BACKGROUND
plant breeding is a mission of continuously discovering and pyramiding desirable genes of agronomic or end-use interest into breeding lines to produce superior cultivars.
molecular markers linked to genes of interest can be developed and applied for marker-assisted selection  to increase the efficiency of genetic improvement  <cit> .
marker development for mas in plant breeding usually requires that a cross be made between two parental plants which differ in genes or traits of interest to produce a segregating progeny population.
the genomes of these segregating plants are then fingerprinted to identify markers linked to the genes of interest.
in the last three decades, a number of generic dna fingerprinting methods, such as restriction fragment length polymorphism   <cit> , random amplified polymorphic dna   <cit> , simple sequence repeat   <cit> , diversity arrays technology   <cit> , amplified fragment length polymorphism   <cit>  and microsatellite-anchored fragment length polymorphism   <cit> , have been used in marker development for molecular plant breeding.
these methods are effective, but are labour-intensive and time-consuming.
at present, the development of markers tightly linked to genes of interest still remains a difficult task.
to expedite marker development, michelmore et al.
<cit>  described the “bulked segregant analysis”  method, in which a small number of informative segregating individual plants  are bulked to form two pools differing only for the selection region before conducting dna fingerprinting for identification of candidate markers linked to the genes of interest.
the identified candidate markers are then tested on a large number of segregating individual plants to confirm the genetic linkage between the markers and the target genes before the markers are implemented in mas.
bsa has been widely used in marker development for molecular plant breeding  <cit> .
in our experience in marker development using the dna fingerprinting method mflp, which is a method based on the combination of the aflp concept with microsatellite motifs  <cit> , we adapted the bsa principle of employing a small number of informative progeny plants, but we kept each individual plant separate in dna fingerprinting.
this approach effectively eliminated the problem of detecting “false positive” candidate markers   <cit> .
using this protocol, we have developed a number of molecular markers linked to various genes of interest applicable to plant breeding  <cit> .
the next-generation sequencing  technology provides a powerful tool for detecting large numbers of dna markers within a short time-frame.
several marker development methods utilising ngs platforms to sequence complexity reduced representations were reported, including reduced-representation libraries   <cit> , complexity reduction of polymorphic sequences   <cit> , restriction-site associated dna sequencing   <cit> , sequence based polymorphic marker technology   <cit> , low coverage multiplexed shotgun genotyping   <cit> , and genotyping by sequencing   <cit> .
“restriction-site associated dna ” was originally described by miller et al.
<cit>  based on microarray platform.
baird et al.
<cit>  adapted the rad on the massively-parallel ngs platform to efficiently detect dna polymorphisms without the requirement of any prior molecular knowledge for the species under investigation.
rad sequencing produces two types of dna markers: one type of markers is from dna variations within the restriction sites which are dominant markers; the other is from sequence variation adjacent to the restriction sites which are co-dominant markers  <cit> .
rad markers have been employed in genetic mapping on fungi  <cit> , fish  <cit> , insects  <cit> , and more recently on plants  <cit> .
narrow-leafed lupin  is a grain legume crop cultivated in australia, europe, america and africa.
anthracnose caused by the fungal pathogen colletotrichum lupini is the most devastating disease of lupin  <cit> .
in australia, a single dominant gene conferring resistance to anthracnose, designated as “lanr1”, is extensively applied in the national lupin breeding program to combat the disease  <cit> .
two molecular markers were established using traditional marker development methods, which were linked to lanr <dig> gene at the genetic distance of  <dig>  and  <dig>  centimorgan , respectively  <cit> .
the objectives of this research were to examine the utility of rad sequencing, applied as dna fingerprinting, for rapid marker development for mas in plant breeding, and to develop molecular markers more closely linked to the disease resistance gene lanr <dig> for molecular breeding in lupin.
