CN104862411A - Molecular markers closely linked to root-knot nematode resistant gene Mel of pepper as well as SSR primers and application of molecular markers - Google Patents

Molecular markers closely linked to root-knot nematode resistant gene Mel of pepper as well as SSR primers and application of molecular markers Download PDF

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CN104862411A
CN104862411A CN201510309297.4A CN201510309297A CN104862411A CN 104862411 A CN104862411 A CN 104862411A CN 201510309297 A CN201510309297 A CN 201510309297A CN 104862411 A CN104862411 A CN 104862411A
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molecular markers
knot nematode
pepper
root
closely linked
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CN104862411B (en
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张宝玺
王立浩
张正海
王学瑛
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract

The invention relates to the field of molecular markers, particularly molecular markers closely linked to a root-knot nematode resistant gene Mel of pepper as well as SSR primers and application of the molecular markers. The molecular markers provided by the invention realize identification of root-knot nematode resistance of pepper plants in the seedling stage, so that the breeding time is greatly shortened. The root-knot nematode resistance of pepper can be identified through the two molecular markers, so that the breeding efficiency is improved. Compared to other molecular markers found at home and abroad, the molecular markers 24-30 and ZY6-13 are closely linked to the root-knot nematode resistant gene Mel of pepper. The genetic distances are respectively 1.825cM and 0.753cM. The molecular markers are of significant in root-knot nematode resistant breeding of pepper.

Description

With the closely linked molecule marker of gene against meloidogyne of capsicum Me1 and SSR primer thereof and application
Technical field
The present invention relates to field of molecular marker, be specifically related to and the closely linked molecule marker of gene against meloidogyne of capsicum Me1 and SSR primer thereof and application.
Background technology
Root knot nematode serious harm capsicum, the capsicum of root knot nematode main harm south China produces, but in recent years along with the increase of northern Protected production area, the raising of multiple crop index, continuous cropping continuous cropping phenomenon are general, makes nematodiasiss north regional rapid spread.In the crop loss that root knot nematode causes, more than 90% is all caused by the most Common Species of Meloidogyne incognita (M.incognita), peanut root-knot nematode (M.arenaria), javanese root knot nematode (M.javanica) and M hapla (M.hapla) four, the heaviest with Meloidogyne incognita harm.The easy contaminate environment of capsicum root knot nematode control chemical prevention, physical control highly energy-consuming DeGrain.Therefore a kind of effective means that disease-resistant variety is control nematode is cultivated.Utilize traditional breeding method general easy affected by environment, efficiency of selection is low.The molecule marker that exploitation is chain with disease-resistant gene, utilizes molecular marker assisted selection breeding to be the high-efficient breeding means that developed recently gets up.At present, capsicum to have been found that and the gene of the nematicide named has 6 at least, gene n, Me1, Me2, Me3, Me4, Me5, be dominant Dominant gene, wherein Me3 and Me4 is chain, and Me3 is to temperature-stable.
The molecular genetic location of Me3 and Me4 completes, is located on same karyomit(e), difference 9.9cM, and develops one with closely linked AFLP and mark, and between genetic distance only 2.7 ± 1.7cM.The molecular genetic location of gene M e1 is not also reported, because Me1 gene discovery comparatively early, disease resistance strong (close to immunity), therefore to the research of the molecule marker of this gene with utilize more meaningful.
Summary of the invention
The object of the present invention is to provide a kind of and the closely linked molecule marker of gene against meloidogyne of capsicum Me1.
Another object of the present invention is to provide the SSR primer pair for the identification of pepper root knot nematode resistance.
The molecule marker that can be used for auxiliary anti-root knot nematode Me1 gene Selection according to the present invention is obtained by following steps:
The present invention with pepper disease resistance kind DH330 (Me1) and susceptible variety 0516 and the BC1 colony that builds thereof for test materials, the molecule marker that the anti-gene M e1 of the auxiliary capsicum root knot nematode of research selects.SNP marker is utilized to screen the molecule marker with Me1 gene linkage, (http://passport.pepper.snu.ac.kr/ in the unigene comparison utilizing chain both sides the most closely SNP marker corresponding to capsicum CM334 and Zunla genomic information No. 9 karyomit(e)s? t=PGENOME/; Http:// peppersequence.genomics.cn/page/species/index.jsp), obtain the DNA sequence dna between two marks and utilize its developing SSR to mark, develop 233 pairs of SSR primers altogether, through primary dcreening operation, wherein 6 show polymorphism in parents and colony.By corresponding with the phenotype of BC1 colony anti-root knot nematode, target gene is positioned at 0.21Mb between SSR marker 24-20 and ZY6-13 (between physical areas 250.08 ~ 250.29Mb), genetic distance is respectively 1.825 and 0.753cM.Utilize these two pairs of molecule markers to carry out anti-root knot nematode qualification in new BC1 colony, effectively can be used for assistant breeding work.
SSR marker 24-20 is DH330 (Me1) amplified fragments size 225bp in the material, and the size in the susceptible material of susceptible material 0516 and segregating population is 210bp.
SSR marker ZY6-13 is DH330 (Me1) amplified fragments size 175bp in the material, and the size in the susceptible material of susceptible material 0516 and segregating population is 181bp.
The SSR primer for specificity screening pepper root knot nematode resistance of the application, its information is as follows.
Advantage of the present invention is not by Disease Resistance Identification, and the DNA detection just in seedling stage to pepper plant, just can judge that capsicum is for root knot nematode.Molecular mark can be widely applied to.
The invention provides the application of molecule marker 24-20 and ZY6-13 in the qualification of pepper root knot nematode resistance.
The invention provides the application of molecule marker 24-20 and ZY6-13 in capsicum annuum marker-assisted breeding.
Molecule marker provided by the invention achieves to be identified the anti-root knot nematode of pepper plant in seedling stage, substantially reduced breeding time, had saved manpower and materials.PCR and gel electrophoresis technology is adopted to detect capsicum genomic dna to be measured by utilizing Auele Specific Primer provided by the invention: amplification SSR marker 24-20, the band of 210bp is amplified, the band of the 225bp that increases in disease-resistant parent and in disease-resistant individual plant of recombinating in capsicum Susceptible parent and susceptible individual plant of recombinating; Amplification SSR marker ZY6-13, amplifies the band of 181bp, the band of the 175bp that increases in disease-resistant parent and in disease-resistant individual plant of recombinating in capsicum Susceptible parent and susceptible individual plant of recombinating.Can be identified pepper root knot nematode resistance by these two molecule markers, improve breeding efficiency.Compare other molecule marker found both at home and abroad, molecule marker 24-20 and ZY6-13 of the present invention and gene against meloidogyne of capsicum Me1 close linkage, genetic distance is respectively 1.825 and 0.753cM, significant in the breeding of capsicum anti-root knot nematode.
Accompanying drawing explanation
Fig. 1 shows five SSR marker at BC1 population segment individual plant amplification.(a)R24，(b)S7，(c)S24，(d)24-20，(e)28-1。Note: M:50bp marker; S: identical with Susceptible parent band; H: containing susceptible and disease-resistant parent's band.
Fig. 2 shows SSR marker and Me1 gene linkage relation.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The present invention to backcross the BC1 colony built with 0516 with pepper disease resistance kind DH330 (Me1) and susceptible variety 0516 and F1 thereof.
Embodiment 1 root knot nematode cultivar identification and the acquisition with closely linked molecule marker 24-20 and ZY6-13 of Me1 gene
1, capsicum Resistance Identification genetic group builds
With the material capsicum strain DH330 (Capsicum annuumL.) (French academy of agricultural sciences A Palloix provides) containing resistant to southern root knot nematode gene M e1 and susceptible material pimento self-mating system 0516 (Capsicum annuum L.) (Vegetable & Flower Inst., Chinese Academy of Agriculture Science's incubation) for parents, F1 is obtained after hybridization, F1 and 0516 BC1 obtained that backcrosses is mapping population, totally 1679 individual plants.
2, the extraction of genomic dna
Extract the genomic dna of BC1 all individual plants, parent and F1 by CTAB method, measure concentration and quality, by concentration dilution to 5ng/ μ l with Biospec-nano micro-spectrophotometer.
3, BC1 population resistance qualification
The plant 5-6 leaf phase carries out Seedling Inoculation, and all plant are watered permeable, treats that water drains, and punches near plant root 2cm, injects 1mL nematode suspension with liquid-transfering gun.Control water control temperature vegetative period, unsuitable overdrying is excessively wet, and temperature controls at 18-28 DEG C.After 60d, clean root system carefully with clear water, note not washing pieces of an egg off, load valve bag 4 DEG C preservation.Contaminate root system about 10min (Roberts et al.1990) with Yihong Huang (0.1g/L), after washing away loose colour, count pieces of an egg number (EM, egg mass).EM>5 is susceptible strain (S), and EM≤5 are disease-resistant strain (R) (source of authentication method).Result statistics twice.DH330,0516, F 1and BC1 finally identifies that strain number is respectively: 40,34,40,1583 strains.According to anti-sense standard, disease-resistant parent DH330 and F 1disease-resistant rate 100%, and pieces of an egg number is 0; Parent 0516 all shows as susceptible; The anti-sense of BC1 is than 1.04:1, χ 2=0.495<3.84, meets the theoretical segregation ratio of dominant gene 1:1.
4, SSR marker exploitation
SNP marker is utilized to screen the molecule marker with Me1 gene linkage, (http://passport.pepper.snu.ac.kr/ in the unigene comparison utilizing chain both sides the most closely SNP marker corresponding to capsicum CM334 and Zunla genomic information No. 9 karyomit(e)s? t=PGENOME/; Http:// peppersequence.genomics.cn/page/species/index.jsp), obtain the DNA sequence dna between two marks and utilize its developing SSR to mark, parameters: 2 ~ 6 bases repeat, dinucleotides multiplicity is no less than 7 times, three, tetranucleotide repeat is no less than 5 times, and five, Hexanucleotide multiplicity least four times.Select above-mentioned simple repeated sequence, intercept each 150bp fragment of tumor-necrosis factor glycoproteins upstream and downstream, design primer, primer amplification fragment 80-270bp according to design of primers principle Primer 3input (http://primer3.ut.ee/).
5, SSR linked marker screening
With parent DNA for template, carry out polymorphism screening to SSR primer, screen 6 altogether to the mark with polymorphism, then analyze BC1 colony with the mark of the polymorphism screened, these 6 pairs of primers all and Me1 gene linkage.Wherein marking 24-20 and ZY6-13 is the molecule marker that anti-root knot nematode Me1 gene linkage is positioned at gene both sides the most closely with capsicum.
PCR amplification system (10 μ L): DNA (5ng/ μ L) 4 μ L, 10x Green Mix 5 μ L, upstream primer (10 μMs/μ L) 0.5 μ L, downstream primer (10 μMs/μ L) 0.5 μ L.
Pcr amplification program: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, and 72 DEG C extend 1min, totally 32 circulations; 72 DEG C extend 8min; 16 DEG C of preservations.
SSR product detects and adopts polyacrylamide gel electrophoresis to detect.
The application of embodiment 2 molecule marker 24-20 and ZY6-13
Molecule marker 24-20 and ZY6-13 that developed is utilized to carry out Resistance Identification in the F2 colony built for parent with capsicum DH330 and 0516, identify 298 individual plants altogether, anti-sense phenotype ratio meets 3:1 segregation ratio, molecule marker 24-20 and ZY6-13 is utilized to identify respectively, molecular data and phenotypic evaluation data are coincide, and two marks detect 6 and 2 restructuring individual plants respectively.
Molecule marker 24-20 and ZY6-13 identifies that the method for root knot nematode resistance is:
(1) DNA of F2 colony to be detected individual plant is extracted;
(2) to F2 colony inoculation Meloidogyne incognita;
(3) with the DNA extracted for template, utilize mark 24-20 and ZY6-13PCR amplified band;
(4) polyacrylamide gel electrophoresis detects amplified production; Genotype is judged according to product band size.
PCR amplification system (10 μ L): DNA (5ng/ μ L) 4 μ L, 10x Green Mix 5 μ L, upstream primer (10 μMs/μ L) 0.5 μ L, downstream primer (10 μMs/μ L) 0.5 μ L.
Pcr amplification program: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, and 72 DEG C extend 1min, totally 32 circulations; 72 DEG C extend 8min; 16 DEG C of preservations.

Claims (3)

1., with the molecule marker of anti-root knot nematode Me1 gene linkage, it is characterized in that, the genetic distance of described molecule marker is respectively 1.825 and 0.753cM.
2. for the identification of the SSR primer pair of pepper root knot nematode resistance, it is characterized in that, described SSR primer pair is respectively:
SSR24-20，
PF：3’–accaataccatgtcaagaaccac-5’,PR:3’–ccagagagcttagacacccc-5’；
SSRZY6-13：
PF：3’-caagatggctaaatcacaagtca-5’,PR3’-accgcgttttcttttcttttgt5’。
3. identify a method for pepper root knot nematode resistance, it is characterized in that, described method comprises the step that the following primer pair of use a pair or two teams carries out increasing:
SSR24-20，
PF：3’–accaataccatgtcaagaaccac-5’,PR:3’–ccagagagcttagacacccc-5’；
SSRZY6-13：
PF：3’-caagatggctaaatcacaagtca-5’,PR3’-accgcgttttcttttcttttgt5’。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105420357A (en) * 2015-12-08 2016-03-23 中国农业科学院蔬菜花卉研究所 Universal SSR marker tightly linked with dominant genic male sterility genes in broccoli
CN114885830A (en) * 2022-04-08 2022-08-12 昆明理工大学 Method for cultivating pepper polygene polymerization germplasm based on combination of molecular marker-assisted selection and ploidy breeding

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1628172A (en) * 2002-03-27 2005-06-15 国立大学法人筑波大学 Novel meloidogyne-resistance gene and utilization thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1628172A (en) * 2002-03-27 2005-06-15 国立大学法人筑波大学 Novel meloidogyne-resistance gene and utilization thereof

Non-Patent Citations (2)

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Title
C. DJIAN-CAPORALINO ET AL.: "Root-knot nematode (Meloidogyne spp.) Me resistance genes in pepper (Capsicum annuum L.) are clustered on the P9 chromosome", 《THEOR APPL GENET》 *
张宇: "与辣（甜）椒抗根结线虫Me1基因紧密连锁的EST-SSR分子标记的开发", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105420357A (en) * 2015-12-08 2016-03-23 中国农业科学院蔬菜花卉研究所 Universal SSR marker tightly linked with dominant genic male sterility genes in broccoli
CN105420357B (en) * 2015-12-08 2020-10-13 中国农业科学院蔬菜花卉研究所 General SSR marker closely linked with dominant nuclear male sterility gene in broccoli
CN114885830A (en) * 2022-04-08 2022-08-12 昆明理工大学 Method for cultivating pepper polygene polymerization germplasm based on combination of molecular marker-assisted selection and ploidy breeding

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