CN106755481B - SSR molecular marker VI for identifying progeny plants of Gala apples and application thereof - Google Patents
SSR molecular marker VI for identifying progeny plants of Gala apples and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of plant genetic breeding and apple germplasm innovation research, and particularly relates to an SSR molecular marker VI for identifying a progeny plant of a Gala apple and application thereof. Identifying the interlocked apple groups where the identified materials are located by using SSR molecular markers for the first time, and simultaneously disclosing SSR molecular markers interlocked with No. 13 and No. 16 chromosomes of the apples; the molecular marker is a codominant marker, and can quickly and accurately identify the Gala apple genetic linkage group, the anther culture plant and the Gala source plant; and the molecular level support is provided for accelerating the utilization of the Gala apple and important agronomic character linked genes and the genetic breeding of apple homozygous plants in the next step.
Description
Technical Field
The invention belongs to the field of plant genetic breeding and apple germplasm innovation research, and particularly relates to an SSR molecular marker VI for identifying a gala apple progeny plant and application thereof.
Background
Apple (A)Malus domesticaBorkh.) has strong ecological adaptability and high nutritive value of fruits, and is one of the fruit tree species with wide cultivation area, large consumption and better economic benefit in the world. China is one of the countries with the widest apple cultivation area and the highest total output. Particularly in northern China, apples are the fruit tree species with the largest cultivation area, and the yield and the area of the apples are the top of fruits in China. The industry becomes the post industry of many provinces and cities in China, and the industry plays a more and more role in improving the income of farmers and promoting the development of local economyThe more important the effect. Apples belong to self-flowering infertile plants, apple varieties in production are heterozygous diploid varieties, apple genomes are highly heterozygous, genetic backgrounds are quite complex, and the childhood period is long, so that the conventional cross breeding period is long, and the efficiency is low.
The breeding efficiency of the homozygous genotype germplasm can be greatly improved. Haploid plants can be obtained by anther culture, and homozygous diploid germplasm can be quickly obtained after chromosome doubling. Since apples are highly heterozygous diploid varieties, they are usually controlled by multiple alleles at a single locus, i.e. the same locus contains two different alleles, whereas haploid varieties contain only one gene. The plant obtained by anther culture should have only one allele of one of its parents if it is of haploid origin. Homozygous diploid germplasm can be obtained through anther culture, and recessive gene materials and mutation breeding materials with excellent characters can be directly obtained, and the materials have important significance for apple genetic breeding research.
Molecular marker technology has been applied in early selection of major agronomic traits of apple, such as red flesh, red skin, scab, woolly apple aphid, fire blight, etc., participating in completing the apple Genome sequencing work and jointly developing 8K (CHAGN É D, etc., Genome-wide SNP detection, identification, and definition of an 8K SNP array for apple [ J ]. P L oS ONE, 2012, 7: e31745. doi: 10.1371/journel.p. 0031745) and 20K (ANCBIO L, etc., Development and identification of a 20K Single Nucleotide Polymorphism (SNP) Genome for apple (Malus × Genome) [ J ]. P2, GS 2014: 10: 1109: 10 cross breeding of apple Genome for apple (GS 10.1371) using the breeding method of breeding in place of the apple Genome breeding program (GS 3977, GS 10.1371).
Compared with other molecular markers such as RF L P, AF L P ISSR and the like, the SSR marker has the characteristics of high polymorphism, co-dominant inheritance, good repeatability, strong specificity and the like, and becomes a marker which is most applied in the fields of genetic diversity research, genetic mapping, important functional gene positioning, molecular assisted breeding and the like in recent years.
SSR markers are widely used in fruit quality trait marker screening, variety Identification and genetic linkage map construction due to their good stability and transferability (L iu, et al. Identification of apple cucumber orientations of simple sequence repeat markers, Genet Mol Res, 2014, 13 (3): 7377 7387; Moriya, et al. Aligned genetic linkage maps of apple rootstock culture 'JM 7' and Malus sieboldii 'Sanashi 63' constrained with novel EST-SSRs. Tree Genet genes, 2012, 8 (4): 709 723.).
So far, no SSR molecular marker of the Gala apple anther culture plant is reported.
Disclosure of Invention
The invention aims to provide an SSR molecular marker VI for identifying the progeny plants of Gala apples and application thereof.
