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A high-throughput method for genotyping S-RNase alleles in apple.

ABSTRACT: We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate. The method was employed to genotype 432 Malus accessions and exposed 25 different S-alleles in a selection of Malus domestica cultivars of mainly Danish origin (402 accessions) as well as a selection of other Malus species (30 accessions). The allele S3 (28 %) was the most common among the Danish cultivars followed by S1 and S7 (both 27 %). The alleles S36 and S40 not previously reported from M. domestica were found in 6 and 17 cultivars, respectively. Complete allelic composition was found in 91 % of the 369 diploid accessions and in 86 % of the 63 triploids concerned. We further identified a relatively high frequency of S33 and S34, which has not been considered by most previous studies. The protocol presented here is easy to adopt and saves both time and work effort compared to previous methods. The robustness is illustrated by the great accuracy and a high number of S-alleles presented.

SUBMITTER: Larsen B

PROVIDER: S-EPMC4760992 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A high-throughput method for genotyping S-RNase alleles in apple.

Larsen Bjarne B Ørgaard Marian M Toldam-Andersen Torben Bo TB Pedersen Carsten C

Molecular breeding : new strategies in plant improvement 20160219

We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate. The method was employed to genotype 432 <i>Malus</i> accessions and exposed 25 different S-alleles in a ...[more]

PMID: 26941563

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Project description:In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site. PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene. Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis. The protocol was refined by evaluating segregating transgenic progeny from nine BC1S1 populations derived from crosses between the transgenic cotton parent 'E-1-7-6' and other cotton cultivars. OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC1S1 population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering. BC1S1 plants identified as homozygous or hemizygous were self-fertilized to produce BC1S2 progeny. For the homozygous class, 19/20 BC1S2 families confirmed the homozygous BC1S1 prediction while 21/21 BC1S2 families confirmed the hemizygous prediction of the original parent. This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs.

Project description:BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or "orphan" crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1-13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Dataset Information

A high-throughput method for genotyping S-RNase alleles in apple.

Publications

A high-throughput method for genotyping S-RNase alleles in apple.

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