RESULTS
generating snp markers by rad sequencing
the marker development procedures employed in this study are illustrated in figure  <dig>  during the rad sequencing stage, a total of  <dig> million raw reads, comprising approximately  <dig> gb of sequencing data, were produced by hiseq <dig> from the two rad-sequencing libraries constructed by the multiplex identifiers  strategy from the  <dig> plants.
after a read grouping procedure within individual plants, each plant had its tag reads for marker discovery.
tag reads from the same restriction association site in the genomes of the two parents were compared.
a total of  <dig> single nucleotide polymorphisms were obtained across the  <dig> plants in the rad sequencing.
the average coverage depth of the nucleotides of the  <dig> snp markers was  <dig> x.
identification of candidate rad markers linked to the lanr <dig> gene
after filtration on the  <dig> snp markers with the parameters for candidate marker identification,  <dig> co-dominant rad markers were obtained .
for each of these  <dig> snp markers, the nine f <dig> ril plants with anthracnose resistance showed the polymorphic nucleotide consistent with that of the disease resistance parent tanjil; while the nine f <dig> ril plants susceptible to anthracnose disease exhibited the marker allele of the polymorphic nucleotide corresponding to the susceptible parent unicrop .
these  <dig> rad markers were considered as candidate markers linked to the disease resistance gene lanr <dig> based on the principles of candidate marker discovery described earlier  <cit> .
*ps = parental cultivar unicrop which is susceptible to anthracnose disease; pr = parental cultivar tanjil which is resistant to anthracnose.
**the nucleotides of the snp markers corresponding to the disease susceptible parent unicrop were recorded as “a”; the nucleotides of the snp markers corresponding to the disease resistant parent tanjil were recorded as “b”; missing data were recorded as “-”.
the dna sequences of the  <dig> rad markers were presented in table  <dig>  the length of the rad reads were all  <dig> base pairs when the first nucleotide “g” from the ecori restriction sites  was included.
the majority of the rad markers contained the snp mutation sites in the middle of the rad sequence reads , which provided enough sequence length to design primer pairs to flank the snp mutation sites in marker conversion.
*candidate marker numbers were consistent to the candidate makers listed in table  <dig>
**the first number was the average coverage depth  of the nucleotide from the  <dig> plants susceptible to anthracnose disease; the second number was the average coverage depth of the nucleotide from the  <dig> plants resistant to anthracnose.
***the first nucleotide of each snp in bracket for each rad sequence read was from parental plant unicrop; the second nucleotide of the snp in bracket for each rad sequence read was from parental plant tanjil.
conversion of selected candidate rad-snp markers into pcr-based markers
each of the five randomly selected snp markers was successfully converted into a sequence-specific, simple pcr-based marker with a pair of sequence-specific primers flanking the snp site .
these pcr-based snp markers exhibited as co-dominant polymorphic bands on the sscp gels .
the five newly established pcr markers were designated as anseq <dig>  anseq <dig>  anseq <dig>  anseq <dig> and anseq <dig> .
*candidate marker numbers were consistent to the candidate makers listed in table  <dig> and table  <dig>
linkage confirmation between the established pcr markers and the disease resistance gene lanr1
marker genotyping data were obtained for the five newly established pcr markers on 186 f <dig> rils from the cross unicrop x tanjil.
linkage analysis using the marker genotyping score data and the anthracnose disease phenotyping data on the 186 f <dig> rils showed that all the five pcr-based markers developed in this study were linked to the disease resistance gene lanr <dig> .
the linked markers reported in this study were on linkage group “nll-11” of the lupin genetic map reported by nelson et al.
