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Background: Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding. Results: Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding. Conclusions: We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands. [ABSTRACT FROM AUTHOR]

Background: Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding. Results: Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding. Conclusions: We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands. [ABSTRACT FROM AUTHOR]

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 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. Results: Twenty 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. Conclusions: We 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. [ABSTRACT FROM AUTHOR]

The reduced pod shattering gene tardus is one of the most important domestication genes in narrow-leafed lupin ( Lupinus angustifolius L.). In development of a molecular marker linked to the tardus gene, we incorporated the concept of marker validation during the initial candidate marker identification stage. Four dominant microsatellite-anchored fragment length polymorphism (MFLP) markers were identified as candidate markers based on their banding patterns in an F recombination inbred line (RIL) population. One specific marker best correlating with phenotypes in the representative germplasm was selected and converted to a simple PCR-based marker. This established marker, designated as 'TaLi', is located at a distance of 1.4 cM from the tardus gene. DNA sequencing revealed six insertion/deletion sites between the non-shattering marker allele and the shattering marker allele. Validation of marker TaLi on 25 domesticated commercial cultivars and 125 accessions of the lupin core collection found a 94% marker and tardus phenotype match. Marker TaLi is the first simple PCR-based marker that can be widely used for non-shattering pod selection in narrow-leafed lupin breeding program. [ABSTRACT FROM AUTHOR]

Selection for anthracnose disease resistance is one of the top priorities in white lupin ( Lupinus albus) breeding programs. A cross was made between a landrace P27174 (resistant to anthracnose) and a cultivar Kiev Mutant (susceptible). The progeny was advanced to F8 recombinant inbred lines (RILs). Disease tests on the RIL population from field trials over 2 years indicated that the disease resistance in P27174 was polygenic controlled. A modified selective genotyping strategy was applied in the development of molecular markers linked to quantitative loci conferring anthracnose diseases resistance. Eight individual plants representing high level of anthracnose resistance (HR), eight plants representing susceptibility (S), together with eight lines representing medium level of anthracnose resistance (MR), were subjected to DNA fingerprinting by Microsatellite-anchored Fragment Length Polymorphisms (MFLP). Six MFLP polymorphisms, which had the banding pattern matching the HR plants and the S plants, were identified as candidate markers linked to quantitative loci conferring anthracnose resistance. The six candidate MFLP markers were delineated into three groups based on their banding variation on the eight MR plants. One candidate MFLP marker each from the three groups was selected, cloned, sequenced, and converted into co-dominant, sequence-specific PCR markers. These three markers, designated as WANR1, WANR2 and WANR3, were tested on a segregating population containing 189 F8 RILs. The disease phenotyping data and the marker genotyping data on the F8 RILs were merged and analysed by the JMP software using the ‘fit-model’ function, which revealed that 71% of the phenotypic variation was controlled by genetic factors, while the other 29% of the phenotypic variation was due to environmental factors and environment × genotype interactions. On individual marker basis, marker WANR1 conditioned 39% of phenotypic variations of anthracnose resistance, followed by marker WANR2 with 8%, and WANR3 with 12%. Further analysis showed that WANR2 and WANR3 were on the same linkage group with a genetic distance of 15.3 cM. The combination of the two markers WANR1 and WANR3 explained 51% out from the 71% of the genetic controlled variations for disease resistance, indicating that the two QTLs working additively for anthracnose disease resistance. A simulation of marker-assisted selection on the F8 RIL population using the two markers WANR1 and WANR3 identified 42 out of the 189 RILs being homozygous for resistance-allele bands for both markers, and 41 of them showed disease severity below 3.0 on the 1 (highly resistant) to 5 (susceptible) scale. The two markers WANR1 and WANR3 have now been implemented for marker-assisted selection for anthracnose resistance in the L. albus breeding program in Australia. [ABSTRACT FROM AUTHOR]

Seeds and plants of wild type Lupinus albus are bitter and contain high level of alkaloids. During domestication, at least three genes conferring low-alkaloid content were identified and incorporated into commercial varieties. Australian lupin breeders exclusively utilize one of these sweetness genes, “ pauper”, in all varieties to prevent possible bitterness contamination via out-crossing. A cross was made between a sweet variety Kiev Mutant (containing pauper gene) and a bitter type landrace P27174, and the population was advanced into F8 recombinant inbred lines (RILs). Twenty-four plants representing sweetness and bitterness were subjected to DNA fingerprinting by the microsatellite-anchored fragment length polymorphism (MFLP) technique. A dominant polymorphism was discovered in an MFLP fingerprint. The MFLP marker was converted into a co-dominant, sequence-specific, simple PCR-based marker. Linkage analysis by the software program MapManager with marker score data and alkaloid phenotyping data from a segregating population containing 190 F8 RILs indicated that the marker is linked to the pauper gene at the genetic distance of 1.4 centiMorgans (cM). This marker, which is designated as “PauperM1”, is capable of distinguishing the pauper gene from the other two low-alkaloid genes exiguus and nutricius. Validation on germplasm from the Australian lupin breeding program showed that the banding pattern of the marker PauperM1 is consistent with the alkaloid genotyping on a wide range of domesticated varieties and breeding lines. The PauperM1 marker is now being implemented for marker assisted selection in the Australian albus lupin breeding program. [ABSTRACT FROM AUTHOR]