Availability of informative molecular markers isa prerequisite for genetic mapping and marker-assisted selection projects.Micro-satellites orSimple Sequence Repeat(SSR)markers arePCR-based and currently the most widel...Availability of informative molecular markers isa prerequisite for genetic mapping and marker-assisted selection projects.Micro-satellites orSimple Sequence Repeat(SSR)markers arePCR-based and currently the most widely usedmarker system in the plant molecular geneticscommunity due to their high degree ofpolymorphism,random distribution throughoutthe genome and their suitability for highthroughput genotyping formats.Despite itsglobal economic importance,cotton has展开更多
While the importance of molecular marker technology was realized more than two decades ago,high-throughput marker development came into vogue only after the availability of hundreds of thousands of sequences in public...While the importance of molecular marker technology was realized more than two decades ago,high-throughput marker development came into vogue only after the availability of hundreds of thousands of sequences in public databases.Many examples now exist where markers are being used routinely in breeding programs for marker-assisted selection(MAS) of traits of interest or marker assisted recovery of genome of interest.Genetic analysis with thousands to tens of thousands of markers is now possible due to the...展开更多
There is considerable interest in quantitatively measuring nucleic acids from single cells to small populations. The most commonly employed laboratory method is the real-time polymerase chain reaction (PCR) analyzed w...There is considerable interest in quantitatively measuring nucleic acids from single cells to small populations. The most commonly employed laboratory method is the real-time polymerase chain reaction (PCR) analyzed with the crossing point or crossing threshold (Ct) method. Utilizing a multiwell plate reader we have performed hundreds of replicate reactions each at a set of initial conditions whose initial number of copies span a concentration range of ten orders of magnitude. The resultant Ct value distributions are analyzed with standard and novel statistical techniques to assess the variability/reliability of the PCR process. Our analysis supports the following conclusions. Given sufficient replicates, the mean and/or median Ct values are statistically distinguishable and can be rank ordered across ten orders of magnitude in initial template concentration. As expected, the variances in the Ct distributions grow as the number of initial copies declines to 1. We demonstrate that these variances are large enough to confound quantitative classi?cation of the initial condition at low template concentrations. The data indicate that a misclassi?cation transition is centered around 3000 initial copies of template DNA and that the transition region correlates with independent data on the thermal wear of the TAQ polymerase enzyme. We provide data that indicate that an alternative endpoint detection strategy based on the theory of well mixing and plate ?lling statistics is accurate below the mis- classi?cation transition where the real time method becomes unreliable.展开更多
Western corn rootworm （WCR, Diabrotica virgifera virgifera LeConte） is highly sensitive to orally delivered double-stranded RNA （dsRNA）. RNAi in WCR is systemic and spreads throughout the insect body. This raises ...Western corn rootworm （WCR, Diabrotica virgifera virgifera LeConte） is highly sensitive to orally delivered double-stranded RNA （dsRNA）. RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA-dependent RNA polymerase （RdRP） activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi- mediated knockdown of Dr v-ATPase C mRNA throughout the WCR gut and other tissues using high-sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v-ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.展开更多
文摘Availability of informative molecular markers isa prerequisite for genetic mapping and marker-assisted selection projects.Micro-satellites orSimple Sequence Repeat(SSR)markers arePCR-based and currently the most widely usedmarker system in the plant molecular geneticscommunity due to their high degree ofpolymorphism,random distribution throughoutthe genome and their suitability for highthroughput genotyping formats.Despite itsglobal economic importance,cotton has
文摘While the importance of molecular marker technology was realized more than two decades ago,high-throughput marker development came into vogue only after the availability of hundreds of thousands of sequences in public databases.Many examples now exist where markers are being used routinely in breeding programs for marker-assisted selection(MAS) of traits of interest or marker assisted recovery of genome of interest.Genetic analysis with thousands to tens of thousands of markers is now possible due to the...
基金partially supported through NSF-DMS 0443855NSF ECS 0601528+1 种基金NIH EB009235the short-lived W.M.Keck Foundation Grant#062014.
文摘There is considerable interest in quantitatively measuring nucleic acids from single cells to small populations. The most commonly employed laboratory method is the real-time polymerase chain reaction (PCR) analyzed with the crossing point or crossing threshold (Ct) method. Utilizing a multiwell plate reader we have performed hundreds of replicate reactions each at a set of initial conditions whose initial number of copies span a concentration range of ten orders of magnitude. The resultant Ct value distributions are analyzed with standard and novel statistical techniques to assess the variability/reliability of the PCR process. Our analysis supports the following conclusions. Given sufficient replicates, the mean and/or median Ct values are statistically distinguishable and can be rank ordered across ten orders of magnitude in initial template concentration. As expected, the variances in the Ct distributions grow as the number of initial copies declines to 1. We demonstrate that these variances are large enough to confound quantitative classi?cation of the initial condition at low template concentrations. The data indicate that a misclassi?cation transition is centered around 3000 initial copies of template DNA and that the transition region correlates with independent data on the thermal wear of the TAQ polymerase enzyme. We provide data that indicate that an alternative endpoint detection strategy based on the theory of well mixing and plate ?lling statistics is accurate below the mis- classi?cation transition where the real time method becomes unreliable.
文摘Western corn rootworm （WCR, Diabrotica virgifera virgifera LeConte） is highly sensitive to orally delivered double-stranded RNA （dsRNA）. RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA-dependent RNA polymerase （RdRP） activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi- mediated knockdown of Dr v-ATPase C mRNA throughout the WCR gut and other tissues using high-sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v-ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.
