CN111705160B - Agave hemp cpPSSR labeled primer and application thereof - Google Patents

Agave hemp cpPSSR labeled primer and application thereof Download PDF

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CN111705160B
CN111705160B CN202010761660.7A CN202010761660A CN111705160B CN 111705160 B CN111705160 B CN 111705160B CN 202010761660 A CN202010761660 A CN 202010761660A CN 111705160 B CN111705160 B CN 111705160B
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金刚
陈涛
覃旭
吴密
黄显雅
覃剑峰
彭欣怡
黄锦媛
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Abstract

The invention discloses development and application of agave sisalana cpPSSR marker primer, wherein the primer is developed based on sequencing of agave sisalana cultivar H.11648 chloroplast genome for cpPSSR marker sequences corresponding to SEQ ID NO. 21-SEQ ID NO.40, and multiple comparison analysis is carried out on the developed cpPSSR marker sequences and homologous sequences of agave published chloroplast genome species, polymorphic cpSR site screening and design of the cpPSSR marker primer are carried out, and the polymorphic primer is applied to agave sisalana resource evaluation through sequencing and comparison of PCR products. The method overcomes the problem of low development efficiency of the traditional cpSSR polymorphic marker, and fills the gap of the current cpSSR marker information of agave americana; the developed cpSSR polymorphic marker primer lays a foundation for researches such as systematic classification, genetic diversity evaluation, germplasm identification and the like of agave.

Description

Ephedra giraldii cpsR marker primer and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to development and application of a cymbidium agaves polymorphism cpSSR marker primer.
Background
Agave, also known as sisal, is a generic name for monocots of the genus Agave (Agave). As is known, there are 257 species of plants in the world, and about 50 species having important economic value. The leaves of some plant species of agave contain rich fiber which has the characteristics of white color, tough texture, high elasticity, strong tension, abrasion resistance, acid and alkali resistance, corrosion resistance, difficult slipping and the like. Due to the characteristics, the agave hemp fiber becomes a hard fiber with the largest use amount and the widest range in the world at present.
High-yield improved variety H.11648 is introduced from east Africa in 1963, and after being suitable for large-area popularization, the high-yield improved variety becomes the current variety of China. The simplification of sisal cultivars brings great hidden danger to the safety production of sisal cultivars, and potential risks are exposed. In recent years, diseases and pests such as agave hemp stem rot, purple leaf roll, pelothyrium bruguinii, new pineapple gray mealy scale and the like are increasingly serious, and the disease and pest are in considerable relation with the simplification of the agave hemp cultivated species. The method promotes gene exchange between species and genus through wide introduction and distant hybridization, and is one of effective ways for accelerating the cultivation of the excellent varieties of agave hemp. For example, agave hybrid No.11648 (hereinafter referred to as H.11648) is a high-yield cold-resistant variety bred by hybridizing pseudobromelia and sisal and backcrossing with sisal; guangxi 76416 is obtained by sexual hybridization with kenaf with thorns (resistant parent) as female parent and H.11648 (high-yield variety) as male parent.
The original place of agave is not in China, so agave germplasm resources in China are relatively deficient, the genetic basis is narrow, and available breeding resources are few. In addition, for a long time, the introduction, collection, preservation and evaluation of sisal hemp germplasm in China are not paid enough attention, so that the germplasm resource innovation work falls behind the embarrassment of production and development requirements.
The chloroplast simple sequence repeats (cpsssr) is an SSR sequence distributed in a plant chloroplast genome, and has important application values in crop genetic diversity analysis, cytoplasmic genetic relationship analysis and germplasm resource identification. At present, the development of the cpssSR markers of agave hemp and the application of the cpSSR markers in the genetic relationship analysis of agave hemp are not related. According to the invention, a batch of agave chloroplast genome sequence information of the cultivar agave is obtained by a high-throughput sequencing technology, and the agave polymorphic cpsSR marker primers are developed and obtained, so that the primers can play an important promoting role in researches such as genetic diversity analysis, germplasm genetic relationship, classification and identification of agave, and provide scientific basis for agave breeding work and innovative utilization of germplasm resources.
Disclosure of Invention
The invention aims to provide a agave cpsssr polymorphism marker primer and application thereof, aiming at the problems. Sequencing chloroplast genomes of agave hemp cultivars H.11648 by using an illumina Hiseq4000 high-throughput sequencing technology, performing multiple comparison with published other agave hemp cpDNA sequences on the basis of digging SSR sites, further developing agave hemp polymorphism cpSSR molecular markers, analyzing cytoplasm genetic diversity of 45 agave hemp materials stored by the cpSSR molecular markers, and performing cytoplasm type typing research. The molecular genetic relationship of sisal hemp germplasm resources based on the cpPSSR difference is analyzed and disclosed from the genetic differentiation angle, so that the protection and the utilization of the sisal hemp germplasm resources are enhanced; and provides basic data and scientific basis for the formulation of the collection and preservation strategy of agave hemp germplasm resources and the breeding work in China. Provides theoretical data for the selection problem of female parent in the hybrid breeding of agave hemp.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the cpPSSR marker primer for agave is composed of 20 pairs of polymorphic primers, and the nucleotide sequences of the primers are as follows:
the cpSSR21 forward primer is shown as SEQ ID NO.1 in the sequence table;
the cpSSR21 reverse primer is shown as SEQ ID NO.2 in the sequence table;
the cpSSR22 forward primer is shown as SEQ ID NO.3 in the sequence table;
the cpSSR22 reverse primer is shown as SEQ ID NO.4 in the sequence table;
the cpSSR23 forward primer is shown as SEQ ID NO.5 in the sequence table;
the reverse primer of the cpPSSR 23 is shown as SEQ ID NO.6 in the sequence table;
the cpSSR24 forward primer is shown as SEQ ID NO.7 in the sequence table;
the cpSSR24 reverse primer is shown as SEQ ID NO.8 in the sequence table;
the cpSSR25 forward primer is shown as SEQ ID NO.9 in the sequence table;
the reverse primer of the cpPSSR 25 is shown as SEQ ID NO.10 in the sequence table;
the cpSSR26 forward primer is shown as SEQ ID NO.11 in the sequence table;
the cpSSR26 reverse primer is shown as SEQ ID NO.12 in the sequence table;
the cpSSR27 forward primer is shown as SEQ ID NO.13 in a sequence table;
the reverse primer of the cpPSSR 27 is shown as SEQ ID NO.14 in the sequence table;
the cpSSR28 forward primer is shown as SEQ ID NO.15 in the sequence table;
the cpSSR28 reverse primer is shown as SEQ ID NO.16 in the sequence table;
the cpSSR29 forward primer is shown as SEQ ID NO.17 in a sequence table;
the reverse primer of the cpPSSR 29 is shown as SEQ ID NO.18 in the sequence table;
the cpSSR30 forward primer is shown as SEQ ID NO.19 in the sequence table;
the cpSSR30 reverse primer is shown as SEQ ID NO.20 in the sequence table;
the forward primer of the cpPSSR 31 is shown as SEQ ID NO.21 in the sequence table;
the cpSSR31 reverse primer is shown as SEQ ID NO.22 in the sequence table;
the cpSSR32 forward primer is shown as SEQ ID NO.23 in the sequence table;
the cpSSR32 reverse primer is shown as SEQ ID NO.24 in the sequence table;
the cpSSR33 forward primer is shown as SEQ ID NO.25 in the sequence table;
the reverse primer of the cpPSSR 33 is shown as SEQ ID NO.26 in the sequence table;
the cpSSR34 forward primer is shown as SEQ ID NO.27 in a sequence table;
the reverse primer of the cpPSSR 34 is shown as SEQ ID NO.28 in the sequence table;
the cpSSR35 forward primer is shown as SEQ ID NO.29 in a sequence table;
the cpSSR35 reverse primer is shown as SEQ ID NO.30 in the sequence table;
the cpSSR36 forward primer is shown as SEQ ID NO.31 in the sequence table;
the reverse primer of the cpPSSR 36 is shown as SEQ ID NO.32 in the sequence table;
the cpSSR37 forward primer is shown as SEQ ID NO.33 in the sequence table;
the reverse primer of the cpPSSR 37 is shown as SEQ ID NO.34 in the sequence table;
the cpSSR38 forward primer is shown as SEQ ID NO.35 in the sequence table;
the cpSSR38 reverse primer is shown as SEQ ID NO.36 in the sequence table;
the cpSSR39 forward primer is shown as SEQ ID NO.37 in the sequence table;
the cpSSR39 reverse primer is shown as SEQ ID NO.38 in the sequence table;
the cpPSSR 40 forward primer is shown as SEQ ID NO.39 in the sequence table;
the cpSSR40 reverse primer is shown as SEQ ID NO.40 in the sequence table.
