Abstract
BackgroundIn 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 linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume 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 fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding.ResultsTwenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program.ConclusionsWe demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.
Highlights
In the last 30 years, a number of DNA fingerprinting methods such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), Diversity Arrays Technology (DArT), have been extensively used in marker development for molecular plant breeding
During the Restriction-site associated DNA (RAD) sequencing stage, a total of 185 million raw reads, comprising approximately 17 Gb of sequencing data, were produced by HiSeq2000 from the two RAD-sequencing libraries constructed by the multiplex identifiers (MID) strategy from the 20 plants
Cluster RAD-tags for each plant by sequence similarity considered as candidate markers linked to the disease resistance gene Lanr1 based on the principles of candidate marker discovery described earlier [11,12,13,14,19,20,21,22,23,24]
Summary
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. In the last three decades, a number of generic DNA fingerprinting methods, such as restriction fragment length polymorphism (RFLP) [4], random amplified polymorphic DNA (RAPD) [5,6], simple sequence repeat (SSR) [7], Diversity Arrays Technology (DArT) [8], amplified fragment length polymorphism (AFLP) [9,10] and microsatellite-anchored fragment length polymorphism (MFLP) [11,12,13,14], have been used in marker development for molecular plant breeding. The development of markers tightly linked to genes of interest still remains a difficult task
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