Development of high-throughput genotyping methods for market-assisted plant breeding

Maxime Paris

Cultivated plant varieties are constantly being improved by selecting progeny for quality and disease resistance traits. Based on the knowledge that DNA sequence variations can cause phenotypic differences, technologies amenable to automation and multiplexing were developed to identify and select progeny carrying superior alleles of economically important genes. A cost-effective DNA extraction method was developed to facilitate the implementation of molecular markers. The method enabled extraction of DNA suitable for PCR from 96 samples simultaneously and was used for routine selection of plant progeny carrying specific traits of interest, including barley yellow dwarf virus resistance (Yd2) and p-amylase activity in barley and stem rust resistance in wheat. PCR assays were multiplexed and robotic equipment was used to increase throughput further. Single Nucleotide Polymorphisms (SNPs) are the most frequent mutations encountered in most genomes. To demonstrate the suitability of SNPs as molecular markers for plant breeding, a high-throughput method was developed to enable their validation and routine implementation. Single and multi-nucleotide primer extension assays enabling co-dominant SNP genotyping from small quantities of plant tissue were developed. In the Single Nucleotide Primer Extension (SNuPE) assay, a genotyping primer with its 3’ end directly flanking a SNP was annealed to target sequence and extended by a single dideoxynucleotide triphosphate (ddNTP) complementary to the polymorphic base. In the multi-nucleotide primer extension assay, designed to facilitate genotyping of heterozygous progeny, the genotyping primer with its 3’ end flanking the SNP was extended by either one or two nucleotides, depending on the allele encountered. Extension products were analysed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) and, making use of the molecular weight difference between DNA bases, the incorporated nucleotides were identified by the increase in mass of the extended primers. In barley, mutation-induced recessive alleles (mlo) of the Mlo locus provide broadspectrum resistance to an important fungal pathogen, powdery mildew. The Mlo gene of susceptible and resistant varieties were sequenced and aligned to identify mutations linked to the resistance. A SNP identified and confirmed from a resistant variety, Alexis (mlo-9), was used to develop single and multiple primer extension assays. Two DH populations were characterised for their response to powdery mildew and used for validation of the molecular marker. The assay enabled accurate co-dominant selection of plants carrying the mlo-9 allele providing ra/o-mediated powdery mildew resistance. MALDI-ToF MS is a platform amenable to multiplexing and automation. An investigation of its use for the analysis of microsatellites, another common form of mutation, resulted in the development of a novel genotyping technique. Following amplification of the target sequence, a primer with its 3’ end directly flanking the microsatellite was extended for its length by the deoxynucleotide triphosphates (dNTPs) complementary to the repeat motif. The extension was interrupted by a single ddNTP complementary to the nucleotide following the repeated region. Extension products were analysed using MALDI-ToF MS and differences in the molecular weights of the products enabled identification of the microsatellite alleles. The technique enabled identification of heterozygous progeny in which alleles differed by a single tri-nucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barley Mlo gene. A novel strategy was designed to develop a perfectly linked marker. In the malting process, P-amylase is a key enzyme involved in the degradation of starch. Four allelic forms of the enzyme are found in barley and each exhibits a different rate of thermal inactivation, or thermostability. The level of thermostability influences starch degradation, which determines the yield of fermentable sugars for alcohol production during brewing. Alignment of the cDNA sequences encoding the four enzyme forms revealed six SNPs (cSNPs). The four alleles could be unambiguously identified by co-dominantly genotyping two of the cSNPs using a duplex SNuPE assay. A Cleaved Amplified Polymorphic Sequence (CAPS) assay enabling broader classification of the alleles was also developed to facilitate the transfer of this marker to other laboratories. These markers were used to select plants carrying alternative P-amylase alleles for barley breeding.

Development of high-throughput genotyping methods for market-assisted plant breeding

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