Further, the primer is obtained by the following steps:
(1) And drawing a chloroplast genome completion diagram of the agave hemp cultivar H.11648 by adopting a high-throughput sequencing method:
the method comprises the following specific steps: extracting high-quality total genome DNA of the agave eaglewood cultivation variety H.11648 by adopting a CTAB method; randomly breaking DNA fragments of the total genomic DNA of agave hemp by a physical method to construct an Illumina sequencing library with the size of 300 fragments; carrying out paired-end sequencing on the sample DNA by adopting an Illumina Hiseq sequencing technology; performing quality filtration on the original data obtained by sequencing; based on the consideration of a small amount of data (2X 150bp, 10G), the chloroplast genome was assembled de novo using SPAdes V3.9.0; finally, obtaining a complete agave lily chloroplast genome sequence through head splicing, sequence comparison and assembly;
(2) Site identification of cpSSR and design of cpSSR primers:
the method specifically comprises the following steps:
1) And developing a cpPSSR marker locus in the chloroplast genome of agave hemp H.11648 by utilizing a search tool SSRIT (http:// www. Grarne. Org/db/searches/ssrtolol), wherein the parameters are respectively set as follows: the number of repeats of a mononucleotide unit is not less than 8, the number of repeats of a dinucleotide unit is not less than 3, the number of repeats of a trinucleotide unit is not less than 3, the number of repeats of a tetranucleotide and a pentanucleotide unit is not less than 3, and the number of repeats of a hexanucleotide unit is not less than 3;
2) Designing a cpSR Primer by using Primer Premier 5 software, and simultaneously combining manual adjustment; following the basic principle of PCR primer design: the length of the primer is 18-22bp, the GC content is 40-55%, the annealing temperature of the primer is 50-60 ℃, the length of the amplified fragment is 250-1000bp, and in order to ensure the accuracy of the sequencing result of a PCR product sanger, the designed site of the primer is at least 40bp from the upstream primer and the downstream primer;
3) By utilizing a method for comparing chloroplast genomics, carrying out homology comparison analysis on a developed agave hemp H.11648cpSSR marker sequence and a chloroplast genome sequence of agave plants published in a GenBank database, carrying out polymorphic cpSSR site screening and cpSSR marker primer design, and obtaining 20 pairs of candidate polymorphic chloroplast genome microsatellite molecular marker primers of agave hemp;
three other published chloroplast genomic species of Agave are Agave Americana, agave attenuata and Agave virginica.
The application of the agave daphne cpsssr labeled primer in the research on the genetic diversity and genetic relationship of agave daphne germplasm resources cytoplasm.