<cit>  as evidenced by the presence of the same r gene  and the previous developed marker “antjm2”.
three of the five markers, anseq <dig>  anseq <dig> and anseq <dig>  were closer to the r gene than the previously developed markers antjm <dig> and antjm <dig>  <cit> .
two of the newly developed markers, anseq <dig> and anseq <dig>  were flanking the r gene at a genetic distance of  <dig>  cm .
discussion
the marker development strategy which we applied in this study consisted of four stages.
firstly, a cross was made between two parental plants to create a segregating progeny population, followed by phenotyping of the gene of interest of the individual progeny plants.
secondly, a small number of informative plants were subjected to dna fingerprinting by ngs based rad sequencing to identify candidate markers linked to the target gene.
thirdly, selected candidate markers were converted into cost-effective, simple pcr-based markers.
fourthly, the converted markers were tested on a large number of individual plants of a segregating population to confirm the genetic linkage between the markers and the gene of interest before the markers were confidently implemented into a molecular breeding program.
in traditional dna fingerprinting methods such as rflp, rapd, aflp and mflp, the dna fingerprints are visualized as dna bands on the gels.
by comparison, the “fingerprints” in rad sequencing are presented as dna sequence reads.
snp markers developed from rad sequencing are suitable for high throughput multiplex implementation in molecular plant breeding on modern snp genotyping platforms.
the most striking advantage in application of ngs based rad sequencing as dna fingerprinting in marker development for molecular plant breeding is the extraordinarily high efficiency.
the massive power of the ngs technology for rapid and large scale marker discovery laid the foundation for the super-fast development of markers linked to the target gene lanr <dig> demonstrated in this study.
in the rad sequencing, we obtained  <dig> snp markers across the  <dig> test plants.
this number of markers obtained from a small portion  of one sequencing run is equivalent to months of investigation with traditional dna fingerprinting methods.
the lupin genome is approximately 1540 cm  <cit> .
the  <dig> snp markers provided an average coverage of about  <dig>  markers for each cm in the genome.
in theory, approximately  <dig> of these snp markers would be distributed on the chromosome at either side of the lanr <dig> gene within the genetic distance of 3 cm, or  <dig> markers at either side of the r gene within the genetic distance of 5 cm.
therefore, it was of no surprise that  <dig> markers were discovered linked to the target lanr <dig> gene in this study.
the large number of molecular markers associated with a target gene should provide breeders with a broad suite of options to choose the markers to suit a wide range of breeding populations to support molecular plant breeding programs  <cit> .
a further major advantage of using ngs technology in marker development is the ease in conversion of candidate markers into cost-effective, simple pcr-based markers.
in mas, molecular markers must be cost-effectively amenable to a large number of samples  <cit> .
in traditional dna fingerprinting such as rapd, aflp and mflp, dna markers recovered from the gels must go through a tedious process of dna fragment isolation, pcr amplification, cloning and sequencing to determine the dna sequences of the marker fragments to enable the design of sequence-specific primers  <cit> .
sometimes marker conversion may still remain problematic even after the marker bands are sequenced, particularly for dominant markers, and for markers resulting from dna variations from the restriction sites targeted by the restriction enzymes employed in dna fingerprinting, in these cases further dna sequence extension after sequencing is required  <cit> .
by contrast, when ngs is used as dna fingerprinting, the dna sequences of candidate markers are known, and ready for primer design.
with the parameters used for candidate marker identification employed in this study, all selected markers are co-dominant markers.
the length of rad sequencing reads in our study was  <dig> base pairs.
the majority of snp mutation sites were in the middle of the sequencing reads, which provided enough sequence length at both ends of the sequence reads to design a pair of sequence-specific primers to flank the snp sites to convert the snp markers into pcr-based co-dominant markers.
anthracnose disease resistance in cultivar tanjil of l. angustifolius is conditioned by a single dominant gene lanr <dig> which is highly heritable  <cit> .
in this study, three out of five established sequence-specific pcr markers, anseq <dig>  anseq <dig> and anseq <dig>  were closer to the target gene lanr <dig> than the other two markers antjm <dig> and antjm <dig> previously developed with conventional dna fingerprinting when  <dig> plants were used  <cit> .
two of the newly developed markers, anseq <dig> and anseq <dig>  are co-dominant markers flanking the lanr <dig> gene in  <dig>  cm.
the accuracy to selection f <dig> plants possessing the lanr <dig> gene using either marker anseq <dig> or anseq <dig> in marker-assisted selection in lupin breeding will be approximately 99%; and the accuracy would be  <dig> % if both markers are applied in mas.
