Progress 09/15/01 to 09/14/05
Outputs
Bt resistance in Heliothis virescens (Hv), one of the major pests of Bt cotton, has not been reported in field populations. Our studies of Bt resistance rely on a laboratory strain called YHD2 that was selected on toxin diet. In 2001 our research group reported in Science that the cadherin gene in YHD2 is interrupted by a retrotransposon. This mutated cadherin gene cannot produce the midgut protein that binds the toxin. This particular mutation can be detected by a DNA diagnostic test that uses multiple DNA primers strategically designed to the DNA sequence around this insertion site. Our concern is whether this specific mutation exists in field populations of Hv and what DNA tests can be developed to detect mutations in the cadherin gene. This grant has accomplished two objectives: the development of DNA based diagnostic methods for reliably detecting any mutation to the cadherin gene that confers Bt-resistance in field population of Hv, and the demonstration that a
mutation at another site in the cadherin gene exists in at least one field individual, but this is not the same mutation found in YHD2. Our DNA based diagnostic methods for detecting any mutation in the cadherin gene are based on the sequence variation in its coding region as well as the size, composition and position of its introns. The cadherin alleles found in seven F1 individuals from a genetic cross between a resistant YHD2 male and susceptible female and their eight parents were sequenced. Using 45 sets of specific primers designed to amplify 200 to 300 base pairs of coding sequence over the total length of the cadherin gene, PCR fragments were amplified, cloned and sequenced from genomic DNA of all 15 individuals. The complete cadherin sequence of each individual was assembled, and all sequences have been aligned with respect to their exons and introns. Sequences range from 17 to 20 kb in length. The conserved DNA bases in the coding sequence can now be identified and primer
sets spanning any exon or adjacent exons of the entire cadherin gene can be designed to detect any mutation conferring Bt resistance anywhere in this gene. The second objective of this grant is to screen many field samples of Hv for the presence of the specific Bt-resistance allele found in YHD2. Genomic DNA from 7104 specimens collected in Texas and Louisiana near Bt-cotton fields from 1996 to 2002 was analysed for the presence of the retransposon in the cadherin gene. Our DNA diagnostic test employs multiple DNA primers strategically designed to the DNA sequence around this insertion site. In addition exons 13 and 18 of the cadherin gene in these samples were examined for the presence of mutations similar to those found in Bt-resistant Pink Bollworm individuals. These tests failed to show these types of mutations to the cadherin gene in these field populations. However, our analysis of one of the males from another field population identified by Fred Gould in 1992 to have a Bt
resistant allele showed its cadherin gene has a large insertion in the membrane binding region of exon 28. This finding demonstrates a second type mutation in the cadherin gene of one Hv individual found in the field.
Impacts
Monitoring resistance to Bt toxin in field populations of Heliothis virescens is presently time-consuming and labor intensive. Current methods offer no way to assess insects possessing one copy of the resistant allele. DNA markers to detect resistance to Bt toxin would greatly facilitate the monitoring of resistance. Currently we have reliable sequence of the cadherin gene from 15 individuals. Conserved regions of this sequence can be used to develop primers for DNA diagnostic tests for resistance that is due to any alteration of the cadherin gene whose product is the midgut receptor protein responsible for binding the toxin.
Publications
- Gahan, Linda J., Fred Gould, Juan Lopez, Steven Micinski, and David G. Heckel. (2006) A PCR screen of field populations of Heliothis virescens for a retrotransposon insertion conferring resistance to Bacillus thuringiensis toxin, Journal of Economic Entomology, submitted.
- Gahan, Linda J., Fred Gould and David G. Heckel. (2006) DNA diagnostic methods for the detection of any mutation to the cadherin gene conferring Bt resistance in Heliothis virescens field populations. In preparation.
- Gahan, Linda J., Fred Gould and David G. Heckel. (2006) A second mutation to the cadherin gene of Heliothis virescens that confers resistance to Bacillus thuringiensis toxin. In preparation.