The application is the application described above, and the genetic diversity analysis is specifically applied, and the specific steps are as follows:
s1, extracting genome DNA: extracting agave hemp high-quality total genome DNA by a CTAB method, measuring the quality and concentration of the DNA by an ultraviolet nucleic acid tester, and detecting and confirming by 1% agarose gel electrophoresis;
s2, performing cpPSSR-PCR reaction;
cpPSSR-PCR reaction System: the amplification reaction was performed in a total volume of 50. Mu.L, containing 3. Mu.L of genomic DNA 50 ng. Mu.L -1 25. Mu.L of 2 XTaq PCR Master Mix, 2.5. Mu.L of forward primer 2. Mu. Mol L -1 2.5. Mu.L of reverse primer 2. Mu. Mol L -1 And 17. Mu.L of ddH 2 O；
cpSSR-PCR reaction program: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 35s, annealing of different cpSSR primers for 40s, extension at 72 ℃ for 45s,32 cycles; extending for 10min at 72 ℃;
s3, sequencing a PCR product sanger: carrying out sanger sequencing on the PCR product by using an ABI3730 sequencer;
s4, data analysis: judging a peak of sanger sequencing by using Bioedit software, and performing multiple comparison on sequencing results of the same SSR marker locus of a plurality of samples of the agave hemp; the allele count, gene diversity and polymorphism information content of each primer pair based on 45 parts of agave hemp material were calculated using PowerMarker3.25 software.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
1. by utilizing a high-throughput sequencing technology, the agave chloroplast whole genome sequence is obtained, and the original data used for primer development is greatly increased.
2. A chloroplast genomics method is utilized, and a candidate polymorphism cpSSR primer is developed based on the homologous comparison of cpSSR sites among different germplasms of agave hemp, so that the development and screening efficiency of the polymorphism primer is greatly improved.
3. Enriches the types of the SSR molecular markers of the agave hemp, and utilizes the developed cpPSSR polymorphic marker primer to analyze and evaluate the polymorphism among agave hemp germplasm resource groups.
Drawings
FIG. 1 is an electrophoresis chart of total DNA extraction of agave hemp H.11648 of the present invention. In the figure, M is a D15000 Marker molecular weight standard, and the bands are 15000bp, 5000bp, 2500bp, 1000bp and 250bp from top to bottom.
FIG. 2 is a physical map of chloroplast genome of agave H.11648 of the present invention.
FIG. 3 shows a map of the cpPSSR marker design site for the cp21 marker polymorphism of the present application.
FIG. 4 is a diagram showing the amplification polymorphism of the cp21 marker site primer in 45 germplasm. In the figure, the left side is marked with 45 parts
Germplasm labels, and the right part is SSR polymorphism comparison results of 45 agave germplasms at cp21 marker sites.
FIG. 5 is a cytoplasmic genetic molecular relationship diagram of 45 agave germplasm of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Examples
1.1 isolation of the Total DNA of agave hemp
Extracting high-quality total DNA of agave hemp cultivar H.11648 by a CTAB method, measuring the absorbances at 230nm, 260nm and 280nm by a NanoDrop2000 micro ultraviolet spectrophotometer, estimating the purity of the extracted total DNA, and detecting the quality of the total DNA by 1.0% agarose gel electrophoresis. The results are shown in FIG. 1.
1.2 complete map drawing of agave Pogostemonis H.11648 chloroplast genome
The total DNA of agave cultivation variety H.11648 is used for constructing PE library. DNA fragmentation DNA was fragmented using a Covaris instrument.
The fragmented DNA fragments were concentrated and recovered using Agencour AMPure XP-nucleic acid purification beads. And constructing an Illumina P150 library.
TABLE 1 detection results of purified qubit2.0 after fragmentation of samples
Sample numbering Concentration/ng/ul Volume/ul
Agave.hybrid No.11648 2.04 30
High-throughput PE (paired end) sequencing is carried out on the total DNA of the agave H.11648 by using Hiseq4000 technology of an illumina platform. Sequencing data were subjected to basic analysis (including Base Calling, vectored, decontaminated) and sequence assembly. And assembling by adopting velvet, and after the assembly is finished, performing Gap filling treatment by adopting GapCloser. The chloroplast genome of agave hemp (cultivar H.11648) is mapped through cpDNA assembly (the result is shown in FIG. 2). GenBank accession No. NC _045534.1.
