Molecular markers are commonly used in genetic diversity analysis, genetic map construction, gene mapping and cloning, and marker assisted selection in plant breeding. Based on detection procedure, most molecular marker technologies can be classified into hybridization-based or PCR-based systems. Restriction fragment length polymorphism (RFLP) is the first hybridization-based molecular marker system that was intensively used at the beginning of the molecular biology era in life science while hybridization-based marker methods such as microarrays and diversity array technology (DArT) are used currently to detect single nucleotide polymorphisms (SNP). In contrast, many PCR-based molecular marker detection methods have been developed. For example, amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) and sequence related amplified polymorphism (SRAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), and sequence characterized amplification region (SCAR), are commonly used in genomic analysis (Jones et al., 2009).
There are advantages and limitations for all molecular marker detection methods. In particular, RFLP probes can be shared in related species so RFLP is advantageous over other molecular markers in comparative genomics. However, the detection procedure in RFLP is complicated and costly. Additionally, RFLP is not easily automated to analyze thousands of individuals for marker assisted selection. AFLP is a commonly used molecular marker system since it can detect multiple genetic loci in a genome. On the other hand, there are many steps in the AFLP detection procedure, which limits its application in marker assisted selection when thousands of individual DNA samples need to be analyzed in a short time. SSRs often have a high level of polymorphism in plant genomes and are commonly used in most genomic applications. Since SSR technology only detects sequence repeats, the number of SSRs in a genome is relatively limited compared with numerous SNPs. RAPD is easily performed in one round of PCR, however, a low level of reproducibility of RAPD amplification limits its wide use in genomic analysis.
As next generation sequencing (NGS) technologies dramatically increase capacity and throughput of DNA sequencing, whole genome sequencing of many plant species has been accomplished and most economically important crop species such as rice, maize, soybean, sorghum, potato, tomato and Chinese cabbage have been fully sequenced. Although it is still challenging to use NGS for assembling a whole complex genome such as barley and wheat, there are thousands of SNPs identified in NGS that can be used to develop molecular markers in species with complex genomes. Furthermore, NGS is directly used in SNP discovery and a few dozen genotypes can be sequenced simultaneously to assemble ultradense genetic maps. Additionally, different strategies are used to produce partial genomes that can be used to directly sequence SNPs using next generation technologies.
2. SRAP technology
We developed and published information on the SRAP marker system in 2001 (Li and Quiros). The original thinking was to simplify the AFLP detection procedure and increase throughput and improve reproducibility compared to RAPD. To produce a simple detection procedure, we skipped restriction enzyme digestion and ligation of target DNA fragments and adapters in the AFLP detection protocol. We designed SRAP primers in sizes similar to those in AFLP, but ran one round of PCR instead of two rounds in AFLP. To detect multiple loci with a pair of SRAP primers, we designed a special PCR running program (940C for 1 min, 350C for 1 min and 720C for 1 min for the first 5 cycles and followed by 30 cycles at the raised annealing temperature of 500C). At the beginning of PCR, the 350C annealing temperature allowed SRAP primers to anneal to multiple loci in target DNA so that the multiple loci were amplified to produce a profile that is similar to that in AFLP. Similar to AFLP, most SRAP markers are dominant while most SNPs and SSRs are co-dominant. Compared with RAPD, SRAP used a pair of primers with 16 to 22 nucleotides instead of 10-mer short primers in RAPD, which gives SRAP a big advantage over RAPD so one SRAP primer can combine with unlimited number of other primers. Although SRAP PCR starts at 350C annealing temperature in the first five cycles, the larger sizes of SRAP primers allowed the increase of annealing temperature to 500C in the following cycles, which significantly improves the reproducibility in SRAP. In contrast, a low level of reproducibility in RAPD is a limitation factor. In addition, SRAP primers can be fluorescently labelled and combined with unlabeled SRAP primers so SRAP PCR products can be separated in capillary instruments such as ABI genetic analyzers.
