Abstract Molecular marker techniques have been widely applied in the fields of genetic diversity analysis, germplasm resources identification, molecular fingerprint and genetic linkage map construction, QTL mapping and molecular assisted breeding. On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions, this study focused on the sorting and induction of single-primer molecular marker techniques, and expounded their derivative development. Finally, the application prospect and future expectation of single-primer molecular marker techniques were described in detail. The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions, so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.
　　Key words Molecular marker techniques; Single primer; Gene-targeted molecular marker techniques; High throughput sequencing
　　Received: November 13, 2020  Accepted: January 8, 2021
　　Supported by the National Natural Science Foundation of China (31960409; 31960416), Guangxi Natural Science Foundation Program (2018GXNSFDA281027; 2018GXNSFDA294004; 2020GXNSFAA297081), Guangxi Academy of Agricultural Sciences Fund Project (GNK2017JZ13; GNK2018YM06; GNK31960409).
　　Junxian LIU (1982-), female, P. R. China, associate researcher, devoted to research about plant biotechnology.
　　*Corresponding author. E-mail: xfq2002@126.com.
　　Molecular markers are genetic markers based on the variation of base sequences between individuals, which can directly reflect the genetic nature of biological individuals and the differences between biological individuals at the DNA level. Molecular markers are molecular techniques that apply the principles of modern molecular biology to reveal biological genetic polymorphisms. They are an extension of genetic markers at the molecular level and are also one of the powerful tools for modern genetic research. Because molecular markers have the characteristics of abundant quantity, high polymorphism, large amount of information, being not affected by seasons, environment and ontogeny stages, and simple and quick detection means, they have been widely used for analysis of genetic diversity and identification of germplasm resources, construction of molecular fingerprints, construction of genetic linkage maps, mapping of QTLs, auxiliary molecular breeding and other research fields［1］. 　　Since the concept of molecular markers was first proposed in 1980, research reports on molecular markers have been constantly appearing. Especially since the 1990s, molecular markers have developed rapidly. The widely used molecular marker techniques mainly include: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), sequence-related amplified polymorphism (SRAP), target region amplified polymorphism (TRAP), start codon targeted polymorphism (SCoT), and single nucleotide polymorphism (SNP)［2-10］.
　　The above-mentioned molecular marker techniques can be divided into two major types of molecular marker techniques that are based on the molecular hybridization technology or the PCR technology. RFLP is a molecular marker technique based on the molecular hybridization technology. The molecular marker techniques based on the PCR technology are divided into molecular marker techniques based on random primer amplification reactions and those based on specific primer amplification reactions according to the amplification specificity of the primers used. The RAPD and ISSR techniques are typical molecular marker techniques based on random primer amplification reactions, while the SSR technique is a typical molecular marker technique based on specific primer amplification reactions. In addition, molecular marker techniques based on the PCR technology can be divided into single-primer molecular marker techniques and double-primer molecular marker techniques according to the number of primers used. The single-primer molecular marker techniques such as RAPD, ISSR and other traditional techniques have been widely used. Moreover, new single-primer functional molecular marker techniques such as SCoT, CDDP and CBDP have also emerged. In view of this, this study on a more complete summary and introduction of the types of single-primer molecular marker techniques, expounded the derivation and development of single-primer molecular marker techniques, and finally described the application prospect and future expectation of single-primer molecular marker techniques in detail, so that researchers can quickly and conveniently select single-primer molecular marker techniques according to their own specific conditions.
　　Types of single-primer molecular marker techniques
　　Single-primer molecular marker techniques are a relatively special class of molecular marker techniques, which refer to the use of only a single primer in a PCR reaction, which acts as both an upstream primer and a downstream primer at the same time. The single primer can bind to two sites not far away in the opposite direction at the same time in the entire genome, and then effectively amplify the sequence between the two sites, thereby generating polymorphic molecular markers. The single primers in the single-primer molecular marker techniques are designed according to the sequences widely existing in genomes. Because the designed single primer has multiple binding sites in an amplified genome, it is possible to reversely bind another part of the effective constituent of the single primer at a site not far, thereby amplifying the sequence between the two binding sites of the primer. Fig. 1 shows the development time course of the more commonly used single-primer molecular marker techniques. 　　DAF
　　Different from the RAPD molecular marker technique, the DAF (DNA Amplification Fingerprinting) molecular marker technique uses single primers with a higher concentration and a shorter length (usually 5 to 8 bases). The PCR amplification only uses two temperature cycles, and the PCR amplification products often need to be separated by polyacrylamide gel electrophoresis, forming a very complex band pattern［11］.
