CN104278097A - SSR molecular marker A003 for identifying sexes of kiwi fruit hybrid populations - Google Patents
SSR molecular marker A003 for identifying sexes of kiwi fruit hybrid populations Download PDFInfo
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Abstract
The invention discloses an SSR molecular marker A003 for identifying sexes of kiwi fruit hybrid populations, and relates to the technical field of molecular genetic breeding. A specific fragment nucleotide sequence of a male plant of the molecular marker is as shown in SEQ ID No1; the specific fragment nucleotide sequence of a female plant is as shown in SEQ ID No2; and primer sequences of the SSR molecular marker A003 are as shown in SEQ ID No3 and SEQ ID No4. The SSR molecular marker A003 can be used for early identification and detection of the sexes of the kiwi fruit, so that great waste caused in breeding and production of a lot of male plants is avoided, sex-related molecular markers can be quickly explored for assisting breeding by using a high-density genetic map, and important practical experience is provided for development of a functional marker. When the SSR molecular marker A003 is used for auxiliary selection of sex marking, the selection efficiency of the female plants in the breeding process is greatly increased, the breeding cycle is shortened, and the SSR molecular marker A003 has great application potential and relatively high economic value.
Description
Technical field
The present invention relates to molecular genetic breeding technical field, particularly relate to a kind of SSR molecular marker A003(A003 for Kiwifruit hybrid Population male and female sex identification and name for distinguishing other SSR molecular marker, A is Kiwifruit latin name
actinidiainitial), namely for the early molecule assisted Selection of Kiwifruit seedling male and female sex character, to improve breeding efficiency, reduce soil and human cost.
Background technology
Kiwifruit (
actinidia chinensisplanch.) being rich in fruit vitamins C, food fibre and multi mineral nutrition, being described as " king of fruit ", is a kind of wholesome emerging high-grade fruit.Along with the rapid raising of living standards of the people, significantly increase the demand of high-quality and new variety Kiwifruit, Kiwifruit breeding efficiency urgently promotes.Kiwifruit is functional dioecian plant, has the similar but Male and female flowers that male and female organ is different of floral shape.The male and female segregation ratio of Kiwifruit filial generation is about 1:1, and in view of the sex function of the different strain of Actinidia, Chinese gooseberry garden configures a small amount of pollination staminiferous plant usually to ensure orchard output and fruit quality, and remaining staminiferous plant is then removed.As perennial fruit tree, Kiwifruit is faced with the problems such as Tong Qichang, floor space be large in traditional breeding method.Kiwifruit seedling just can bloom after down planting the several years, utilizes the difference of Kiwifruit floral shape structure can differentiate the male and female sex of plant, and therefore in several years before flowering, a large amount of not result staminiferous plants causes great waste to breeding work.Owing to lacking the molecule marker that available Sex Determination in Actinidia is differentiated, the early sex qualification work of kiwi fruit plant is difficult to carry out always.The advantages such as the high and schedule of operation of SSR marker tool high polymorphism, locus specificity, stability is simple are a kind of codominant inheritance easy to detect marks.In conjunction with genetics and genomics achievement in research, carry out the research of Kiwifruit molecular mark, build economic, efficient molecular breeding technology system, become the important research direction of Kiwifruit breeding.
Summary of the invention
The object of the invention is to the shortcoming and defect overcoming prior art existence, a kind of SSR molecular marker A003 for Kiwifruit hybrid Population male and female sex identification is provided.
Specific requirement is:
Filter out the SSR molecular marker A003 for the qualification of Kiwifruit early sex;
The preparation method of above-mentioned SSR molecular marker A003 is provided;
The application of above-mentioned SSR molecular marker A003 in Kiwifruit sex identification and marker-assisted breeding is provided.
The object of the present invention is achieved like this:
Utilize Kiwifruit dense genetic map to compose, floral trait phenotypic character site, location, in sex determination interval screening SSR marker, finally obtain the SSR molecular marker A003 that can be used for the qualification of Kiwifruit early sex, form the technical scheme of this invention.The qualification of kiwi fruit plant early sex reduce to a great extent breeding early stage in soil, the waste of the production means and human cost, have important theoretical academic significance and higher economic worth.
One, SSR molecular marker A003
SSR molecular marker A003(for Kiwifruit hybrid Population male and female sex identification is called for short SSR molecular marker A003), its staminiferous plant increases the specific fragment nucleotide sequence that obtains as shown in SEQ.ID No1; Its female plant increases the specific fragment nucleotide sequence that obtains as shown in SEQ.ID No2.
Molecule marker develops based on the simple sequence repeat polymorphisms in Kiwifruit sex determination region.According to the sequence information of sex relevant range design Auele Specific Primer, the pcr amplification product of SSR molecular marker A003 shows as staminiferous plant and female plant and respectively increases and obtain two bands of a spectrum, and wherein a bands of a spectrum clip size is identical.The staminiferous plant specific spectruming belt size obtained that increases is 287bp, and specific fragment nucleotide sequence is as shown in SEQ.ID No1; The female plant specific spectruming belt size obtained that increases is 304bp, and specific fragment nucleotide sequence is as shown in SEQ.ID No2.Sex Determination in Actinidia can be distinguished by the size of specific fragment and specific nucleotide sequence.
Present invention also offers the primer of the above-mentioned molecule marker that increases.
The primer sequence of described S001 mark is as shown in SEQ.ID No3 and SEQ.ID No4.
Two, SSR molecular marker A003 preparation method
Molecule marker preparation method comprises the following steps:
1. Kiwifruit dense genetic map spectrum is built;
2. collect sex phenotype and be converted to phenotypic markers;
3. navigation watch phenotypic marker on genetic map;
4. determine on genome sequence between sex-determining region;
5. SSR marker in sex determination interval is screened.
Its working mechanism is:
There is many simple sequence repeats (SSR) unit in genome, there is highly divergent isolate in its number.Flanking sequence due to specific SSR is all the unique sequence that conservative property is stronger usually, thus can carry out pcr amplification according to flanking sequence synthetic primer, thus increase out by single microsatellite locus.Due to the variation quantitatively of single microsatellite locus repeating unit, individual amplified production just produces the polymorphism of length, detects allelic polymorphism with this.
Three, SSR molecular marker A003 application
The application of SSR molecular marker A003 in sorb Kiwifruit and A.chinensis Planch. ' No. 4, sea, osmanthus ' hybrid Population
Utilize female plant genomic dna and staminiferous plant genomic dna in the primer amplification Kiwifruit hybrid Population of above-mentioned SSR molecular marker A003, pcr amplification product shows as staminiferous plant and female plant and respectively increases and obtain two bands of a spectrum, and wherein a bands of a spectrum clip size is identical.Staminiferous plant increases the specific fragment nucleotide sequence that obtains as shown in SEQ.ID No1; Female plant increases the specific fragment nucleotide sequence that obtains as shown in SEQ.ID No2.Sex Determination in Actinidia can be identified by the size of specific fragment and specific nucleotide sequence.
In molecule marker primer amplification Kiwifruit genomic dna, PCR reacts amplification system and is:
10 μ L reaction systems comprise each 0.2 μM of forward and reverse primer, 0.2mM dNTPs, 2mM MgCl
2, 1 μ l 10X Taq buffer, 0.5 units Taq polysaccharase, 50ng DNA template.
In molecule marker primer amplification Kiwifruit genomic dna, PCR amplification program is:
95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 35s, 72 DEG C extend 50s, 33 circulations; 72 DEG C extend 5-10min.
Pcr amplification product is with polyacrylamide gel electrophoresis (8%).
The present invention has following advantages and positively effect:
1. this SSR molecular marker A003 can be used for the Forepart identification detection of Sex Determination in Actinidia, avoids a large amount of staminiferous plant in breed and production, cause great waste;
2. utilize dense genetic map to compose, can excavate the relevant molecule marker of sex fast for assistant breeding, the exploitation for functional label provides important practical experience;
3. this SSR molecular marker A003 is used for male and female Sex-linked marker assisted Selection, will greatly improve the efficiency of selection of female plant in breeding, shortens breeding cycle, has very large application potential and higher economic worth.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of this SSR molecular marker A003 in the 9th linkage group;
Fig. 2 is the polyacrylate hydrogel electrophorogram that this SSR molecular marker A003 increases in mass screening;
Fig. 3 is one of polyacrylate hydrogel electrophorogram of increasing in hybrid Population female plant and staminiferous plant of this SSR molecular marker A003;
Fig. 4 is the polyacrylate hydrogel electrophorogram two that this SSR molecular marker A003 increases in hybrid Population female plant and staminiferous plant;
english to Chinese
1, SSR(Simple Sequence Repeats) mark: be a kind of molecular marking technique based on specific primer PCR that development in recent years is got up, also referred to as microsatellite DNA (Microsatellite DNA), it is the tandem repetitive sequence reaching tens Nucleotide that a class is made up of for repeating unit several Nucleotide (being generally 1 ~ 6).
2, PCR:Polymerase Chain Reaction, polymerase chain reaction is the isothermal DNA amplification that the mid-80 grows up.
Embodiment
Describe in detail below in conjunction with drawings and Examples:
If do not specialize, the conventional means that technique means used in embodiment is well known to the skilled person.
One, the acquisition of SSR molecular marker A003
Test site: Dawu County of Hubei China province; Test period: in April, 2014
Material: test selects 174 parts ' sorbs ' and ' osmanthus extra large No. 4 ' hybrid Population to be the colony of Kiwifruit Molecular and Genetic Study, comprising 87 parts of Kiwifruit staminiferous plants and 87 parts of Kiwifruit female plants.Sorb (
a. rufa) extremely low, cold-resistant, the resistance to storage of Kiwifruit Vitamin C content, carpopodium base portion does not generate absciss layer, has the characteristic of not shedding of surviving the winter.' No. four, sea, osmanthus ' (
a. chinensiscv. ' GuiHai No.4 ') Vitamin C content is high, wide adaptability, strong stress resistance, high temperature resistant arid, and the ability of anti-anthrax, sunscald is strong, susceptible gene is low.
1. Kiwifruit dense genetic map spectrum is built
Utilize and there is respective good character and genetic affinity ' sorb ' far away and ' No. four, sea, osmanthus ' structure hybrid Population.Based on the RAD-seq technology of s-generation order-checking, simplification order-checking is carried out to genome, the SNP genotype of rapid detection parent and segregating population, build genotypic database; JoinMap 4.1 mapping software is used to build the high-density genetic linkage maps of Kiwifruit.
2. collect sex phenotype and be converted to phenotypic markers
Collect the male and female sex of each individual plant in hybrid Population, set up sex phenotype's database, the sex phenotype of female plant and staminiferous plant is converted to a phenotype molecule marker.
3. navigation watch phenotypic marker on genetic map
In conjunction with genotype SNP marker and phenotype molecule marker, sex phenotype's mark is positioned at (see figure 1) in No. 9 linkage group on genetic linkage map by utilization JoinMap 4.1 mapping software.
4. determine between sex-determining region on genome sequence
Arrange sex phenotype and mark neighbouring SNP marker, by the nucleotide sequence comparison of SNP marker to Kiwifruit genome sequence; Comparison result display sex phenotype marks contiguous SNP marker and all comes from No. 25 karyomit(e), and particular location is near 2Mb; Therefore, Kiwifruit sex relevant portions is locked in No. 25 chromosomal 1.5-2.5Mb interval.
5. SSR marker in sex determination interval is screened
Extract the nucleotide sequence in 1.5-2.5Mb interval on Kiwifruit genome No. 25 karyomit(e), search the simple repeated sequence 30 of function; Utilize Primer5.0 software, for simple repeated sequence design of amplification primers; Utilize the primer of design and synthesis, pcr amplification is carried out to Kiwifruit female plant and staminiferous plant, gropes suitable amplification condition, ensure sharpness and the stability of amplified band; Screening obtains this molecule marker and respectively can increase in staminiferous plant and female plant and obtain a key band (see figure 2).
Two, the application of SSR molecular marker A003
1, one of SSR molecular marker A003 application in sorb Kiwifruit and A.chinensis Planch. ' No. 4, sea, osmanthus ' hybrid Population
Gather the leaflet tablet of 87 parts of female plants and 87 portions of staminiferous plants in Hubei China province Dawu County's sorb Kiwifruit and A.chinensis Planch. ' No. 4, sea, osmanthus ' hybrid Population, extract plant genomic dna; Molecule marker primer SEQ.ID No3 and SEQ.ID No4 is adopted to increase to 174 parts of Kiwifruit DNA samples.
PCR reacts amplification system: 10 μ L reaction systems comprise each 0.2 μM of forward and reverse primer, 0.2mM dNTPs, 2mM MgCl
2, 1 μ l 10X Taq buffer, 0.5 units Taq polysaccharase, 50ng DNA template;
PCR amplification program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 35s, 72 DEG C extend 50s, 33 circulations; 72 DEG C extend 5-10min.174 parts of sample standard deviations increase two bands of a spectrum, and wherein one has bands of a spectrum for male and female plant.In all 87 portions of staminiferous plants, all obtain staminiferous plant specific amplified bands of a spectrum (SEQ.ID No1); In all 87 parts of female plants, all obtain female plant specific amplified bands of a spectrum (SEQ.ID No2); Fig. 3 shows the polyacrylate hydrogel electrophorogram that this SSR molecular marker A003 increases in hybrid Population female plant and staminiferous plant.
