Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

Background:
In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP,SSR, DArT, have been extensively used in marker development for molecular plant breeding.However, it remains a daunting task to identify highly polymorphic and closely linkedmolecular markers for a target trait for molecular marker-assisted selection. The nextgenerationsequencing (NGS) technology is far more powerful than any existing genericDNA fingerprinting methods in generating DNA markers. In this study, we employed a grainlegume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprintingfor rapid, cost-effective marker development tagging a disease resistance gene for molecularbreeding.
Results:
Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin weresubjected to RAD single-end sequencing by multiplex identifiers. The entire RADsequencing products were resolved in two lanes of the 16-lanes per run sequencing platformSolexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencingdata, were collected. Sequence comparison among the 20 test plants discovered 8207 SNPmarkers. Filtration of DNA sequencing data with marker identification parameters resulted inthe discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Fiverandomly selected markers were converted into cost-effective, simple PCR-based markers.Linkage analysis using marker genotyping data and disease resistance phenotyping data on aF8 population consisting of 186 individual plants confirmed that all these five markers werelinked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and arenow replacing the markers previously developed by a traditional DNA fingerprinting methodfor marker-assisted selection in the Australian national lupin breeding program.
Conclusions:
We demonstrated that more than 30 molecular markers linked to a target gene of agronomictrait of interest can be identified from a small portion (1/8) of one sequencing run onHiSeq2000 by applying NGS based RAD sequencing in marker development. The markersdeveloped by the strategy described in this study are all co-dominant SNP markers, whichcan readily be converted into high throughput multiplex format or low-cost, simple PCRbasedmarkers desirable for large scale marker implementation in plant breeding programs.The high density and closely linked molecular markers associated with a target trait help toovercome a major bottleneck for implementation of molecular markers on a wide range ofgermplasm in breeding programs. We conclude that application of NGS based RADsequencing as DNA fingerprinting is a very rapid and cost-effective strategy for markerdevelopment in molecular plant breeding. The strategy does not require any prior genomeknowledge or molecular information for the species under investigation, and it is applicableto other plant species.

Source: http://www.biomedcentral.com/1471-2164/13/318

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