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Progress 01/01/04 to 12/31/04
Outputs
The overall goal of this grant is to develop DNA based diagnostic methods for reliably detecting and accurately quantifying low levels of Bt resistance in field population of Heliothis virescens (Hv), one of the major cotton pests of Bt cotton. Objective 1 focuses on designing gene specific methods of Bt-resistance detection in field populations of these insect pests. A recent discovery showed that a laboratory strain called YHD2 is resistant to Bt toxin because its cadherin gene is interrupted by a retrotransposon. Its presence can be detected by a DNA diagnostic test that uses multiple DNA primers strategically designed to the DNA sequence around this insertion site. Because the retransposon could occupy other positions in the cadherin gene, it is necessary to design many sets of multiple primers that would span the total cadherin gene. Before this can be accomplished, we must assess the sequence variation in its coding region as well as the size, composition and
position of its introns. To meet this objective, 35 sets of specific primers have been designed to amplify 200 to 300 base pairs of coding sequence over the total length of the cadherin gene. PCR fragments from genomic DNA range in size from 600 to 3400 base pairs, depending on the size of the intron. The 35 sets of PCR fragments of the cadherin gene are also overlapping so that the total sequence for a cadherin allele can be reconstructed. These primers sets have been used to identify the location of 30 introns and to sequence the cadherin gene from seven susceptible Hv individuals plus their parents, four of which are resistant males. The complete cadherin sequence of each individual has been reconstructed, and all sequences have been aligned with respect to their exons and introns. Sequences range from 17 to 20 KB in length. The conserved regions in the coding sequence have been identified and primer sets spanning the entire cadherin gene have been designed to detect any mutation
anywhere in this gene. The second objective of this grant is to screen large field samples of Hv for specific Bt-resistance alleles. We have sampled 7500 specimens collected from the Red River Valley in Louisiana from 1996 to 2002. We have isolated the genomic DNA from each sample and bulked it in samples of 8. We analysed these bulks for the presence of the retransposon in the cadherin gene using our DNA diagnostic test that uses multiple DNA primers strategically designed to the DNA sequence around this insertion site. In addition we are examining exons 13 and 18 of the cadherin gene in these samples for the presence of deletion mutations similar to those found in Pink bollworm individuals that are resistant to Bt toxin. Finally we have begun to analyse three male individuals from field populations identified by Fred Gould in 1992. These males when mated to YHD2 females produced offspring that were able to grow on Bt toxin. We want to know where the mutation in the cadherin gene is
found. A manuscript is in preparation.
Impacts
Monitoring resistance to Bt toxin in field populations of Heliothis virescens is presently time-consuming and labor intensive. Current methods offer no way to assess insects possessing one copy of the resistant allele. DNA markers to detect resistance to Bt toxin would greatly facilitate the monitoring of resistance. Currently we are developing a diagnostic test for resistance that is due to a alteration of the cadherin gene whose product is the midgut receptor protein responsible for binding the toxin.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The overall goal of this grant is to develop DNA based diagnostic methods for reliably detecting and accurately quantifying low levels of Bt resistance in field population of Heliothis virescens (Hv), one of the major cotton pests of Bt cotton. Objective 1 focuses on designing gene specific methods of Bt-resistance detection in field populations of these insect pests. A recent discovery showed that a laboratory strain called YHD2 is resistant to Bt toxin because its cadherin gene is interrupted by a retrotransposon. Its presence can be detected by a DNA diagnostic test that uses multiple DNA primers strategically designed to the DNA sequence around this insertion site. Because the retransposon could occupy other positions in the cadherin gene, it is necessary to design many sets of multiple primers that would span the total cadherin gene. Before this can be accomplished, we must assess the sequence variation in its coding region as well as the size, composition and
position of its introns. To meet this objective, 35 sets of specific primers have been designed to amplify 200 to 300 base pairs of coding sequence over the total length of the cadherin gene. PCR fragments from genomic DNA range in size from 600 to 3400 base pairs, depending on the size of the intron. The 35 sets of PCR fragments of the cadherin gene are also overlapping so that the total sequence for a cadherin allele can be reconstructed. These primers sets have been used to identify the location of 30 introns and to sequence the cadherin gene from seven susceptible Hv individuals. Since each individual possesses two cadherin alleles and we desire to know the sequence of a single allele, we are analyzing F1 females obtained from a cross between a resistant YHD2 male and a susceptible female. The variation in size of the thirty different introns in the susceptible strain as opposed to no variation in the resistant strain YHD2 allows us to distinguish the different allele fragments.
The genomic DNA from the F1 female and each of her parents has been isolated and PCR amplified with each set of primers to generate fragments of the cadherin gene. These PCR fragments have been sized on an agarose gel then cloned and sequenced. To date the entire cadherin gene has been sequenced for all seven F1 susceptible individuals plus their parents, four of which are resistant males. The complete cadherin sequence of each individual has been reconstructed, and all sequences have been aligned with respect to their exons and introns. Sequences range from 17 to 20 KB in length. The conserved regions in the coding sequence have been identified and primer sets spanning the entire cadherin gene have been designed to detect any mutation anywhere in this gene. The second objective of this grant is to screen large field samples of Hv for specific Bt-resistance alleles. We have sampled 7500 specimens collected from the Red River Valley in Louisiana from 1996 to 2002. We are in the process
of isolating the genomic DNA from each sample. DNA from 8 samples will be bulked and analysed for the presence of the retransposon in the cadherin gene using our DNA diagnostic test. To date this is 10% complete.