Qualification result:
2, the application two of SSR molecular marker A003 in sorb Kiwifruit and A.chinensis Planch. ' No. 4, sea, osmanthus ' hybrid Population
Gather the leaflet tablet of 100 parts of female plants and 100 portions of staminiferous plants in Hubei China province Wuhan City's sorb Kiwifruit and A.chinensis Planch. ' No. 4, sea, osmanthus ' hybrid Population, extract plant genomic dna; Molecule marker primer SEQ.ID No3 and SEQ.ID No4 is adopted to increase to 200 parts of Kiwifruit DNA samples.
PCR reacts amplification system: 10 μ L reaction systems comprise each 0.2 μM of forward and reverse primer, 0.2mM dNTPs, 2mM MgCl
2, 1 μ l 10X Taq buffer, 0.5 units Taq polysaccharase, 50ng DNA template;
PCR amplification program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s; 58 DEG C of annealing 35s, 72 DEG C extend 50s, 33 circulations; 72 DEG C extend 5-10min.200 parts of sample standard deviations increase two bands of a spectrum, and wherein one has bands of a spectrum for male and female plant.In all 100 portions of staminiferous plants, all obtain staminiferous plant specific amplified bands of a spectrum (SEQ.ID No1); In all 100 parts of female plants, all obtain female plant specific amplified bands of a spectrum (SEQ.ID No2); Fig. 4 shows this SSR molecular marker A003 sex identification and is marked at the polyacrylate hydrogel electrophorogram increased in hybrid Population female plant and staminiferous plant.
Qualification result
sequence table
<110> Wuhan Botanical Garden, Chinese Acadmey of Sciences
The SSR molecular marker A003 of <120> Kiwifruit hybrid Population male and female sex identification
<140>
<141>
<160>?4
<210>?1
<211>?287
<212> molecule marker
<400>?XXXXXXXGGGGTAAATTTGTACAGTACGTATACAGTTGTCTTATCCACAATACCTCTCTCTCTCACACACAAGCGCACACAGGCTGTGGATATCCATCTTCACGTTCAATGACAGTCTGACCAACCCCTCTCTCTCTCTCTCTCCATATCTCCAAATATCTCAATCTACAATGGCGATGTCGTTCTCACCCCCCAAACCCAGTACGGACAACGTACCACCAGCCCAAGCATGGCCTAAACCCTCCCAAAGCCATTCTCTCCCTCCGAGGTCCGTAAAGCTCCTCCTA；
<210>?2
<211>?304
<212> molecule marker
TGCAAGCGGGGGTAAATTTGTACAGTACGTATACAGTTGTCTTATCCACAATACCTCTCTCTCTCTCACACACAAGCGCACACAGGCTGTGGATATCCATCTTCACGTTCAATGACAGTCTGACCAACCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCATATCTTCAAATATCTCAATCTACAATGGCGATGTCGTTCTCACCCCCCAAACCCAGTACGGACAACGTACCACCAGCCCAAGCATGGCCTAAACCCTCCCAAAGCCATTCTCTCCCTCCGAGGTCCGTAAAGCTCCTCCTA；
<210>?3
<211>?24
<212> primer-1
<400>?GCAAGCGGGGGTAAATTTGTACAG；
<210>?4
<211>?24
<212> primer-2
<400>?GGATAGGAGGAGCTTTACGGACCT。
sequence table
<110> Wuhan Botanical Garden, Chinese Acadmey of Sciences
The SSR molecular marker A003 of <120> Kiwifruit hybrid Population male and female sex identification
<140>
<141>
<160>?4
<210>?1
<211>?287
<212> molecule marker
<400>?XXXXXXXGGGGTAAATTTGTACAGTACGTATACAGTTGTCTTATCCACAATACCTCTCTCTCTCACACACAAGCGCACACAGGCTGTGGATATCCATCTTCACGTTCAATGACAGTCTGACCAACCCCTCTCTCTCTCTCTCTCCATATCTCCAAATATCTCAATCTACAATGGCGATGTCGTTCTCACCCCCCAAACCCAGTACGGACAACGTACCACCAGCCCAAGCATGGCCTAAACCCTCCCAAAGCCATTCTCTCCCTCCGAGGTCCGTAAAGCTCCTCCTA；
<210>?2
<211>?304
<212> molecule marker
TGCAAGCGGGGGTAAATTTGTACAGTACGTATACAGTTGTCTTATCCACAATACCTCTCTCTCTCTCACACACAAGCGCACACAGGCTGTGGATATCCATCTTCACGTTCAATGACAGTCTGACCAACCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCATATCTTCAAATATCTCAATCTACAATGGCGATGTCGTTCTCACCCCCCAAACCCAGTACGGACAACGTACCACCAGCCCAAGCATGGCCTAAACCCTCCCAAAGCCATTCTCTCCCTCCGAGGTCCGTAAAGCTCCTCCTA；
<210>?3
<211>?24
<212> primer-1
<400>?GCAAGCGGGGGTAAATTTGTACAG；
<210>?4
<211>?24
<212> primer-2
<400>?GGATAGGAGGAGCTTTACGGACCT。
Claims (3)
1., for a SSR molecular marker A003 for Kiwifruit hybrid Population male and female sex identification, it is characterized in that:
Staminiferous plant specific fragment nucleotide sequence is as shown in SEQ.ID No1;
Female plant specific fragment nucleotide sequence is as shown in SEQ.ID No2;
The primer sequence of described SSR molecular marker A003 is as shown in SEQ.ID No3 and SEQ.ID No4.
2., by the preparation method of SSR molecular marker A003 according to claim 1, it is characterized in that comprising the following steps:
1. Kiwifruit dense genetic map spectrum is built;
2. collect sex phenotype and be converted to phenotypic markers;
3. navigation watch phenotypic marker on genetic map;
4. determine on genome sequence between sex-determining region;
5. SSR marker in sex determination interval is screened.
3., by the application of SSR molecular marker A003 according to claim 1, it is characterized in that:
The application of molecule marker in sorb Kiwifruit and A.chinensis Planch. ' No. 4, sea, osmanthus ' hybrid Population.
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Cited By (7)
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| CN104561332A (en) * | 2015-01-20 | 2015-04-29 | 黑龙江省林业科学研究所 | SSR molecular marker for identifying aspen gender and application of SSR molecular marker |
| CN106048064A (en) * | 2016-08-12 | 2016-10-26 | 西南林业大学 | SSR molecular marker used for identifying female Pistacia chinensis Bunge and primer pair thereof |
| CN107338318A (en) * | 2017-08-25 | 2017-11-10 | 中国科学院武汉植物园 | Molecular labeling Geo101 primers and application for Kiwi berry mill mountain system row Male cultivar identification |
| CN107385071A (en) * | 2017-08-25 | 2017-11-24 | 中国科学院武汉植物园 | Molecular labeling primer and application for Kiwi berry mill mountain system row Male cultivar identification |
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| CN111394495A (en) * | 2020-03-02 | 2020-07-10 | 中国科学院武汉植物园 | General molecular marker primer for sex identification of commercial varieties of kiwi fruits and application |
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| CN104561332B (en) * | 2015-01-20 | 2016-10-05 | 黑龙江省林业科学研究所 | A kind of SSR molecular marker identifying Populus davidiana sex and application thereof |
| CN106048064A (en) * | 2016-08-12 | 2016-10-26 | 西南林业大学 | SSR molecular marker used for identifying female Pistacia chinensis Bunge and primer pair thereof |
| CN107338318A (en) * | 2017-08-25 | 2017-11-10 | 中国科学院武汉植物园 | Molecular labeling Geo101 primers and application for Kiwi berry mill mountain system row Male cultivar identification |
| CN107385071A (en) * | 2017-08-25 | 2017-11-24 | 中国科学院武汉植物园 | Molecular labeling primer and application for Kiwi berry mill mountain system row Male cultivar identification |
| CN109055597A (en) * | 2018-09-27 | 2018-12-21 | 中国科学院武汉植物园 | Molecular labeling primer and application for No. 1 cultivar identification of Kiwi berry Mi jujube |
| CN109055597B (en) * | 2018-09-27 | 2021-08-03 | 中国科学院武汉植物园 | Molecular marker primer for identifying kiwi fruit and kiwi fruit variety No. 1 and application |
| CN109554503A (en) * | 2019-01-31 | 2019-04-02 | 中国农业科学院郑州果树研究所 | A kind of tara vine seedling gender identification molecular labeling and its application in early days |
| CN111394495A (en) * | 2020-03-02 | 2020-07-10 | 中国科学院武汉植物园 | General molecular marker primer for sex identification of commercial varieties of kiwi fruits and application |
| CN111394495B (en) * | 2020-03-02 | 2021-06-18 | 中国科学院武汉植物园 | General molecular marker primer for sex identification of commercial varieties of kiwi fruits and application |
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