　　RAPD
　　The RAPD (random amplified polymorphic DNA) technique is the first molecular marker technique based on the PCR technology and single primer amplification, developed by Williams et al. from the American DuPont company in 1990［3］. The RAPD molecular marker technique uses genomic DNA as a template (or cDNA as a template for differential gene display research), and uses one (sometimes two) random short oligonucleotide sequence (10 bp long) as a primer. Through such technique, multiple DNA bands are amplified by PCR at low annealing temperature, and then separated by agarose gel electrophoresis, and polymorphic bands can be displayed after staining. The principle of amplification is that a single primer acts as two single primers in the entire amplification reaction. A single primer binds to multiple sites on the upstream and downstream of the DNA template. Only when the distance between the two binding sites is within the PCR amplification capacity, effective amplification can be achieved, then obtaining multiple bands. The polymorphism is mostly caused by the variation of primer binding sites, so it is a type of dominant marker.
　　The RAPD molecular marker technique has the following advantages: ① the RAPD technique only requires a small amount of DNA template (5-50 ng), and the requirement for the quality of DNA template is not high. ② The RAPD technique does not require DNA probes, and primer design does not need to know DNA sequence information, so the cost of primer synthesis is low, there is no species limitation for primers, and synthesizing a set of primers can satisfy the genome analysis and research of different species. ③ The RAPD technique is fast and efficient, and can amplify 6-12 bands with one primer. ④ The RAPD technique does not involve complicated techniques such as Southern hybridization and autoradiography, and has high safety, simple experimental equipment, and high sensitivity. It is precisely because of these characteristics that the RAPD molecular marker technique is still preferred and used by many scientific researchers, but its main disadvantages are as follows: ① the stability and repeatability of the RAPD technical analysis is poor, and the analysis results lack good comparability among different laboratories; ② the RAPD molecular markers are generally dominantly inherited (a few are codominantly inherited), and cannot distinguish between heterozygous genotypes and homozygous genotypes; and ③ the bands produced by the RAPD technique have co-migration problems, and bands of the same molecular weight appear between different individuals, but there is no guarantee that these individuals have the same (homologous) fragment. Meanwhile, a single band seen on the gel may also contain different amplification products, because the agarose gel electrophoresis used can only distinguish bands with different molecular weights, but cannot distinguish bands with the same molecular weight but different base sequences. 　　ISJ
　　The ISJ (Intron Spice Junction) technique is a molecular marker technique based on the PCR technology and single primer amplification proposed by Weining and Langridge in 1991［12］. The molecular marker technique uses a semi-specific (semi-random) single primer designed according to the intron-exon splice junction sequence for PCR amplification. The amplified products are usually separated by agarose gel electrophoresis, but due to more and more complex band patterns, the separation by polyacrylamide gel electrophoresis is better. Compared with the RAPD molecular marker technique, the ISJ molecular marker technique produces more complex and polymorphic bands, and the polymorphic bands are likely to come from genes and are linked to traits［13-14］.
　　Intron-exon splice junction sequences are short conserved sequences that connect introns and exons of genes, and are related to the effective splicing of introns. They are widely present in plant genomes and also very conservative among different plant species, and have consensus sequences. Furthermore, because the sequences and lengths of adjacent introns have great variability, intron-exon splice junction sequences are suitable for the development of molecular markers, and are good sequence resources for primer design and molecular marker development (Fig. 2).
　　DAMD
　　The DAMD (directed amplification of minisatellite region DNA) technique was a molecular marker technique developed by Heath et al. in 1993 by designing single primers based on published core sequences of small satellite DNA and directly amplifying the variable regions of tandem repeats by the PCR technology［15］. Tandem repeat sequences result in the length polymorphism of amplified fragments due to the change in the number of repeats［16］, and the amplified products are generally separated by agarose gel electrophoresis［14］. This molecular marker technique is somewhat similar to the ISSR molecular marker technique.
　　Minisatellite DNA is also called variable number of tandem repeats (VNTR) or hypervariable repeats (HVR), which mainly exists in the proximal telomeres of chromosomes and has a length from a few hundred to several thousand bases. The repeat unit is generally 10-70 bp, and the number of repeat units in series has "individual" characteristics and "species" specificity. The DAMD technique is one of the important molecular markers to efficiently reveal differences between organisms.
　　ISSR 　　The ISSR (inter-simple sequence repeats) technique is a molecular marker technique based on the PCR technology and single-primer amplification developed by Zietkiewicz et al. in 1994［4］. The ISSR molecular marker technique uses genomic DNA as a template and one (sometimes two) anchor primers designed according to SSR as PCR amplification primers to perform PCR of the sequence between the SSR sites on the genomic DNA at a higher annealing temperature, obtaining the amplified products, which are usually separated by agarose gel or polyacrylamide gel electrophoresis, and EB or AgNO3 staining shows polymorphic DNA bands. The principle is to add 2-4 random bases to the 3 or 5 end of the SSR sequence, then use this as a primer to perform PCR amplification on the DNA sequence between the SSR sequences that are arranged in reverse and not far apart instead of amplifying the SSR sequence itself, and analyze the DNA polymorphisms between different samples based on the presence or absence of bands.
　　The single primer design of the ISSR molecular marker technique is simpler than that of the SSR molecular marker technique. Amplification can be performed with primers without the need to know the DNA sequence in advance, and more DNA polymorphisms can be revealed than the RFLP, RAPD and SSR molecular marker techniques. The ISSR molecular marker technique has the advantages of simple and efficient operation, low primer cost, good repeatability, and rich   polymorphism, and the produced markers are also dominant, which is the same as the RAPD molecular marker technique. It is precisely because of these characteristics that the ISSR molecular marker technique is still one of the molecular marker techniques that many scientific researchers prefer and use.
　　IRAP
　　The IRAP (Inter-Retrotransposon Amplified Polymorphism) technique is a molecular marker technique based on retrotransposons and single-primer amplification developed by Kalendar et al. on barley in 1999. The principle is to design primers based on the conserved sequence of a retrotransposon LTR or the relatively conserved sequence of a reverse transcriptase (RT). These primers are annealed with the corresponding conserved region of the LTR retrotransposons during the PCR amplification process, and the fragment between two adjacent retrotransposon members of the same family is obtained through amplified (Fig. 3). The amplified products are generally separated by polyacrylamide gel electrophoresis［17］. The IRAP molecular marker technique as a kind of retrotransposon molecular marker technique, can detect the polymorphism between retrotransposon insertion sites, and has the advantages of simple operation, high sensitivity, high detection efficiency and rich polymorphism. It has been widely used in the fields of germplasm identification and genetic diversity analysis. 　　Agricultural Biotechnology2021
　　IMP
　　The IMP (inter MITE polymorphisms) technique is a molecular marker technique based on the PCR technology and single primer amplification developed by Chang et al. in 2001. The single primers used are designed based on the inverted repeat TIR sequences of MITE (miniature inverted repeat transposable element) transposons. The single primer PCR amplification detects the DNA polymorphisms between two adjacent MITE transposon sites, and the amplification products are generally separated by acrylamide gel electrophoresis［18］. The results of the IMP molecular marker technique are stable, reliable and reproducible. Successful amplification can be performed in corn, wheat, barley, oat and sugarcane, and obtain high DNA polymorphisms.
　　MITEs are a special class of non-autonomous DNA transposons discovered in the 1990s, which have a terminal inverted repeat (TIR) of about dozens of bases and a target site duplication (TSD) at both ends, and are generally less than 500 bp in length. They are widely distributed in the genome of plants in a high-copy form. Their universality, high copy number and insertion polymorphism make them very suitable for developing molecular markers (Fig. 4).
　　URP
　　The URP (universal rice primers) technique is a molecular marker technique based on the PCR technology and single primer amplification developed by Kang et al. on rice in 2002［19］. They screened a fragment sequence (pKRD) from the genomic library of wild rice, which is widely distributed on five chromosomes of rice, with a copy number between 1 500 and 4 500. Such high-copy sequence provides an opportunity for the development of molecular markers. According to this, they randomly designed 40 single primers based on the entire fragment sequence of pKRD. The single primers were used for PCR amplification under strict conditions at a high annealing temperature of 55 ℃, and the amplified products were generally separated by agarose gel electrophoresis. In the end, 19 single primers can produce complex and rich band patterns after amplification. Among them, 12 single primers could successfully amplify and produce band polymorphisms in many different species, and these 12 single primers are called universal rice primers［19］. Although the single primers of URP molecular marker technique originally come from rice, they can be used in different species (including plants, animals and microorganisms). The single primers allow amplification at high temperatures, and the amplification results are reproducible and stable, providing scientific researchers with a choice of molecular marker technique［14,19］. 　　SCoT
　　The start codon targeted polymorphism technique (SCoT) is a molecular marker technique based on the PCR technology and single primer amplification developed by Collard and Mackill on rice in 2009. The SCoT molecular marker technique uses genomic DNA as a template (or cDNA can be used as a template for differential gene separation), and perform PCR amplification using single primers designed based on the sequences near the conservative ATG translation start sites in plant genes at a unified temperature of 50 ℃, and the amplified products are generally separated by agarose gel electrophoresis to produce polymorphic molecular markers derived from functional genes. The polymorphisms are mainly derived from point mutations at primer binding sites. Therefore, most of the molecular markers produced by the SCoT technique are dominant molecular markers, but there are also a few co-dominant molecular markers of length polymorphism caused by insertion-deletion mutations［9,20-21］.
　　Studies have shown that sequences near start codons of genes are very conservative and had consensus sequences［22-23］. The single primers used in the SCoT technique are designed based on the conservation of the flanking regions of the ATG translation start sites in plant genes. In the SCoT technique, single primers play almost the same role as the single primers of RAPD and ISSR techniques, and also serve as upstream and downstream primers. The difference is that the single primers used in the SCoT technique can simultaneously bind to the ATG translation start site regions on the positive and negative strands of double-stranded DNA, thereby amplifying the sequence between the two binding sites (Fig. 5). The primer design of the SCoT technique should meet the following requirements: ① the single primers are designed according to the conservation of the sequences near the ATG translation start sites; ② the single primers take A in ATG as the downstream +1 position, +4 position must be G, the +7 position must be A, and the +8 and +9 positions must be C; and ③ the single primers have a length of 18 bp, include no merging bases, and show a GC content in the range of 50%-72%, and no dimer and hairpin structure formation is the best［9,20-21］.
　　The SCoT molecular marker technique has several major characteristics: ① this technique is based on single-primer PCR, which is simple to operate, reproducible, and easy to establish; ② compared with the ISSR molecular marker technique, this technique is a functional molecular marker technique which can effectively produce molecular markers associated with traits, and is convenient for molecular marker-assisted breeding; ③ this technique uses longer primers and theoretically has better repeatability than RAPD molecular marker technique; and ④ the single primer design of this technique is simple, and can produce more new primers with few modifications based on the original primer sequences, and the designed single primers can be used universally among different plants due to their conservative nature. 　　CDDP
　　The CDDP (conserved DNA derived polymorphism) technique is a molecular marker technique based on conserved DNA sequences and single primer amplification developed by Collard and Mackill on rice in 2009［24］. The primers used in this molecular marker technique are designed based on a class of common conserved and consistent protein family sequences (such as transcription factors WRKY, MYB, ERF, KNOX, flowering control genes MADS-BOX and auxin-binding protein gene ABP1)  (Fig. 6), and the protein family also exists in other plants.  This type of sequences is relatively conserved in different plants. The former ensures that the designed single primers can have multiple binding sites in a plant genome, which makes it possible for a single primer to perform PCR amplification separately. The latter allows the designed single primers to be transferred between different plants, that is to say, the single primers designed by the molecular marker technique can be used as universal primers, but the binding affinity of single primers on different plants is different, the amplification efficiency will also be different, so it is necessary to design a large number of single primers and screen them. The CDDP molecular marker technique bridges the gap between the utilization of protein families conserved sequences and the development of molecular markers. The specific design requirements of the single primers used in the CDDP molecular marker technique are as follows:  ① multiple comparison softwares (Clustal W, Genedoc, DNAman, etc.) are used to make multiple comparisons of functional protein sequences or functional gene sequences from different species to clarify the conserved protein sequences or DNA sequences co-existing in different species, so as to provide anchor sites for single primers; ② the length of a single primer should range from 15 to 19 bp, its GC content should be greater than 60%, which is conducive to binding the single primer to gene exon regions (because exons are usually rich in GC content), and moreover, single primers with a GC content greater than 60% also have good amplification repeatability; and ③ generally, the conservative DNA sequences of genes or gene families related to biotic and abiotic stress and plant development were selected as anchor sites for single primer development, because most of these genes are multi-gene families, providing more anchor sites for the single primer designed, and making performing PCR amplification with a single primer lone possible. 　　IPBS
　　The iPBS (inter primer binding site) technique is a molecular marker technique based on single primer amplification developed by Kalendar et al. in 2010 that can simultaneously isolate LTR retrotransposons in plant genomes. The iPBS molecular marker technique uses genome DNA as a template, designs single primers according to the conserved tRNA binding sites (primer binding sites) adjacent to the 5 LTR sequences and performs PCR to amplify the two PBS intervals in LTR retrotransposons. The amplified products are generally separated by agarose gel electrophoresis, and the DNA polymorphisms generated by the amplification are derived from the insertion of LTR retrotransposons (Fig. 7). The iPBS technique can not only be used as a molecular marker technique, but also be used to isolate LTR retrotransposons. Theoretically, using this technique can isolate LTR retrotransposons in the genome of any species, and the isolated PBS-LTR sequences can also be used directly to design primers and develop molecular markers. This technique is simple and efficient, and can save a lot of manpower and material resources. This technique is different from the earlier SSAP, IRAP and REMAP molecular marker techniques based on LTR retrotransposons in that it can directly screen primers without the need to know the relevant LTR sequence information in advance［25］.
　　HFO-TAG
　　The HFO-TAG (high-frequency oligonucleotides-targeting active genes) technique was proposed by Levi et al. on watermelon in 2010 that uses oligonucleotides (8-10 bp) having high GC bases (>85%) and frequently appearing in expressed sequence tags (EST) as single primers for PCR amplification. The PCR amplification annealing temperature used in this molecular marker technique is 52 ℃, and the amplified products are generally separated by agarose gel electrophoresis, so the operation is simple and the amplification stability is strong. The GC content of the single primers used is high, and the polymorphic bands produced by amplification are likely to be related to functional genes. According to the application results on watermelon, the HFO-TAG molecular marker technique can distinguish four cultivars with similar genetic backgrounds, and it produces more polymorphic bands than RAPD and ISSR molecular marker techniques［26］.
　　BPS
　　For the BPS technique, like the RAPD and ISSR techniques, its technical core is to design single primers based on short conserved and consistent branch point signal sequences. The design of a single primer can be from 5 to 3 or from 3 to 5, and the single primer is used for PCR amplification. The single primer acts as both forward and reverse primers in the entire reaction, by which only the region where the distance between the forward and reverse primers is not very far can be effectively amplified, and the amplified products are separated by agarose gel electrophoresis. Compared with the RAPD technique, the BPS technique uses longer primers (15-18 bp), thereby greatly improving repeatability and stability, and the polymorphic bands generated by amplification are likely to come from the intron regions of genes［27］. 　　The branch point signal sequences are short conserved sequences with a length of 5-8 bp, the function of which is to provide signals for the formation of lasso structures required for intron cleavage. They are widely distributed in eukaryotic genes, and usually located in introns at a position 10-50 bp from the upstream receptor splicing sites［28］ or other regions except introns［29］. Therefore, the branch point signal sequences will be widespread in entire eukaryotic genomes, and are suitable for designing single primers, which can provide a large number of binding sites for single primers used in PCR amplification［27］.
　　CBDP
　　The CBDP (conserved DNA derived polymorphism) technique is a molecular maker technique based on the PCR technology and single primer amplification developed by Singh et al. on jute, cotton and flax. The single primers used in this molecular marker technique are designed according to CAAT box sequences in plant promoters. They have a length of 18 bp, and contain GC between 39% and 55%. The single primer sequences includes three parts, of which the 5 end is a filled sequence of 10 (TGAGCACGAT) or 11 bp (CTGAGCACGAT), the middle is a core sequence of 5 bp (CCAAT), and the 3 end is a random combination base sequence of 2 or 3 bp. The PCR amplification is carried out by touchdown PCR (5 cycles of denaturation at 94 ℃ for 1 min, annealing at 35 ℃ for 1 min and extension at 72 ℃ for 1 min; 35 cycles of denaturation at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min and extension at 72 ℃ for 1 min; final extension at 72 ℃ 10min), and the amplified products can be separated by agarose gel electrophoresis［30］,  producing polymorphic molecular markers, which include both dominant molecular markers caused by point mutations at primer binding sites, and co-dominant molecular markers of length polymorphisms caused by insertion-deletion mutations (Fig. 8).
　　Derivative development of single-primer molecular marker techniques
　　Different from the complete randomness of single primers used in the RAPD molecular marker technique, the single primers used in single-primer molecular marker techniques are mostly designed based on sequence units widely present in genomes, and are semi-random (or semi-specific). They tend to bind to sequence units. The single primers of the ISSR technique are designed based on microsatellite sequences; the single primers of the DAMD technique are designed based on minisatellite sequences; the single primers of the ISJ technique are designed based on the conservative splicing site sequences between exons and introns; the single primers of the SCoT technique are designed based on the conserved start codon sequence of plants; the single primers of the CDDP technique are designed based on the conservation of homologous sequences between species; and the single primers of the IMP technique are designed according to the conserved TIR sequences in MITEs. Strictly speaking, the amplification of these single primers in genomes is semi-random, has a strong anchor effect, and tends to amplify specific regions. With the continuous deepening of research, researchers continue to consciously combine single primers designed according to different genomic components into double primers to see if they can amplify new band sites or cover new ones in genomes. The RAMP technique uses shorter 5 anchored ISSR single primers in combination with RAPD single primers［31］. The REMAP technique used ISSR single primers in combination with single primers designed based on LTRs conserved sequences of retrotransposons［17］. The principle of the R-ISSR technique is basically the same as the RAMP technique, except that the primers used are short 3-anchored ISSR single primers［32］. The RMAPD technique was developed by Zhang Yongde in 2005 in the Nipponia nippon and then further applied in goats. This technique was developed under the inspiration of the RAMP technique. It combines any one of the two primers designed based on SSR sites in combination with RAPD single primers［33-34］. The FluoMEP technique was developed in animals by Chang et al. in 2007. The most important characteristic of this technique is that it have primers designed based on widespread and conserved motifs of genomes (such as intron-exon splicing site sequences, GATA/GACA repeats, vertebrate SINE sequences) and uses fluorescently labeled "common primers" in combination with RAPD single primers［35］. The ISAP and IT-ISJ techniques were developed in cotton in 2008 by Lu et al. and Zheng Liang. In both techniques, the upstream and downstream primers are designed based on the conservativeness of intron-exon splice junction sequences to amplify genomes. Generally, the principles and specific operation methods of the two techniques are basically the same, and the difference lies in the design philosophy of specific primers. The primer design of the IT-ISJ molecular marker technique uses the primer design ideas of the AFLP molecular marker technique and SRAP molecular marker technique, adding 3 selective bases to the 3, and in the primer design, for primers of sufficient length, the technique adds restriction site sequences to the 5 of primers［36-37］. The MITE-TRAP technique is a new type of molecular marker technique developed by Ma Zhongyou on rice in 2007. This technique was inspired by the SRAP molecular marker technique and the TRAP molecular marker technique. It changes the designing of fixed primers in the TRAP molecular marker technique based on EST to designing of fixed primers in the MITE-TRAP molecular marker technique based on the conserved sequences in MITEs［38］. The PAAP-RAPD technique was developed in cotton by Pang in 2009. The technique designs promoter anchor primers based on the conserved sequences of promoter regions and combines them with the RAPD single primers. It provides a method for the purposeful production of molecular markers from promoter regions［39］. 　　