genotyping based selection using these markers is capable of distinguishing the homozygous resistant plants  from heterozygous resistant plants  among the f <dig> progeny plants resulting from rxs crosses.
this leads to the selection and fixation of the desirable gene at the early generation in the breeding cycle  <cit> .
by comparison, plants selected based on traditional disease phenotyping would contain both the rr genotype and the rr genotype, where further disease resistance selection in the following breeding cycle is still required due to segregation from plants with rr genotype.
therefore, genotyping based marker-assisted selection is much more cost-effective than traditional phenotyping based selection.
the two markers anseq <dig> and anseq <dig> are now replacing the previously developed markers antjm <dig> and antjm <dig> for marker-assisted selection in the australian national lupin breeding program.
CONCLUSIONS
we have demonstrated that the ngs-based rad sequencing technology can be cohesively integrated into the marker development protocol for molecular plant breeding.
the sequencing reads generated from the rad sequencing have the same function and effects as the dna fingerprints produced by traditional dna fingerprinting methods for marker development in molecular plant breeding.
the application of ngs-based technology in marker development provides several significant advantages over tradition methods.
firstly, marker development with ngs is very rapid.
the entire rad sequencing work can be completed in days.
secondly, dozens of molecular markers linked to a target gene can be discovered in one sequencing run, which is in sharp contrast to traditional dna fingerprinting methods in which only one or a few markers can be found after working for months.
the large number of linked markers not only provides the luxury for the molecular geneticist to choose the marker most closely linked to the gene, but also offers plant breeders the option to select markers applicable to a wide range of crosses in their breeding programs.
thirdly, dna markers obtained by our marker development strategy are all co-dominant, which can readily be converted into cost-effective, simple pcr-based markers desirable for high throughput implementation on modern snp genotyping platforms for marker-assisted selection in molecular plant breeding.
the marker development strategy applied in this study does not require any prior genome knowledge or genetic mapping information.
this will potentiate its utilization across a wide range of plant species.

 plant breeding is a mission of continuously discovering and pyramiding desirable genes of agronomic or end-use interest into breeding lines to produce superior cultivars. molecular markers linked to genes of interest can be developed and applied for marker-assisted selection to increase the efficiency of genetic improvement. marker development for mas in plant breeding usually requires that a cross be made between two parental plants which differ in genes or traits of interest to produce a segregating progeny population. the genomes of these segregating plants are then fingerprinted to identify markers linked to the genes of interest. in the last three decades, a number of generic dna fingerprinting methods, such as restriction fragment length polymorphism, random amplified polymorphic dna, simple sequence repeat, diversity arrays technology, amplified fragment length polymorphism and microsatellite-anchored fragment length polymorphism, have been used in marker development for molecular plant breeding. these methods are effective, but are labour-intensive and time-consuming. at present, the development of markers tightly linked to genes of interest still remains a difficult task. to expedite marker development, michelmore et al. described the “bulked segregant analysis” method, in which a small number of informative segregating individual plants are bulked to form two pools differing only for the selection region before conducting dna fingerprinting for identification of candidate markers linked to the genes of interest. the identified candidate markers are then tested on a large number of segregating individual plants to confirm the genetic linkage between the markers and the target genes before the markers are implemented in mas. bsa has been widely used in marker development for molecular plant breeding. in our experience in marker development using the dna fingerprinting method mflp, which is a method based on the combination of the aflp concept with microsatellite motifs, we adapted the bsa principle of employing a small number of informative progeny plants, but we kept each individual plant separate in dna fingerprinting. this approach effectively eliminated the problem of detecting “false positive” candidate markers. using this protocol, we have developed a number of molecular markers linked to various genes of interest applicable to plant breeding. the next-generation sequencing technology provides a powerful tool for detecting large numbers of dna markers within a short time-frame. several marker development methods utilising ngs platforms to sequence complexity reduced representations were reported, including reduced-representation libraries, complexity reduction of polymorphic sequences, restriction-site associated dna sequencing, sequence based polymorphic marker technology, low coverage multiplexed shotgun genotyping, and genotyping by sequencing. “restriction-site associated dna ” was originally described by miller et al. based on microarray platform. baird et al. adapted the rad on the massively-parallel ngs platform to efficiently detect dna polymorphisms without the requirement of any prior molecular knowledge for the species under investigation. rad sequencing produces two types of dna markers: one type of markers is from dna variations within the restriction sites which are dominant markers; the other is from sequence variation adjacent to the restriction sites which are co-dominant markers. rad markers have been employed in genetic mapping on fungi, fish, insects, and more recently on plants. narrow-leafed lupin is a grain legume crop cultivated in australia, europe, america and africa. anthracnose caused by the fungal pathogen colletotrichum lupini is the most devastating disease of lupin. in australia, a single dominant gene conferring resistance to anthracnose, designated as “lanr1”, is extensively applied in the national lupin breeding program to combat the disease. two molecular markers were established using traditional marker development methods, which were linked to lanr gene at the genetic distance of and centimorgan, respectively. the objectives of this research were to examine the utility of rad sequencing, applied as dna fingerprinting, for rapid marker development for mas in plant breeding, and to develop molecular markers more closely linked to the disease resistance gene lanr for molecular breeding in lupin.  generating snp markers by rad sequencing the marker development procedures employed in this study are illustrated in figure  during the rad sequencing stage, a total of million raw reads, comprising approximately gb of sequencing data, were produced by hiseq from the two rad-sequencing libraries constructed by the multiplex identifiers strategy from the plants. after a read grouping procedure within individual plants, each plant had its tag reads for marker discovery. tag reads from the same restriction association site in the genomes of the two parents were compared. a total of single nucleotide polymorphisms were obtained across the plants in the rad sequencing. the average coverage depth of the nucleotides of the snp markers was x. identification of candidate rad markers linked to the lanr gene after filtration on the snp markers with the parameters for candidate marker identification, co-dominant rad markers were obtained. for each of these snp markers, the nine f ril plants with anthracnose resistance showed the polymorphic nucleotide consistent with that of the disease resistance parent tanjil; while the nine f ril plants susceptible to anthracnose disease exhibited the marker allele of the polymorphic nucleotide corresponding to the susceptible parent unicrop. these rad markers were considered as candidate markers linked to the disease resistance gene lanr based on the principles of candidate marker discovery described earlier. *ps = parental cultivar unicrop which is susceptible to anthracnose disease; pr = parental cultivar tanjil which is resistant to anthracnose. **the nucleotides of the snp markers corresponding to the disease susceptible parent unicrop were recorded as “a”; the nucleotides of the snp markers corresponding to the disease resistant parent tanjil were recorded as “b”; missing data were recorded as “-”. the dna sequences of the rad markers were presented in table  the length of the rad reads were all base pairs when the first nucleotide “g” from the ecori restriction sites was included. the majority of the rad markers contained the snp mutation sites in the middle of the rad sequence reads, which provided enough sequence length to design primer pairs to flank the snp mutation sites in marker conversion. *candidate marker numbers were consistent to the candidate makers listed in table  **the first number was the average coverage depth of the nucleotide from the plants susceptible to anthracnose disease; the second number was the average coverage depth of the nucleotide from the plants resistant to anthracnose. ***the first nucleotide of each snp in bracket for each rad sequence read was from parental plant unicrop; the second nucleotide of the snp in bracket for each rad sequence read was from parental plant tanjil. conversion of selected candidate rad-snp markers into pcr-based markers each of the five randomly selected snp markers was successfully converted into a sequence-specific, simple pcr-based marker with a pair of sequence-specific primers flanking the snp site. these pcr-based snp markers exhibited as co-dominant polymorphic bands on the sscp gels. the five newly established pcr markers were designated as anseq anseq anseq anseq and anseq. *candidate marker numbers were consistent to the candidate makers listed in table  and table  linkage confirmation between the established pcr markers and the disease resistance gene lanr1 marker genotyping data were obtained for the five newly established pcr markers on 186 f rils from the cross unicrop x tanjil. linkage analysis using the marker genotyping score data and the anthracnose disease phenotyping data on the 186 f rils showed that all the five pcr-based markers developed in this study were linked to the disease resistance