In general, there is a difference of GC content between gene coding sequences and other sequences in plant genomes. We used this difference to design two sets of SRAP primers. The forward primers contained a GGCC cassette closing the 3’ end of SRAP primers that might preferentially anneal to the GC-rich regions while the reverse SRAP primer set was incorporated with an AATT cassette that would preferentially anneal SRAP primers to introns and gene spacers so that SRAP could preferentially amplify gene-rich regions in a genome. After sequencing SRAP fragments and constructing a SRAP genetic map in
There is wide flexibility in the design of SRAP primers. After testing the primers we used for gene cloning, we found that most of these primers worked well in SRAP amplification. In the construction of an ultra-dense genetic map in
To enhance the capacity and effectiveness of SRAP technology, we combined SRAP with Illumina’s Solexa sequencing to directly integrate genetic loci on the
3. Genetic map construction
Genetic maps are extensively used in gene mapping, QTL mapping and assembly of whole genome sequence. High density molecular markers in genetic maps are advantageous and necessary in most applications. The detection of multiple loci in a SRAP PCR reaction can be automated through fluorescently labelled SRAP primers so it is feasible to construct a high density genetic map using SRAP technology.
Using cDNA-SRAP technology, we first constructed a transcriptome map based on
SRAP technology can be combined with other markers to construct genetic maps. For example, Yu et al., (2007) constructed a high-density genetic map in a cultivated allotetraploid cotton population using SSR, SRAP, AFLP, and target region amplification polymorphism (TRAP). This high density cotton genetic map consists of 697 SSR, 171 TRAP, 129 SRAP, 98 AFLP, and two morphological markers, covering a genetic distance of 4,536.7 cM with the average genetic distance of 4.1 cM per marker. Gulsen et al., (2010) reported a new citrus linkage map using SRAP, RAPD, SSR, ISSR, peroxidase gene polymorphism (POGP), resistant gene analog (RGA), and a morphological marker, Alternaria brown spot resistance gene. In total, they assembled 385 SRAP, 97 RAPD, 95 SSR, 18 ISSR, 12 POGP, and 2 RGA markers on the citrus genetic map.
In the Cucurbitaceae family, Yeboah et al., (2007) constructed genetic maps in cucumber using SRAP and ISSR markers. They developed pseudo-testcross F1 segregating populations from a cross between two diploid parents and constructed male and female parental genetic maps separately with 164 SSR and 108 SRAP markers. More recently, Zhang et al., (2012) constructed a high density consensus genetic map in an inter-subspecific mapping population in cucumber. The consensus map contained over a thousand molecular markers including 1,152 SSR, 192 SRAP, 21 SCAR and one STS. In another cucurbit species, Levi et al., (2006) constructed an extended genetic map for watermelon using five PCR-based molecular markers SRAP, AFLP, SSR, ISSR and RAPD. As suggested by the authors, low polymorphism is often observed in watermelon cultivars, combining several marker systems is necessary to construct a high density genetic map covering the whole genome.
SRAP markers have been used to construct genetic maps in a wide range of plant species. In Dendrobium plants that are used as Chinese herbs, Xue et al., (2010) constructed two genetic maps in two Dendrobium species,
4. QTL mapping
A common application of genetic maps is QTL mapping of complex traits. Since QTL are often underpinned by multiple genes in a genome, it is difficult to tag QTL using procedures for tagging Mendelian loci. In general, if a complex trait is changed into a simple Mendelian trait and the underlying QTL is Mendelized, many strategies are available to map and clone the Mendelized genes. In fact, most cloned QTL have been accomplished through such a Mendelized strategy by developing near-isogenic lines (NILs). However, most QTL are not easily Mendelized, so it is necessary to construct genetic maps first and then perform QTL mapping.
In canola, we used SRAP and SSR to construct a genetic map in a doubled haploid (DH) line population that was developed from a synthetic yellow-seeded line and a conventional canola cultivar through microspore culture (Chen et al., 2009). Data for three complex traits including days to flowering, oil content and seed yield at three locations for three years were collected and used in QTL mapping. For oil content, 27 QTL on 14 linkage groups and for seed yield, 18 QTL on 11 linkage groups were identified while days to flowering was suggested to be controlled by a single genetic locus in this mapping population. In rapeseed, Chen et al., (2007) used 208 SSR and 189 SRAP markers to construct a genetic map for a DH line population and performed QTL mapping of yield-related traits in
In cotton, Lin et al., (2005) developed a mapping population by crossing
Similarly, Zhang et al., (2009b) reported on QTL mapping in cotton using SRAP and other markers. They assembled a genetic map containing 509 SSR, 58 intron targeted intron/exon splice junction (IT-ISJ), 29 SRAP and 8 morphological loci in 60 linkage groups. Among these 60 linkage groups, 54 were assigned into 26 chromosomes. This genetic map was used to identify QTL for fiber quality traits in five environments. In total, thirteen QTL including four QTL for fiber length, two QTL for fiber strength, two QTL for fiber fineness, three QTL for fiber length uniformity, and two QTL for fiber elongation were identified. Eleven out of 13 QTL were assigned into the A-subgenome and other two QTL, into the D-subgenome.
In chrysanthemum (
In radish, Xu et al., (2012) recently constructed a genetic map with 592 molecular markers including 287 SRAP, 135 RAPD, 78 SSR, 49 ISSR, 29 randomly amplified microsatellite polymorphism (RAMP), and 14 resistant gene analogs (RGA). They used this genetic map to analyze QTL that controlled root cadmium accumulation. They mapped four QTL on linkage groups 1. 4. 6 and 9. The QTL on linkage group 9 was a major one that accounted for 48.64% of phenotypic variance, suggesting that this QTL might be applied for marker assisted selection to improve radish root quality by reducing cadmium concentration.
5. Gene tagging and cloning
SRAP technology has several merits for gene tagging. Since SRAP detection uses unlimited primer combinations and there are multiple loci detected in a single SRAP PCR reaction, SRAP technology is advantageous over other molecular marker systems for gene tagging. After many genetic loci in a genome are screened quickly, closely linked SRAP markers to a trait of interest can be identified easily. We intensively used SRAP to perform gene tagging and cloning in Brassica species and worked on several economically important traits such as yellow-seeded canola and rapeseed, disease resistance and glucosinolates.
Yellow-seeded oilseeds in Brassica species are suggested to be related to high oil content so it is worthwhile to characterize the genes controlling seed coat color. Using SRAP technology, we cloned and characterized a gene controlling seed coat color and plant hairiness traits in
Similarly, we used SRAP to tag other genes controlling the seed-coat color trait in yellow sarson, another yellow-seeded
SRAP technology is an effective molecular marker system to analyze qualitative and quantitative resistance to plant diseases. In general, qualitative and quantitative resistances are conferred by oligogenic or multigenic loci, respectively. In canola, blackleg is a major disease and qualitative resistance is available. We used the previously described ultradense genetic map to tag resistance genes to blackleg in
In several reports, SRAP markers were used to map genes controlling resistance to plant diseases in several crop species. For instance, Yi et al., (2008) used SRAP, STS and SSR markers to tag a resistance gene (
In rice, Zhao et al., (2010) searched for SSR markers linked to a dominant resistance gene (RSV1) to rice stripe virus and then used the SRAP method to find closely linked markers. They located RSV1 into a region flanked by SSR and SRAP markers. In maize, a new dominant resistance gene to maize head smut was tagged by SSR-BSA and SRAP-BSA methods (Li et al., 2012). Closely linked molecular markers were identified and used to transfer the resistance gene from the resistant source to elite lines via marker assisted selection to breed head smut resistant hybrid cultivars in maize.
In eggplant, Mutlu et al., (2008) tagged a Fusarium wilt resistance gene using SRAP, SRAP-RGA, RAPD, and SCAR markers. They used 2316 primer combinations to identify molecular markers linked to the resistance gene, of which two SRAP markers were closely linked to the resistance gene. The SRAP markers were converted into SCAR markers and used in marker assisted selection of the Fusarium wilt resistance in eggplant.
Besides plant disease resistance, genes underpinning other traits have been tagged using SRAP technology, For instance, genes controlling two important traits, sex determination and tuberculate fruit in cucumber were tagged using SRAP technology (Li et al., 2008; Zhang et al., 2010a). In cucumber, there are three major gene loci, F/f, M/m, and A/a that determine various sex types. Li et al., (2008) analyzed M/m gene locus and identified 8 SRAP markers linked to this gene locus. Additionally, they used SRAP markers to perform chromosome walking and converted some SRAP markers to co-dominant SCAR markers through sequencing SRAP fragments. Eventually they identified very closely linked SRAP markers at a genetic distance of less than one cM. Similarly, Zhang et al., (2010a) performed gene tagging of cucumber tuberculate fruit. They found that the tuberculate fruit (Tu) was controlled by a dominant gene and used a BSA strategy to identify molecular markers linked to this dominant gene. After testing 736 SRAP primer combinations, they found 9 SRAP markers that were linked to the Tu gene and used SSR markers to anchor this gene on chromosome 5, further indicating that they would use the mapping results to clone the Tu gene later.
