Amaranthus plant species identification method based on high-throughput sequencing data

Abstract

The invention provides an amaranthus plant species identification method based on high-throughput sequencing data. A specific variation region in an amaranthus chloroplast genome is screened based on a high-throughput sequencing data system, and a standardized amaranthus plant species rapid identification process is developed by combining Pi value screening and specific variation region marking. Compared with a traditional DNA bar code fragment, the identification precision of the targeted specific variation region is remarkably improved, so that species with highly overlapped forms can be effectively distinguished, and the identification accuracy is improved. Meanwhile, the method greatly reduces the technical threshold and cost, PCR amplification is performed on the DNA of the sample to be detected by designing a specific primer of a targeted specific variation region, whole genome sequencing and assembly are avoided, the detection cost and detection period of a single sample are reduced, and the method is compatible with a conventional PCR platform and has high practicability. And on-site rapid detection of scenes such as a medicinal material market and a customs port becomes possible.

Description

Technical Field

[0001] This invention belongs to the field of bioinformatics, specifically relating to a plant molecular identification method based on bioinformatics, and more specifically to a method for identifying Amaranth species based on high-throughput sequencing data. Background Technology

[0002] The genus *Amaranthus* L., first named by Linnaeus in 1753, belongs to the family Amaranthaceae within the order Caryophyllales. It comprises approximately 74 species, primarily distributed in tropical, subtropical, and warm temperate regions worldwide. These plants are diverse in function, categorized by use into grain-producing species (such as *A. cruentus*, *A. hypochondriacus*, and *A. caudatus*) and traditional foliage-producing species (such as *A. tricolor* and *A. cruentus*), all possessing high nutritional value. Some species are also rich in bioactive substances with medicinal value. Amaranthus plants are highly adaptable, exhibiting significant resilience, including drought tolerance and heavy metal tolerance, thus being considered a potential alternative food crop in the context of climate change. However, this genus also includes many invasive plants that seriously threaten native ecosystems and agricultural production. These species typically possess extremely high reproductive capacity, efficient dispersal mechanisms, and broad ecological adaptability, enabling them to spread rapidly and gain ecological dominance, thus competing with native plants or crops for resources. According to the U.S. Department of Agriculture (USDA), there are currently about 20 widely distributed amaranth weeds globally, including *Amaranthus palmeri*, *Amaranthus powellii*, *Amaranthus retroflexus*, *Amaranthus spinosus*, *Amaranthus tuberculatus*, and *Amaranthus viridis*. These invasive species can significantly suppress the yields of crops such as cereals and vegetables, having a profound impact on both agricultural and non-agricultural systems. Therefore, clarifying their phylogenetic relationships is of great significance for species classification, genetic diversity assessment, and sustainable resource management.

[0003] Traditional taxonomy primarily divides the genus Amaranth into three subgenera based on morphological characteristics such as inflorescence position, number of perianth segments, fruit dehiscence, and sex: Amaranthus, Amaranthus, Acnida, and Albersia. However, due to extensive interspecific hybridization, high phenotypic plasticity, and the lack of clear boundaries for some traits, species delineation is often highly uncertain, resulting in long-standing ambiguities in phylogenetic relationships among subgenera.

[0004] The development of molecular systematics has provided new tools for resolving complex taxonomic relationships. In recent years, with the advancement of high-throughput sequencing technology, new methods, such as genomic skimming, have been able to efficiently and cost-effectively acquire single-copy nuclear gene data. These methods are applicable to a wide range of materials, from fresh samples to ancient specimens, demonstrating broad application potential. In the genus *Amaranthus*, using molecular data to improve phylogenetic resolution not only helps clarify taxonomic relationships among species but also provides important evidence for assessing phylogenetic and interspecific relationships among the three subgenera. Previous molecular phylogenetic studies have employed various molecular markers, including chloroplast DNA (such as matK and trnL), internal transcribed spacers (ITS) of nuclear genes, low-copy nuclear genes, and biallelic SNPs. Although some molecular results are consistent with traditional morphological classification, further research indicates that convergent evolution of morphological characteristics, high phenotypic plasticity, and frequent hybridization in *Amaranthus* may mask true phylogenetic signals, thus limiting the accuracy of morphology-based classification. In addition, existing molecular systems research still faces many challenges, such as insufficient sample coverage, unclear species identification, and low resolution of genetic markers, which lead to problems with accuracy and universality.

[0005] Therefore, there is an urgent need to develop a method for identifying Amaranth species based on high-throughput sequencing data, systematically screening specific highly variable regions and specific SSR markers, and applying it to rapid and low-cost identification of Amaranth species. Summary of the Invention

[0006] To address the issues of low resolution of universal DNA markers and insufficient accuracy and universality of identification results in existing methods for identifying Amaranth species, this invention provides a method for identifying Amaranth species based on high-throughput sequencing data.

[0007] The technical solution adopted in this invention is: a method for species identification of Amaranth species based on high-throughput sequencing data, comprising the following steps:

[0008] S1. Obtain the chloroplast genome database of Amaranth species and extract specific variant regions and specific SSR loci;

[0009] S2. Obtain the chloroplast genome of the sample to be tested, and align it to the chloroplast genome database based on specific variant regions and specific SSR sites to identify the species.

[0010] Preferably, step S1 includes:

[0011] S1-1. Collect multiple Amaranth samples, extract DNA, perform genome-level sequencing, assemble and annotate them to obtain the chloroplast genomes of the corresponding samples, and construct a chloroplast genome database;

[0012] S1-2. Perform multiple sequence alignment, nucleotide polymorphism analysis, and SSR analysis on the obtained chloroplast genome to screen for specific variant regions and specific SSR sites.

[0013] Preferably, the screening method for the specific mutation region includes:

[0014] Based on the chloroplast genome database, tandem sequence alignment was performed on each gene and spacer region to construct the corresponding matrix;

[0015] Based on the obtained matrix, the nucleotide diversity Pi value of the CDS sequence of the protein-coding gene and the IGS sequence of the intergenic region is calculated, and specific variant regions are obtained by screening based on the nucleotide diversity Pi value.

[0016] Preferably, the nucleotide diversity Pi value of the specific variant region is ≥0.01.

[0017] Preferably, the specific mutation region includes at least one of atpA+–atpFexon2-, rpl22+–rps19-, psbN-–psbT-_, ccsA+–rpl32-, rpl22, ndhD+–psaC-, ndhF+–rpl32+, ccsA-–ndhD-, rpl33-–rps18+, psaJ-–rpl33+, psbI+–psbK-, psaI-–ycf4+, ndhE-–psaC+, rps15+–ycf1-, rps4-–ycf3exon1+, and ndhA_exon2-–ndhI+.

[0018] Preferably, the screening method for the specific SSR sites includes:

[0019] Based on the chloroplast genome database, SSR sites were searched for in the chloroplast genome using a multi-level repeat threshold standard.

[0020] The multi-level repeat threshold criteria include: single nucleotide repeats ≥ 10 times, dinucleotide and trinucleotide repeats ≥ 5 times, and tetranucleotide, pentanucleotide and hexanucleotide repeats ≥ 3 times.

[0021] Preferably, the specific SSR site includes sites with single nucleotide repeats A / T ≥ 10 times.

[0022] Preferably, the specific SSR site includes at least one of rpoC2-–rpoC2+, rpoB-–rpoB+, trnM-CAU-–atpE-52, atpB-–atpB+, petL-–petG+, ​​and rpoA-–rpoA+.

[0023] This invention also provides a reference library for species identification of Amaranth species, including specific variant regions and specific SSR loci; the specific variant regions include atpA+–atpFexon2-, rpl22+–rps19-, psbN-–psbT-, ccsA+–rpl32-, rpl22, ndhD+–psaC-, ndhF+–rpl32+, ccsA-–ndhD-, rpl33-–rps18+, psaJ-–rpl33+, psbI+ –psbK-, psaI-–ycf4+, ndhE-–psaC+, rps15+–ycf1-, rps4-–ycf3exon1+, ndhA_exon2-–ndhI+; the specific SSR site includes at least one of rpoC2-–rpoC2+, rpoB-–rpoB+, trnM-CAU-–atpE-52, atpB-–atpB+, petL-–petG+, ​​rpoA-–rpoA+.

[0024] The present invention also provides the application of the reference library for species identification of the genus Amaranth in the identification of species of the genus Amaranth.

[0025] The beneficial effects of this invention are as follows: Based on high-throughput sequencing data, this invention assembles the chloroplast genome of Amaranth species from low-coverage or conventional resequencing data, screens and extracts 16 identified specific variant regions and species-specific SSR loci, and develops a simple and efficient species identification process that can quickly identify the source of samples without complete assembly. Compared with traditional morphological identification methods or single-marker identification methods that rely on a single or a few molecular markers, this invention utilizes whole-genome information to screen multiple chloroplast genome-specific variant regions and specific SSR loci, achieving higher resolution, effectively improving identification speed and accuracy, and adapting to data from different sequencing platforms (such as Illumina PE150) and under different DNA sample quality conditions. Compared with traditional PCR amplification systems limited by primer design, this invention directly extracts target fragments based on sequencing data, offering better flexibility and adaptability. Therefore, this invention has strong promotional value and practicality, and can simultaneously address the rapid identification of both food-type Amaranth crops and invasive Amaranth weeds, possessing broad potential for agricultural applications and ecological management.

[0026] Instruction manual illustrations

[0027] Figure 1The nucleotide polymorphism Pi value analysis is an embodiment of the present invention; wherein (a) is a nucleotide polymorphism (Pi) analysis based on 80 CDS genes, and (b) is a nucleotide polymorphism (Pi) analysis based on the intergenic regions of these 80 CDS genes; the gene and intergenic regions are arranged in a counterclockwise direction starting from the junction of SSC and IRa. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0029] To improve the accuracy of Amaranth germplasm resource identification, this embodiment integrates chloroplast genome data from 32 Amaranth species, including 30 resequencing assemblies from this embodiment and 2 from the NCBI database. Samples from 20 Amaranth species were collected through field sampling; voucher specimens are stored at the Shanghai Chenshan Botanical Garden Herbarium (CSH). Sampling information is shown in Table 1. In addition, resequencing data from 10 published Amaranth species and 2 complete chloroplast genome sequences were downloaded from the NCBI database; raw data information is shown in Table 1.

[0030] Table 1. Experimental materials information and chloroplast genome sequence accession numbers

[0031] Number Species Genus Family Source NCBI Accession Number 1 A. acanthochiton Amaranthus Amaranthaceae ------------- SRR19158648 2 A. albus Amaranthus Amaranthaceae CSHO143430 --------------- 3 A. arenicola Amaranthus Amaranthaceae ------------- SRR19158645 4 A. australis Amaranthus Amaranthaceae ------------- SRR19158645 5 A. blitoides Amaranthus Amaranthaceae CSHO130585 --------------- 6 A. blitum Amaranthus Amaranthaceae CSHO092277 --------------- 7 A. bouchonii Amaranthus Amaranthaceae RQHZ10613 --------------- 8 A. cannabinus Amaranthus Amaranthaceae ------------- SRR19158644 9 A. caudatus Amaranthus Amaranthaceae CSHO136194 --------------- 10 A. crispus Amaranthus Amaranthaceae ------------- SRR19158640 11 A. cruentus Amaranthus Amaranthaceae CSHO125858 --------------- 12 A. cuspidifolius Amaranthus Amaranthaceae ------------- SRR8666185 13 A. deflexus Amaranthus Amaranthaceae ------------- NC053787 14 A. dubius Amaranthus Amaranthaceae RQHD03140 --------------- 15 A. floridanus Amaranthus Amaranthaceae ------------- SRR19158641 16 A. greggii Amaranthus Amaranthaceae ------------- SRR19158640 17 A. hybridus Amaranthus Amaranthaceae CSHO125997 --------------- 18 A. hypochondriacus Amaranthus Amaranthaceae CSHO130664 --------------- 19 A. palmeri Amaranthus Amaranthaceae CSH0158648 --------------- 20 A. polygonoides Amaranthus Amaranthaceae CSHO112465 --------------- 21 A. powellii Amaranthus Amaranthaceae CSHO138875 --------------- 22 A. quitensis Amaranthus Amaranthaceae ERR3021435 --------------- 23 A. retroflexus Amaranthus Amaranthaceae CSHO132444 --------------- 24 A. roxburghianus Amaranthus Amaranthaceae ------------- SRR8666185 25 A. rudis Amaranthus Amaranthaceae ------------- SRR19158642 26 A. spinosus Amaranthus Amaranthaceae CSHO125842 --------------- 27 A. standleyanus Amaranthus Amaranthaceae CSHO181627 --------------- 28 A. tenuifolius Amaranthus Amaranthaceae CSHO181632 --------------- 29 A. tricolor Amaranthus Amaranthaceae CSHO112310 --------------- 30 A. tuberculatus Amaranthus Amaranthaceae ------------- NC065855 31 A. viridis Amaranthus Amaranthaceae CSHO112199 --------------- 32 A. watsonii Amaranthus Amaranthaceae ------------- NC065854 33 Achyranthes aspera Achyranthes Amaranthaceae ------------- NC050063 34 Alternanthera sessilis Alternanthera Amaranthaceae ------------- PP239384 35 Bassia scoparia Bassia Amaranthaceae ------------- NC086517 36 Celosia argentea Celosia Amaranthaceae ------------- MZ636551 37 Chenopodium album Chenopodium Amaranthaceae ------------- MW417304 38 Cyathula officinalis Cyathula Amaranthaceae ------------- OP936078 39 Froelichia latifolia Froelichia Amaranthaceae ------------- MH286309 40 Gomphrena globosa Gomphrena Amaranthaceae ------------- NC069834 41 Oxybasis glauca Oxybasis Amaranthaceae ------------- MN422308 42 Phaulothamnus spinescens Phaulothamnus Achatocarpaceae ------------- MH286322 43 Ptilotus polystachyus Ptilotus Amaranthaceae ------------- NC046575 44 Suaeda salsa Suaeda Amaranthaceae ------------- NC045302

[0032] Specifically, the method for species identification of Amaranth plants based on high-throughput sequencing data provided in this embodiment mainly includes the following steps:

[0033] S1-1. Collect multiple Amaranth samples, extract DNA, perform genome-level sequencing, assemble and annotate them to obtain the chloroplast genomes of the corresponding samples, and construct a chloroplast genome database;

[0034] S1-2. Perform multiple sequence alignment, nucleotide polymorphism analysis, and SSR analysis on the obtained chloroplast genome to screen for specific variant regions and specific SSR sites;

[0035] S2. Obtain the chloroplast genome of the sample to be tested, and align it to the chloroplast genome database based on specific variant regions and specific SSR sites to identify the species.

[0036] Further, in step S1-1, the DNA extraction method is as follows: Genomic DNA is extracted using a modified CTAB method, namely PlantZol reagent. The extracted DNA is dissolved in TE buffer for subsequent experiments. The quality and concentration of the DNA are detected using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA), and the DNA is diluted to the working concentration. Simultaneously, the integrity of the DNA is detected by 1% agarose gel electrophoresis.

[0037] Further, in step S1-1, the library construction and DNA sequencing methods for the chloroplast genome are as follows. DNA samples meeting quality standards are sent to the Illumina HiSeq™ platform (Majorbio, Shanghai, China) for whole-genome resequencing for chloroplast genome assembly.

[0038] Further, in step S1-1, the chloroplast genome assembly and annotation methods are as follows: De novo chloroplast genome assembly was performed on paired-end sequencing data from 30 samples using GetOrganelle v1.7.5.0, with the parameter set to -Fembplant_pt (plant chloroplast mode), iteratively optimizing sequence integrity. The assembly results were aligned using NCBI BLAST, and the published chloroplast genome (GenBank) with the highest nucleotide similarity (usually >99%) was selected as the reference sequence for subsequent annotation correction. Gene prediction and functional annotation were performed using CPGMAVAS2. The annotation results were visualized and verified using the CPGview-RSG online tool (http: / / 47.96.249.172:16085 / cpgview / home), and gene boundaries and orientations were adjusted. A chloroplast genome circular map was constructed in Geneious Prime, labeling gene locations and regulatory elements. Geneious v2022.1.5 software was used to perform statistical analysis on the basic characteristics of the Amaranth chloroplast genome sequences, including total genome length, four-part (LSC, SSC, IRa, IRb) length, and GC content. This embodiment successfully assembled the chloroplast genomes of 32 Amaranth species, including 8 species reported for the first time. The genome size ranged from 149,949 bp to 150,818 bp, with GC content ranging from 36.52% to 36.63%. Structural comparisons showed that the Amaranth chloroplast genomes have a highly conserved four-part structure, with consistent gene content and arrangement, containing 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes, and no rearrangements or inversions were detected.

[0039] Further, in steps S1-2, the chloroplast genome multiple sequence alignment method is as follows. To analyze structural variations among Amaranth chloroplast genomes, this embodiment employs a whole-sequence alignment method. Using the Amaranth retroflexus chloroplast genome (accession number MW646089) indexed in the NCBI database as a reference sequence, the Biopython toolkit in the Python programming environment was used to perform standardized preprocessing on the chloroplast whole-genome sequences of 30 Amaranth samples obtained in the experiment, including sequence orientation correction and redundant base removal. Annotation files in GB_MVISTA_ANNOTATION format were generated based on the annotation information of the reference genome. Subsequently, the preprocessed FASTA format sequence files and annotation files were uploaded to the mVISTA online analysis platform (https: / / genome.lbl.gov / vista / mvista / submit.shtml), and the Shuffle-LAGAN algorithm, suitable for long sequence alignment, was selected for multiple sequence alignment analysis. The visual alignment map generated by this platform allows for an intuitive assessment of sequence similarity differences among chloroplast genomes of different Amaranth species. The analysis results indicate that the overall chloroplast genome of the genus *Amaranthus* exhibits high conservation, with no genomic rearrangements or inversions detected. Further comparison revealed that the variation frequency in non-coding regions was significantly higher than that in coding regions. Specifically, large single-copy regions (LSCs) and small single-copy regions (SSCs) showed higher levels of variation, while inverted repeat regions (IRs) were relatively conserved with lower levels of variation. Collinearity analysis further supports this finding, as no gene rearrangements or inversions were observed in the chloroplast genomes of 27 *Amaranthus* species, fully confirming the conservation characteristics of the *Amaranthus* chloroplast genome.

[0040] Further, in steps S1-2, the nucleotide polymorphism analysis method is as follows. To identify highly variable genetic markers in the chloroplast genome of Amaranth species, this embodiment analyzes the nucleotide polymorphism (Pi) of genes and intergenic regions of 32 representative species. First, the sequence information of protein-coding regions (CDS) and non-coding regions (nCDS) are accurately extracted from the chloroplast genome annotation files of each species using Perl scripts (source: https: / / github.com / quxiaojian / Bioinformatic_Scripts). To ensure sequence quality, all extracted sequences undergo rigorous manual verification and standardization. In the sequence alignment stage, the latest version of MAFFT v7 is used for alignment, and all parameters are kept at default settings to ensure the comparability of results. DnaSP v.5.10 is used. The software analyzes the nucleotide diversity of the entire chloroplast genome sequence and coding regions, with a step size of 300 bp and a window size of 1,000 bp. For example... Figure 1As shown, the Pi value in the coding region ranges from 0 to 0.0128, while the intergenic region exhibits a wider range of polymorphism, extending from 0 to 0.0325. Notably, the Pi value in the intergenic region (0.03256) is 2.5 times that in the coding region (0.0128) (0.03256 / 0.0128 = 2.5), indicating that the nucleotide polymorphism level in the intergenic region is significantly higher than that in the coding region, and it exhibits heterogeneous variation characteristics. In this embodiment, genes with a Pi value greater than 0.010 are defined as specifically variant genes. Sort by Pi value from highest to lowest, the most significant specific variant regions include: atpA+–atpFexon2-, rpl22+–rps19-, psbN-–psbT-_, ccsA+–rpl32-, rpl22, ndhD+–psaC-, ndhF+–rpl32+, ccsA-–ndhD-, rpl33-–rps18+, psaJ-–rpl33+, psbI+–psbK-, psaI-–ycf4+, ndhE-–psaC+, rps15+–ycf1-, rps4-–ycf3exon1+, and ndhA_exon2-–ndhI+. Further analysis shows that these specific variant regions are mainly enriched in large single-copy regions (LSCs) and small single-copy regions (SSCs), while the degree of variation in inverted repeat regions (IRs) is relatively low.

[0041] Further, in steps S1-3, the screening method for specific SSR loci is as follows. The MICROSATELLITE (MISA) online tool was used to search for SSR loci in the chloroplast sequences of 32 Amaranth samples. A multi-level repeat threshold standard was adopted: single nucleotide repeats ≥ 10 times, dinucleotide and tertiary nucleotide repeats ≥ 5 times, and tetranucleotide, pentanucleotide, and hexanucleotide repeats ≥ 3 times. This parameter setting referenced experience in studying SSR characteristics of plant chloroplast genomes, effectively identifying polymorphic SSR loci while avoiding false positives. In this example, a total of 2414 SSR loci were identified, with relatively small differences in their number across species (71–785). Microsatellite (SSR) analysis revealed significant polymorphic characteristics in the Amaranth chloroplast genome. Among the 2414 identified SSR loci, single nucleotide repeats (1668) were dominant (69.1%), exhibiting a clear A / T preference, with only a few species (9) containing C / G repeats. There are 746 polynucleotide repeats (two to six nucleotides), among which tetranucleotide repeats (348) are the most abundant. Therefore, in this embodiment, sites with single nucleotide repeats A / T ≥ 10 times, namely rpoC2-–rpoC2+, rpoB-–rpoB+, trnM-CAU-–atpE-52, atpB-–atpB+, petL-–petG+, ​​and rpoA-–rpoA+, were selected as specific SSR sites.

[0042] S2. Obtain the chloroplast genome of the sample to be tested, and align it to the chloroplast genome database based on specific variant regions and specific SSR sites to identify the species.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for species identification of Amaranth species based on high-throughput sequencing data, characterized in that, Includes the following steps: S1. Obtain the chloroplast genome database of Amaranth species and extract specific variant regions and specific SSR loci; S2. Obtain the chloroplast genome of the sample to be tested, and align it to the chloroplast genome database based on specific variant regions and specific SSR sites to identify the species.

2. The method as described in claim 1, characterized in that, Step S1 includes: S1-1. Collect multiple Amaranth samples, extract DNA, perform genome-level sequencing, assemble and annotate them to obtain the chloroplast genomes of the corresponding samples, and construct a chloroplast genome database; S1-2. Perform multiple sequence alignment, nucleotide polymorphism analysis, and SSR analysis on the obtained chloroplast genome to screen for specific variant regions and specific SSR sites.

3. The method as described in claim 1 or 2, characterized in that, The screening method for the specific mutation region includes: Based on the chloroplast genome database, tandem sequence alignment was performed on each gene and spacer region to construct the corresponding matrix; Based on the obtained matrix, the nucleotide diversity Pi value of the CDS sequence of protein-coding genes and the IGS sequence of intergenic regions is calculated, and specific variant regions are obtained by screening based on the nucleotide diversity Pi value.

4. The method as described in claim 3, characterized in that, The nucleotide diversity Pi value of the specific variant region is ≥0.

01.

5. The method as described in claim 1, characterized in that, The specific mutation region includes atpA +– atpF exon2-、 rpl22 +– rps19 -、 psbN -– psbT -_、 ccsA +– rpl32 -、 rpl22 , ndhD +– psaC -、 ndhF +– rpl32 +、 ccsA -– ndhD -、 rpl33 -– rps18 +、 psaJ -– rpl33 +、 psbI +– psbK -、 psaI -– ycf4 +、 ndhE -– psaC +、 rps15 +– ycf1 -、 rps4 -– ycf3 exon1+ ndhA _exon2-– ndhI + at least one of them.

6. The method as described in claim 1, characterized in that, The screening method for the specific SSR sites includes: Based on the chloroplast genome database, SSR sites were searched for in the chloroplast genome using a multi-level repeat threshold standard. The multi-level repeat threshold criteria include: single nucleotide repeats ≥ 10 times, dinucleotide and trinucleotide repeats ≥ 5 times, and tetranucleotide, pentanucleotide and hexanucleotide repeats ≥ 3 times.

7. The method as described in claim 1, characterized in that, The specific SSR sites include sites with single nucleotide repeats A / T ≥ 10 times.

8. The method as described in claim 1, characterized in that, The specific SSR sites include rpoC2 -– rpoC2 +、 rpoB -– rpoB +、 trnM - CAU -– atpE - 52 , atpB -– atpB +、 petL -– petG +、 rpoA -– rpoA + at least one of them.

9. A reference library for species identification of the genus Amaranth, characterized in that, Including specific variant regions and specific SSR sites; The specific mutation region includes atpA +– atpF exon2-、 rpl22 +– rps19 -、 psbN -– psbT -_、 ccsA +– rpl32 -、 rpl22 , ndhD +– psaC -、 ndhF +– rpl32 +、 ccsA -– ndhD -、 rpl33 -– rps18 +、 psaJ -– rpl33 +、 psbI +– psbK -、 psaI -– ycf4 +、 ndhE -– psaC +、 rps15 +– ycf1 -、 rps4 -– ycf3 exon1+ ndhA _exon2-– ndhI At least one of the following; The specific SSR sites include rpoC2 -– rpoC2 +、 rpoB -– rpoB +、 trnM - CAU -– atpE - 52 , atpB -– atpB +、 petL -– petG +、 rpoA -– rpoA + at least one of them.

10. The application of the reference library for species identification of Amaranth as described in claim 9 in the identification of species of Amaranth.

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