In addition, researchers continue to use single primers designed according to the widely existing sequence components in genomes in combination with the linker anchor primers in the traditional classic AFLP molecular maker technique. The AFLP molecular maker technique is a very powerful molecular tool, which was identified in the form of a patent in 1993, but many people used it around the world. In 1995, the inventor had no choice but to publish it in the journal Nucleic Acids Research in the form of a paper, which led to the development of many new composite molecular maker techniques established on the AFLP molecular maker technique.     The five molecular maker techniques, SAMPL(1994), MFLP(2001), MITE-AFLP(2003), DArT(2001), and (MITE)-DArT(2008), were developed by combining the traditional classic AFLP molecular maker technique and single-primer amplification reactions, and can be said to be the extension and development of the AFLP molecular maker technique. The SAMPL technique was invented by Morgante and Vogel in 1994 and published in the form of a patent. This technique uses primers designed based on the composite microsatellite sequence in the AFLP technique［40］. The MFLP technique was developed by Yang et al. in lupin in 2001. It uses the primers designed based on microsatellite sequences into the AFLP technique［41］. The MITE-AFLP technique combines the AFLP technique with the IMP technique. Due to the use of primers designed according to the conserved TIR sequences in MITEs for selective amplification, it can produce polymorphic markers caused by MITE site and flanking sequence mutations and length changes between MITE sites in addition to polymorphic markers caused by restriction site mutations［42］.  The DArT technique is a new molecular marker technique based on the AFLP technique and microarray technique, which has the characteristics of high throughput, no need for genome information, automated analysis, etc. It was developed in rice in 2001 by Jaccoud et al.［43］. The (MITE)-DArT technique is an improvement on the DArT technique by Bonin et al. in 2008 and introduced primers designed according to the conserved sequences in the transposon MITEs. The introduction of such primers can effectively generate genome representatives with reduced genome complexity［44］.
　　Conclusions
　　With the rapid development of high-throughput sequencing technology and bioinformatics, the cost and price of sequencing and bioinformatics analysis are also falling rapidly. Combined with the strong support of the state and departments at all levels in research funding, many of todays ordinary researchers can use high-throughput sequencing technology and bioinformatics to quickly and efficiently develop large quantities of SSR, InDel, and SNP markers, and then accurately and efficiently conduct research including genetic diversity analysis, germplasm resource identification, molecular fingerprinting, molecular genetics linkage map construction, gene/QTL mapping and molecular marker-assisted breeding, laying a solid technical foundation for related basic research, applied basic research and even applied research. 　　However, single-primer molecular marker techniques, as a relatively special class of multi-site dominant molecular marker techniques, will not be replaced by the large-scale SSR, InDel and SNP markers that are quickly and efficiently developed with the help of high-throughput sequencing technology and bioinformatics. They have their own advantages unmatched by SSR, InDel and SNP markers. Most of them have the advantages of simple operation, high amplification efficiency, large number of amplified bands, rich polymorphism, simple primer design, strong primer versatility, low cost of use, and low requirements for operators, and are suitable for many common laboratory research and use, such as germplasm resource, variety purity and hybrid authenticity identification which has no need for the use of high-throughput sequencing technology and bioinformatics, as these single-primer molecular marker techniques have great advantages and practical feasibility. In recent years, such single-primer molecular maker techniques have been continuously developed and have also been widely used.
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