePlus