Efficient screening for expressed sequence tag polymorphisms (ESTPs) by DNA pool sequencing and denaturing gradient gel electrophoresis (DGGE) in spruces

Abstract

There is an urgent need to accelerate the development of informative codominant markers of coding regions such as ESTPs (expressed sequence tag polymorphisms) to estimate map synteny within and among taxa. A set of primer pairs for 207 ESTs or cDNAs from Picea and Pinus taxa was screened on three distantly-related taxa in the genus Picea, P. mariana (Mill.) B.S.P., P. glauca (Moench) Voss and P. abies (L.) Karst. Of these, 118 (57%) resulted in positive amplification of single-locus gene products in the first two species. To detect polymorphism, these 118 markers were further screened on a panel of 10 pedigree parents for each of P. mariana and P. glauca, either by agarose gel electrophoresis (AGE) or by parallel denaturing gradient gel electrophoresis (DGGE) with standard conditions of 15-45% urea-formamide. Of these, 87 and 74 were found polymorphic in P. mariana and P. glauca, respectively, and 65 were polymorphic in both species. DNA pool sequencing has been explored as a possible strategy to increase economically the detection throughput of SNPs and small indels, and to characterize the types of DNA polymorphism detected by DGGE. Different DNA samples of known sequences were pooled in different ratio mixtures before and after PCR amplifications to determine their minimum relative abundance for detection of DNA polymorphisms by sequencing. For detection of a polymorphism in the DNA pools, the minimum level of relative abundance was 10%. Pooling DNA samples before or after PCR amplification had no effect on the detection of polymorphism by sequencing. For each species panel, the DNAs were pooled and then amplified and sequenced for the 118 primer pairs. With this strategy, the number of ESTPs increased to 107 in P. mariana and 106 in P. glauca, and the number of ESTPs shared by both species increased to 99. About half of the ESTP markers displayed both SNP and indel polymorphisms while the other half displayed only SNPs. Most of the additional ESTPs were amenable to detection by DGGE or CAPS (Cleaved Amplified Polymorphic Sequence) for mapping purposes.