Male sterility is a commonly used method to produce hybrid seeds for exploiting heterosis in crops. Since genic male sterility is usually controlled by a few genes, SRAP technology is useful to tag the genes underpinning male sterility. For example, Zhang et al., (2011c) used SRAP and SSR markers to tag a dominant genic male sterile gene in
SRAP technology has also been used to tag quantitative traits using the same approaches as described previously in qualitative traits. In alfalfa, Castonguay et al., (2010) used SRAP to identify polymorphic genetic loci that controlled superior tolerance to freezing. Through BSA analysis, they found four SRAP markers that were associated with freezing tolerance and the frequency of their occurrence reflected changes in response to selection. In another report, SRAP was used to tag a major QTL controlling cadmium accumulation in oat (Tanhuanpaa et al 2007). The concentration of toxic cadmium in oat grains is often over the accepted limit and must be reduced. SRAP, RAPD and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were used to perform BSA analysis in an F
6. Genetic diversity
Genetic diversity analysis is necessary in plant breeding, plant systematics and evolution, plant pathology. SRAP is an adequate molecular marker system for genetic diversity analysis in plants and fungi. Since SRAP has many features such as simplicity, reliability, flexibility, detection of multiple loci and cost-effectiveness, which allows beginners and experienced people to perform SRAP routinely with limited facilities or in well-established genomics labs. Since genome sequence information is not necessary for SRAP detection, SRAP can be used to perform genetic diversity analysis in a wide range of living organisms. We first used SRAP to analyze the genetic diversity of parental lines that were used to produce hybrid cultivars in
In melons, Ferriol et al., (200) used SRAP and AFLP to analyze 69 accessions selected from morphotypes and unclassified types that belong to two subspecies,
In grasses, Budak et al., (2004a; 2004b; 2005) used SRAP to analyze genetic diversity and ploidy complexity in buffalograss. They found that SRAP markers were abundant and that they could distinguish genetic diversity among closely related cultivars. Their data showed that among several molecular markers (SSRs, ISSRs, SRAPs, and RAPDs), SRAP estimated the highest mean genetic dissimilarities in buffalograss. Additionally, they used SRAP and other markers to analyze ploidy complex and geographic origin of the
Similarly, Gulsen et al., (2009) used SRAP, peroxidase gene polymorphism (POGP), ISSR and RAPD to study the relationship of ploidy levels, geographic locations and genetic diversity in bermudagrass. They found that there was a significant correlation between ploidy levels in diploids, triploids, tetraploids, pentaploids, and hexaploids and band frequencies of molecular markers (r = 0.62, P < 0.001), suggesting that ploidy levels resulted in genome variation and genetic diversity. Geographic locations of Cynodon accessions also contributed to genetic diversity based on molecular marker analysis. They suggested that combining several molecular markers would be more efficient to evaluate genetic diversity and genetic structure in bermudagrass and eventually broaden genetic basis for developing new cultivars.
In elephant grass, Xie et al., (2009) used SRAP markers to study the genetic diversity and relationships of commonly used cultivars in China. They generated 1,395 genetic loci with 62 SRAP primer combinations with an average of 22.5 genetic loci per primer combination. They found that SRAP loci were very polymorphic (72.8%) and used these SRAPs to estimate the genetic diversity within and between elephant grass cultivars. The results showed the genetic diversity within cultivars was less than that among tested cultivars and the relationship of those tested cultivars was also estimated.
In cereal crops, Zaefizadeh and Goleiv (2009) analyzed genetic diversity and relationships among durum wheat landraces by SRAP marker and phenotypic differences. They used 65 SRAP markers and 27 traits to perform cluster analysis of 40 subconvars of
In rice, Dai et al., (2012) developed indica- and japonica-specific markers using SRAP, TRAP, and SSR markers and performed genetic diversity analysis of Asian
In alfalfa, Vandemark et al.,(2006) used SRAP markers to analyze genetic relationships among historical sources of alfalfa germplasm in North American. Their results showed that SRAP detected highly polymorphic loci (>90%) in alfalfa, which distinguished nine original sources of Medicago germplasm based on genetic similarity calculated with SRAP markers. They suggested that SRAP technology is an adequate marker system for detecting polymorphisms in alfalfa.
In sesame, Zhang et al., (2010b) performed genetic diversity analysis using SRAP and SSR markers. They analyzed 404 landraces from a sesame collection in China. Using11 SRAP and 3 SSR markers, they produced 175 fragments, of which 126 were polymorphic with an average polymorphism rate of 72%. They calculated several parameters such as Jaccard’s genetic similarity coefficients, Nei’s gene diversity and Shannon’s information index and constructed a dendrogram with all the 404 landraces. According to the dendrogram, landraces from different agro-ecological zones did not cluster together, suggesting that geographical locations did not represent the greater genetic variation among the sesame landraces. They concluded that SRAP and SSR markers would be useful to study sesame genetic diversity and understand the relationship of those indigenous landraces, which would guide the collection, protection and utilization of sesame landraces in breeding purposes.
In banana and plantain, Youssef et al., (2011) used SRAP and AFLP markers to analyze 40 Musa accessions including commercial cultivars and wild species. They developed 353 SRAP and 787 AFLP markers to perform cluster analysis using an unweighted pair-grouping method with arithmetic averages (UPGMA) and principal coordinate (PCO) analysis. They eventually assigned all the 40 accessions into corresponding
In grape, Guo et al., (2012) used SRAP markers to study genetic variability and relationships of cultivated wine-type
In medicinal plants, Ortega et al., (2007) analyzed genetic diversity of cultivated and non-cultivated mashua,
In ornamental plants, Hao et al., (2008) used SRAP technology to perform genetic diversity analysis of 29 ornamental and medicinal Paeonia. Dendrogram and principle component analysis indicated that SRAP markers well characterized the genetic relationships of these 29 peony cultivars, which is useful to guide parent selection and molecular marker assisted selection in Paeonia breeding. In another ornamental plant, Feng et al., (2009b) performed genetic analysis of diversity and population structure of
In chrysanthemum, Zhang et al., (2011a) did a genetic diversity study on two flowering traits of chrysanthemum, initial blooming time and the duration of flowering. They identified two pairs of major genes with high levels of inheritance. Using SRAP technology, they performed association mapping of these two traits and identified SRAP markers that were significantly associated with phenotypes, suggesting that SRAP markers might be useful in chrysanthemum breeding. In another report on ornamental plants, Soleimani et al., (2012) used wild, cultivated, and ornamental pomegranates (
In woody plants, Li et al., (2010) did genetic diversity analysis of sea buckthorn which is grown as a nutritious berry crop. They produced 191 polymorphic loci using SRAP technology to perform cluster analysis of 77 accessions, of which 73
In fungi, Sun et al., (2006) used SRAP markers to classify
7. Other applications
SRAP amplification is actually a small portion of all possible sampling of a genome. So SRAP can be used to produce a reduced genome samples when multiple SRAP reactions are pooled. As described previously, pooled SRAP produces can be directly sequenced using next generation sequencing technologies. When replacing genomic DNA with cDNA samples, SRAP is adequate to perform gene expression profiling and also construct cDNA genetic maps.
More recently, Yu et al., (2012) used SRAP markers to distinguish fertile somatic hybrids of
In aquaculture, Ding et al., (2010) used SRAP and SCAR markers to differentiate two cultured populations in grass carp (
8. Summary remarks
SRAP was first used to construct a genetic map and tag genes in
There is a wide range of applications of SRAP technology such as genetic map construction, genetic diversity analysis, gene tagging and cloning. Since SRAP detects multiple loci in one reaction, it is feasible to construct ultradense genetic maps with over 10,000 SRAP molecular markers. SRAP has advantages over other molecular detection techniques in gene tagging and cloning and allows screening thousands of loci shortly to pinpoint the genetic position underlying the trait of interest. Sequencing SRAP products enhances the applications of SRAP technology. In well characterized genomes, SRAP sequences are used to identify the chromosomal region of mapped genes while in species without a known whole genome sequence, sequences of SRAP markers on a genetic map allow arranging sequence contigs and assembly of a whole genome sequence.
SRAP molecular technology is very useful in plant breeding. In QTL mapping, common QTL for the same trait of interest can be effectively identified. Since SRAP has a high throughput feature, multiple mapping populations can be analyzed effectively to construct several genetic maps. In addition, the same set of SRAP primers allows detection of the same genetic loci, which can used to align several genetic maps. SRAP is effective and efficient in marker assisted selection in plant breeding since thousands of samples can be analyzed inexpensively. SRAP technology has been commonly used in analysis of genetic diversity of many plant species. Currently, SRAP are used in most crops, tree species, ornamental and medicinal plants.