1.3 annotation of the agave chloroplast genome
After sequencing is completed on the chloroplast genome of the agave H11648, the structural characteristics of the agave H11648 are analyzed. The length of chloroplast genome of agave H.11648 is 157, 274bp, and the average sequencing depth is 1065.6X. The agave cultivar h.11648 chlorophyll genome prompted us to further analyze the characteristics of agave plants and provide clues to the evolution trajectory of monocots.
TABLE 2 H.11648 chloroplast genome gene annotation table
Figure SMS_1
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Figure SMS_2
See table 2 for an explanation: wherein, the protein coding genes are 99, the rRNA genes are 8, and the tRNA genes are 40.
1.4 development of cpPSSR of agave
1.4.1 cpsR site screening
The SSR locus of the sisal hemp chloroplast genome is developed by utilizing a search tool SSRIT (http:// www. Gramene.org/db/searches/ssrtolo). The chloroplast genome refers to agave H.11648cpDNA sequence.
The parameters are set as follows: the number of the single nucleotide units is more than or equal to 8, the number of the dinucleotide units is more than or equal to 3, the number of the trinucleotide units is more than or equal to 3, the number of the tetranucleotide and pentanucleotide units is more than or equal to 3, and the number of the hexanucleotide units is more than or equal to 3. The study was mainly searching for completely repetitive SSR sites, with SSRs that circularly permuted or complement in reverse phase being considered as the same species, e.g. AAG repeat motifs including AAG, AGA, GAA, CTT, TCT and TTC. The polymorphic microsatellite marker loci were directly analyzed and discovered from the alignment results by multiple alignments of cpDNA sequences of different species within agave (Table 3, FIG. 3). The Agave species of cpDNA comparison pairs are Agave virginica, H.11648, agave attenuata and Agave Americana (the GenBank accession numbers are, respectively, agave virginica: NC-032707.1, H.11648, NC 045534.1.
And identifying 45 parts of the conventional agave germplasm resources by using 20 pairs of developed polymorphic primers, carrying out sanger sequencing on a PCR product, analyzing polymorphic sites according to a sequencing result, and constructing a phylogenetic tree by using powermarker3.25 software.
TABLE 3 genetic information for the 20 cpPSSR loci
Figure SMS_3
1.4.2 cpPSSR primer design
According to the sequence information of the candidate polymorphism cpSSR locus, the primers are designed as follows:
SSR primers for cytoplasmic genetic polymorphism analysis developed in Table 4
Figure SMS_4
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Figure SMS_5
1.5 cytoplasmic genetic relationship analysis of agave hemp germplasm resources
1.5.1 analytical methods
The developed 20 pairs of polymorphic primers are used for identifying the existing 45 agave germplasm resources, the PCR product is subjected to sanger sequencing, and polymorphic sites are analyzed according to the sequencing result (for example, FIG. 4 illustrates an amplification polymorphic diagram of the cp21 marker site in 45 germplasms). Genetic diversity parameters including allele counts, allele frequencies, polymorphism Information Content (PIC), and genetic diversity were calculated using the PowerMarker. The Nei-based method calculates genetic distances and constructs a clustering plot of sisal germplasm (fig. 5).