Impacts
Monitoring resistance to Bt toxin in field populations of Heliothis virescens is presently time-consuming and labor intensive. Current methods offer no way to assess insects possessing one copy of the resistant allele. DNA markers to detect resistance to Bt toxin would greatly facilitate the monitoring of resistance. Currently we are developing a diagnostic test for resistance that is due to a alteration of the cadherin gene whose product is the midgut receptor protein responsible for binding the toxin.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The overall goal of this grant is to develop DNA based diagnostic methods for reliably detecting and accurately quantifying low levels of Bt resistance in field population of Heliothis virescens, one of the major cotton pests of Bt cotton. Objective 1 focuses on designing gene specific methods of Bt-resistance detection in field populations of these insect pests. A recent discovery showed that a laboratory strain called YHD2 is resistant to Bt toxin because its cadherin gene is interrupted by a retrotransposon. Its presence can be detected by a DNA diagnostic test that uses multiple DNA primers strategically designed to the DNA sequence around this insertion site. Because the retransposon could occupy other positions in the cadherin gene, it is necessary to design many sets of multiple primers that would span the total cadherin gene. Before this can be accomplished, we must assess the sequence variation in its coding region as well as the size, composition and
position of its introns. To meet this objective, 23 sets of specific primers have been designed to amplify 200 to 300 base pairs of coding sequence over the total length of the cadherin gene. PCR fragments from genomic DNA range in size from 600 to 2000 base pairs, depending on the size of the intron. The 23 sets of PCR fragments of the cadherin gene are also overlapping so that the total sequence for a cadherin allele can be reconstructed. These 23 sets of primers have been used to locate the position of 30 introns in the cadherin gene. These 23 sets of primers are being used to sequence the cadherin gene from seven susceptible Hv individuals. Since each individual possesses two cadherin alleles and we desire to know the sequence of a single allele, we are analyzing F1 females obtained from a cross between a resistant YHD2 male and a susceptible female. The variation in size of the thirty different introns in the susceptible strain as opposed to no variation in the resistant strain
YHD2 allows us to distinguish the different allele fragments. The genomic DNA from the F1 female and each of her parents has been isolated and PCR amplified with each set of primers to generate fragments of the cadherin gene. These PCR fragments are separated by electrophoresis on an agarose gel and the respective susceptible and resistant alleles can be identified according to size. These PCR fragments from each F1 female and her parents have also been cloned and sequenced. To date 70% of the cadherin gene has been sequenced for all seven F1 susceptible individuals plus their parents. The second objective of this grant is to screen large field samples of Heliothis virescens for specific Bt-resistance alleles. We have obtained approximately 5000 specimens collected in the Red River Valley in Louisiana from 1996 to 2002 and preserved in ethanol. We have processed these samples by removing one half of the abdomen of each individual moth and placing it in a well of a 96 rack of culture
tubes. A quick method of genomic DNA isolation has been developed that uses only one phenol extraction followed by a chloroform extraction and then DNA precipitation in the presence of salt and ethanol.
Impacts
Monitoring resistance to Bt toxin in field populations of Heliothis virescens is presently time-consuming and labor intensive. Current methods offer no way to assess insects possessing one copy of the resistant allele. DNA markers to detect resistance to Bt toxin would greatly facilitate the monitoring of resistance. Currently we are developing a diagnostic test for resistance that is due to a alteration of the cadherin gene whose product is the midgut receptor protein responsible for binding the toxin.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The initial phase of the project requires the investigators to develop new methods to analyze large number of insects for the presence or absence of a resistant DNA marker to Bt toxin. First, the insects must be ground in DNA isolation buffer. This will require some new equipment. The investigator has secured demonstration models of the Mixer Mill 300 by Retsch and the Geno/Grinder 2000 by SPEX Certiprep Inc. The latter is preferred because its design offers greater safety features and allows the use of any type of deep 96 well plates. The methodology for isolating DNA with high throughput has also been developed. InstaGene Matrix by BioRad can be utilized to bind genomic DNA after the insect is ground in the buffer. The concentrations of the grinding solution and the matrix have been determined that will produce an acceptable template for PCR with the primers used to detect Bt toxin resistance. Alternatively, the DNA can be purified using a phenol extraction, and a
chloroform extraction followed by precipitation with ethanol in the presence of NaCl. Another phase of the project requires primers to be designed to various increments of the cadherin-like gene from Heliothis virescens (Hv). Five primer sets have been crafted and used to amplify DNA fragments of the cadherin-like gene from the Hv genomic DNA. Subsequently, these PCR products have been cloned and sequenced. Progress will continued along this same line in the coming year until all of the gene is sequenced from genomic DNA and the position of the introns is established.
Impacts
Monitoring resistance to Bt toxin in field populations of Heliothis virescens is presently time-consuming and labor intensive. Current methods offer no way to assess insects possessing one copy of the resistant allele. DNA markers to detect resistant to Bt toxin would greatly facilitate the monitoring of resistance. Currently we are developing a diagnotic test for resistance that is due to a alteration of the cadherin gene whose product is the midgut receptor protein responsible for binding the toxin.
Publications
- No publications reported this period
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