gene lanr. the linked markers reported in this study were on linkage group “nll-11” of the lupin genetic map reported by nelson et al. as evidenced by the presence of the same r gene and the previous developed marker “antjm2”. three of the five markers, anseq anseq and anseq were closer to the r gene than the previously developed markers antjm and antjm. two of the newly developed markers, anseq and anseq were flanking the r gene at a genetic distance of  cm. discussion the marker development strategy which we applied in this study consisted of four stages. firstly, a cross was made between two parental plants to create a segregating progeny population, followed by phenotyping of the gene of interest of the individual progeny plants. secondly, a small number of informative plants were subjected to dna fingerprinting by ngs based rad sequencing to identify candidate markers linked to the target gene. thirdly, selected candidate markers were converted into cost-effective, simple pcr-based markers. fourthly, the converted markers were tested on a large number of individual plants of a segregating population to confirm the genetic linkage between the markers and the gene of interest before the markers were confidently implemented into a molecular breeding program. in traditional dna fingerprinting methods such as rflp, rapd, aflp and mflp, the dna fingerprints are visualized as dna bands on the gels. by comparison, the “fingerprints” in rad sequencing are presented as dna sequence reads. snp markers developed from rad sequencing are suitable for high throughput multiplex implementation in molecular plant breeding on modern snp genotyping platforms. the most striking advantage in application of ngs based rad sequencing as dna fingerprinting in marker development for molecular plant breeding is the extraordinarily high efficiency. the massive power of the ngs technology for rapid and large scale marker discovery laid the foundation for the super-fast development of markers linked to the target gene lanr demonstrated in this study. in the rad sequencing, we obtained snp markers across the test plants. this number of markers obtained from a small portion of one sequencing run is equivalent to months of investigation with traditional dna fingerprinting methods. the lupin genome is approximately 1540 cm. the snp markers provided an average coverage of about markers for each cm in the genome. in theory, approximately of these snp markers would be distributed on the chromosome at either side of the lanr gene within the genetic distance of 3 cm, or markers at either side of the r gene within the genetic distance of 5 cm. therefore, it was of no surprise that markers were discovered linked to the target lanr gene in this study. the large number of molecular markers associated with a target gene should provide breeders with a broad suite of options to choose the markers to suit a wide range of breeding populations to support molecular plant breeding programs. a further major advantage of using ngs technology in marker development is the ease in conversion of candidate markers into cost-effective, simple pcr-based markers. in mas, molecular markers must be cost-effectively amenable to a large number of samples. in traditional dna fingerprinting such as rapd, aflp and mflp, dna markers recovered from the gels must go through a tedious process of dna fragment isolation, pcr amplification, cloning and sequencing to determine the dna sequences of the marker fragments to enable the design of sequence-specific primers. sometimes marker conversion may still remain problematic even after the marker bands are sequenced, particularly for dominant markers, and for markers resulting from dna variations from the restriction sites targeted by the restriction enzymes employed in dna fingerprinting, in these cases further dna sequence extension after sequencing is required. by contrast, when ngs is used as dna fingerprinting, the dna sequences of candidate markers are known, and ready for primer design. with the parameters used for candidate marker identification employed in this study, all selected markers are co-dominant markers. the length of rad sequencing reads in our study was base pairs. the majority of snp mutation sites were in the middle of the sequencing reads, which provided enough sequence length at both ends of the sequence reads to design a pair of sequence-specific primers to flank the snp sites to convert the snp markers into pcr-based co-dominant markers. anthracnose disease resistance in cultivar tanjil of l. angustifolius is conditioned by a single dominant gene lanr which is highly heritable. in this study, three out of five established sequence-specific pcr markers, anseq anseq and anseq were closer to the target gene lanr than the other two markers antjm and antjm previously developed with conventional dna fingerprinting when plants were used. two of the newly developed markers, anseq and anseq are co-dominant markers flanking the lanr gene in  cm. the accuracy to selection f plants possessing the lanr gene using either marker anseq or anseq in marker-assisted selection in lupin breeding will be approximately 99%; and the accuracy would be % if both markers are applied in mas. genotyping based selection using these markers is capable of distinguishing the homozygous resistant plants from heterozygous resistant plants among the f progeny plants resulting from rxs crosses. this leads to the selection and fixation of the desirable gene at the early generation in the breeding cycle. by comparison, plants selected based on traditional disease phenotyping would contain both the rr genotype and the rr genotype, where further disease resistance selection in the following breeding cycle is still required due to segregation from plants with rr genotype. therefore, genotyping based marker-assisted selection is much more cost-effective than traditional phenotyping based selection. the two markers anseq and anseq are now replacing the previously developed markers antjm and antjm for marker-assisted selection in the australian national lupin breeding program.  