TABLE 5 lists 45 parts of agave germplasm resources
Reference numerals Germplasm name Reference numerals Germplasm name
1 A.attenuata 23 A.americana.wy
2 A.cantula 24 A.americana.yc
3 A.viridis 25 A.GF4
4 A.franzosinii 26 A.americana.hy
5 A.fourcroydes 27 A.potatorum
6 A.amaniensis 28 A.desmettiana.jblz
7 A.americana 29 A.ssp.yxlsl
8 A.ssp.fxm 30 A.sisalana
9 A.ssp.my1 31 A.datylio
10 A.americana.jblsl 32 A.dasylirioides
11 A.angustifolia 33 A.bracteosa
12 A.chiapensis 34 A.albopilosa
13 A.ellemeetiana 35 A.cerulata
14 A.felgeri 36 A.albescens
15 A.densiflora 37 A.angustiarum
16 A.colorata 38 A.chazaroi
17 A.andreae 39 A.avellanidens
18 A.deserti 40 A.calodonta
19 A.azurea 41 A.chrysantha
20 A.durangensis 42 A.tequilana
21 A.abisaii 43 A.titanota
22 A.hybrid.76416 44 A.horrida
45 A.havardiana
TABLE 6 statistics of genetic diversity for 45 agave germplasm
Figure SMS_6
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Figure SMS_7
20 pairs of cpSSR primers are applied to detect 20 candidate polymorphic SSR sites in 45 parts of agave hemp resources, and the 20 candidate polymorphic sites have polymorphism in 45 parts of germplasm and account for 100.00 percent of the polymorphic sites. The high efficiency of the method for developing the cpsSR polymorphic marker site is shown. PIC index is between 0.3508 and 0.7381; the gene diversity is between 0.4357 and 0.7733. Among them, the cp40 marker locus obtained the largest number of alleles (9), a PIC value of 0.6351, and a gene diversity of 0.6616 (table 6).
The above results indicate that the cpSSR marker used in this study meets the basic requirements for genetic diversity analysis.
From the cluster map 45 germplasm could be grouped into three groups from cpDNA genetic level, a. Calodotta, a. Chrysanthha, a. American. Hy, a. Attenuata and a. Densiflora clustered as a first group; felgeri and a. Colorata cluster into a second group; other germplasm clusters are in a third group; and germplasm which has important application in production at present is clustered in a third group. The clustering result can better reflect the genetic relationship among various germplasms. Research shows that the cpPSSR polymorphism primer developed by the invention can be used for genetic relationship analysis and germplasm identification of agave germplasm.
There was some genetic differentiation within 45 germplasm from the cluster map (fig. 5). Some germplasms GF4, 76416, A.amaniensis, A.angustifolia, A.cantala, A.tequila, A.fourcroydes, A.sisalana, A.virilias and the like which have important application in production are grouped into a class and have remarkable acclimation characteristics.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the present invention.
Sequence listing
<110> Guangxi Zhuang nationality autonomous region subtropical zone crop research institute (Guangxi subtropical agricultural product processing research institute)
<120> agave cpsSR marker primer and application thereof
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aaattggatt gactacggag 20
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Claims (3)

1. The cymbidium agave cpssSR labeled primer group is characterized by consisting of 20 pairs of polymorphic primers, wherein the nucleotide sequences of the primers are as follows:
the cpSSR21 forward primer is shown as SEQ ID NO.1 in the sequence table;
the reverse primer of the cpPSSR 21 is shown as SEQ ID NO.2 in the sequence table;
the cpSSR22 forward primer is shown as SEQ ID NO.3 in the sequence table;
the reverse primer of the cpPSSR 22 is shown as SEQ ID NO.4 in the sequence table;
the cpSSR23 forward primer is shown as SEQ ID NO.5 in the sequence table;
the cpSSR23 reverse primer is shown as SEQ ID NO.6 in the sequence table;
the cpSSR24 forward primer is shown as SEQ ID NO.7 in the sequence table;
the reverse primer of the cpPSSR 24 is shown as SEQ ID NO.8 in the sequence table;
the cpSSR25 forward primer is shown as SEQ ID NO.9 in the sequence table;
the reverse primer of the cpPSSR 25 is shown as SEQ ID NO.10 in the sequence table;
the cpSSR26 forward primer is shown as SEQ ID NO.11 in the sequence table;
the cpPSSR 26 reverse primer is shown as SEQ ID NO.12 in the sequence table;
the cpSSR27 forward primer is shown as SEQ ID NO.13 in the sequence table;
the cpSSR27 reverse primer is shown as SEQ ID NO.14 in the sequence table;
the cpSSR28 forward primer is shown as SEQ ID NO.15 in the sequence table;
the reverse primer of the cpPSSR 28 is shown as SEQ ID NO.16 in the sequence table;
the cpSSR29 forward primer is shown as SEQ ID NO.17 in the sequence table;
the cpSSR29 reverse primer is shown as SEQ ID NO.18 in a sequence table;
the cpSSR30 forward primer is shown as SEQ ID NO.19 in a sequence table;
the reverse primer of the cpPSSR 30 is shown as SEQ ID NO.20 in the sequence table;
the cpSSR31 forward primer is shown as SEQ ID NO.21 in a sequence table;
the reverse primer of the cpPSSR 31 is shown as SEQ ID NO.22 in the sequence table;
the cpSSR32 forward primer is shown as SEQ ID NO.23 in a sequence table;
the cpSSR32 reverse primer is shown as SEQ ID NO.24 in the sequence table;
the cpSSR33 forward primer is shown as SEQ ID NO.25 in the sequence table;
the cpSSR33 reverse primer is shown as SEQ ID NO.26 in the sequence table;
the cpSSR34 forward primer is shown as SEQ ID NO.27 in the sequence table;
the reverse primer of the cpPSSR 34 is shown as SEQ ID NO.28 in the sequence table;
the cpSSR35 forward primer is shown as SEQ ID NO.29 in the sequence table;
the cpSSR35 reverse primer is shown as SEQ ID NO.30 in the sequence table;
the cpSSR36 forward primer is shown as SEQ ID NO.31 in the sequence table;
the cpSSR36 reverse primer is shown as SEQ ID NO.32 in the sequence table;
the cpSSR37 forward primer is shown as SEQ ID NO.33 in the sequence table;
the cpSSR37 reverse primer is shown as SEQ ID NO.34 in the sequence table;
the cpSSR38 forward primer is shown as SEQ ID NO.35 in the sequence table;
the cpSSR38 reverse primer is shown as SEQ ID NO.36 in the sequence table;
the cpSSR39 forward primer is shown as SEQ ID NO.37 in the sequence table;
the reverse primer of the cpPSSR 39 is shown as SEQ ID NO.38 in the sequence table;
the cpPSSR 40 forward primer is shown as SEQ ID NO.39 in the sequence table;
the reverse primer of the cpPSSR 40 is shown as SEQ ID NO.40 in the sequence table.
2. The use of the cpsssr labeled primer set according to claim 1 for genetic diversity analysis and genetic relationship research of agave germplasm resources.
3. The use according to claim 2, wherein the genetic diversity analysis comprises the following specific steps:
s1, extracting genome DNA: extracting agave hemp high-quality total genome DNA by a CTAB method, measuring the quality and concentration of the DNA by an ultraviolet light-splitting nucleic acid measuring instrument, and detecting and confirming by 1% agarose gel electrophoresis;
s2, performing cpPSSR-PCR reaction;
cpSSR-PCR reaction System: the amplification reaction was performed in a total volume of 50. Mu.L, including a concentration of 50 ng. Mu.L -1 3. Mu.L of genomic DNA, 25. Mu.L of 2 XTAQA PCR Master Mix, and 2. Mu. Mol L of the concentration -1 2.5. Mu.L of the forward primer of (2. Mu. Mol L) -1 2.5. Mu.L of the reverse primer of (1), ddH 2 O 17 μL；
cpSSR-PCR reaction program: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 35s, annealing of different cpSSR primers 40s, extension at 72 ℃ for 45s,32 cycles; extending for 10min at 72 ℃;
s3, sequencing a PCR product sanger: carrying out sanger sequencing on the PCR product by using an ABI3730 sequencer;
s4, data analysis: judging a peak of sanger sequencing by using Bioedit software, and performing multiple comparison on sequencing results of the same SSR marker locus of a plurality of samples of the agave hemp; the allele count, gene diversity and polymorphism information content of each primer pair based on 45 parts of agave hemp material were calculated using PowerMarker3.25 software.
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CN107201408A (en) * 2017-07-15 2017-09-26 中国热带农业科学院南亚热带作物研究所 A kind of method that exploitation sisal hemp SSR primers are sequenced based on transcript profile

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