we have demonstrated that the ngs-based rad sequencing technology can be cohesively integrated into the marker development protocol for molecular plant breeding. the sequencing reads generated from the rad sequencing have the same function and effects as the dna fingerprints produced by traditional dna fingerprinting methods for marker development in molecular plant breeding. the application of ngs-based technology in marker development provides several significant advantages over tradition methods. firstly, marker development with ngs is very rapid. the entire rad sequencing work can be completed in days. secondly, dozens of molecular markers linked to a target gene can be discovered in one sequencing run, which is in sharp contrast to traditional dna fingerprinting methods in which only one or a few markers can be found after working for months. the large number of linked markers not only provides the luxury for the molecular geneticist to choose the marker most closely linked to the gene, but also offers plant breeders the option to select markers applicable to a wide range of crosses in their breeding programs. thirdly, dna markers obtained by our marker development strategy are all co-dominant, which can readily be converted into cost-effective, simple pcr-based markers desirable for high throughput implementation on modern snp genotyping platforms for marker-assisted selection in molecular plant breeding. the marker development strategy applied in this study does not require any prior genome knowledge or genetic mapping information. this will potentiate its utilization across a wide range of plant species. 
we demonstrated that more than molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion of one sequencing run on hiseq by applying ngs based rad sequencing in marker development. 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. linkage analysis using marker genotyping data and disease resistance phenotyping data on a f population consisting of individual plants confirmed that all these five markers were linked to the r gene. 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. the next-generation sequencing technology is far more powerful than any existing generic dna fingerprinting methods in generating dna markers. twenty informative plants from a cross of rxs in lupin were subjected to rad single-end sequencing by multiplex identifiers. 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 this study, we employed a grain legume crop lupinus angustifolius as a test case, and examined the utility of an ngs-based method of rad sequencing as dna fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. 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. two of these newly developed sequence-specific pcr markers, anseq and anseq flanked the target r gene at a genetic distance of centimorgan, 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. the entire rad sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform solexa hiseq a total of million raw reads, approximately gb of sequencing data, were collected. however, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. filtration of dna sequencing data with marker identification parameters resulted in the discovery of molecular markers linked to the disease resistance gene lanr five randomly selected markers were converted into cost-effective, simple pcr-based markers.
 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 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 as a test case, and examined the utility of an ngs-based method of rad sequencing as dna fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding.  twenty informative plants from a cross of rxs 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 hiseq a total of million raw reads, approximately gb of sequencing data, were collected. sequence comparison among the test plants discovered snp markers. filtration of dna sequencing data with marker identification parameters resulted in the discovery of molecular markers linked to the disease resistance gene lanr 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 f population consisting of individual plants confirmed that all these five markers were linked to the r gene. two of these newly developed sequence-specific pcr markers, anseq and anseq flanked the target r gene at a genetic distance of centimorgan, 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.  we demonstrated that more than molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion of one sequencing run on hiseq 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. 