The invention is realized by the following technical scheme: an SSR molecular marker VI for identifying the progeny plants of the Gala apples is used simultaneously in the detection process, and the molecular marker is 2 pairs of SSR primers with nucleotide sequences shown as follows:
l G13 labeled GD 147:
upstream: 5，- TCCCGCCATTTCTCTGC -3，
Downstream: 5，- GTTTAAACCGCTGCTGCTGAAC -3，
L G16 marker Hi22f 06:
upstream: 5，- CAATGGCGTCTGTGTCACTC -3，
Downstream: 5，- GTTTACGACGGGTAAGGTGATGTC -3，。
An application of an SSR molecular marker VI for identifying a Gala apple progeny plant comprises the following steps:
(1) using Gala apple genome DNA as PCR amplification template, respectively using the above-mentioned SSR molecular marker as primer pair to make PCR amplification, its reaction system is 15 mu L, in which it contains 10 × PCR Buffer 1.5 mu × 0, 2.5 mM dNTPs mix 1.2 mu × 1, 10 ng/mu × 2 Primers F1.5 mu L, 10 ng/mu L Primers R1.5 mu L, 5U Taq polymerase 0.15 mu L, 100 ng/mu L DNA template 0.75 mu L and deionized water to make up it to 15 mu L, and (2) its amplification program is that 94 deg.C predisposing 2 min30 s, 94 deg.C denaturing 30 s, 60 deg.C annealing 30 s, 72 deg.C extending 40s, 35 cycles, 72 deg.C 10 min, 4 deg.C preserving for stand-by, (3) detecting PCR product, using 8% nondenaturing polyacrylamide electrophoresis, adding each non-denaturing reaction product of 1/2 to L of 1/2 deg.C denaturing voltage, uniformly mixing them, fixing 150V, fixing the constant No. 150, and freezing NO3Dyeing and photographing; (4) when the method is used for genetic linkage identification, the to-be-detected apple can amplify any one specific strip corresponding to the 2 pairs of SSR primers, so that the genetic material contained in the germplasm of the to-be-detected apple is located in the corresponding genetic linkage group, and otherwise, the genetic material contained in the germplasm of the to-be-detected apple does not have the corresponding genetic linkage group; (5) when the kit is used for identifying anther culture plants, the apple to be detected can amplify a corresponding single specific strip in the 2 pairs of SSR primers, so that the apple plant to be detected is a homozygous material, and if two strips appear, the apple plant to be detected is not the homozygous material; (6) when the method is used for variety source identification, the apple to be detected can amplify any one specific strip corresponding to the 2 pairs of SSR primers, which indicates that the apple plant to be detected is from the Gala apple, and if no specific strip is amplified, the apple plant to be detected is not a progeny variety cultured by the Gala apple.
Compared with the prior art, the invention has the following advantages:
1. the invention reports that SSR molecular markers are used for identifying the linkage group of the apples where the identification material is located for the first time at home and abroad, and reports the SSR molecular markers linked on No. 13 chromosomes and No. 16 chromosomes of the apples at the same time;
2. the molecular marker is a co-dominant marker, and can quickly and accurately identify the Gala apple genetic linkage group, the anther culture plant and the Gala breeding progeny variety;
3. the research results provide a molecular level support for accelerating the utilization of the Gala apple and important agronomic character linked genes and the genetic breeding of the apple homozygous plants in the next step;
4. provides a method for quickly identifying the molecular level verification of the Gala cultivated plants.
By identifying 2 pairs of SSR molecular markers linked on chromosomes 13 and 16 of the flower drug cultured plant of the Gala apple, the genotype type and the genetic diversity of the Gala apple can be systematically and accurately known, a foundation is laid for enriching and developing an apple allele system, a theoretical basis is provided for scientific utilization of the Gala apple in future, and the genotype and the homozygosity of the flower drug cultured plant of the Gala apple are identified. The invention has positive promoting effect on the breeding of new apple varieties and molecular marker-assisted breeding. Meanwhile, the 2 pairs of SSR molecular markers screened by the method also provide support for source verification of the progeny varieties of the Gala apples on the molecular level.
Drawings
FIG. 1 is a capillary electropherogram of L G13-labeled GD147, and FIG. 2 is a capillary electropherogram of L G16-labeled Hi22f 06.
Detailed Description
The homozygous genotype has important roles In higher Plant genetic mechanism research and Breeding application (Murovec, et al. Haploids and double Haploids In Plant Breeding. In: Abdurakhmonov I (ed) Plant Breeding: 2012: 87-106.). Apple is a fruit tree species with a highly heterozygous genome, and the difficulty of genome assembly can be greatly reduced by using a haploid genome (Dunwell. Haploids aerating plants: origins and ex-ploitation. Plant Biotechnol J, 2010, 8 (4): 377-424.). The apple is a perennial herb, the reproductive cycle is long, and the self-incompatibility causes that the method for obtaining the homozygous plant through multi-generation self-crossing is difficult to realize. The induction of embryoid production by anther Culture to obtain homozygous genotype lines is of great significance for breeding and genetic analysis of apples with highly heterozygous genotypes (German. economic embryo and dhaproid technology as available competent to Plant breeding. Plant Cell Rep,2011, 30(5): 839-. The regenerated plant is obtained by inducing embryoid through anther culture, and the ploidy and the source of the obtained regenerated plant are accurately identified, so that the method has important significance for innovative germplasm genetic analysis. In order to better utilize the germplasm materials, the invention identifies the linkage group and the genotype of the Gala apple and the plant obtained by the anther culture of the Gala apple, and provides a molecular level support for accelerating the utilization of the Gala apple and the important agronomic character linkage gene and the genetic breeding of the apple homozygous plant.
Gala apple flower medicine culture
After the Gala apples have buds in the last ten days of 4 months, selecting the mature Gala apples by a mixed sampling method, collecting well-developed buds, sealing the buds by a sealing bag, and placing the buds in a refrigerating chamber at 4 ℃ of a refrigerator for low-temperature pretreatment. After low-temperature treatment, sterilizing the flower buds in an ultra-clean workbench by using 0.1% sodium hypochlorite, taking out the anthers from the flower buds by using a pair of tweezers, inoculating the anthers to an embryoid induction culture medium, carrying out dark culture at 25 ℃, transferring the embryoids to a regeneration culture medium for regeneration after 3-5 months until the embryoids grow to 8-10 mm, carrying out subculture, carrying out rooting culture, domestication and indoor transplantation to obtain regenerated plants.
Second, SSR molecular marker analysis
1. Total DNA extraction of genome
Selecting 6 plants of the Gala apple and Gala flower cultured regeneration plants, and respectively extracting the total DNA of the anther by adopting a CTAB method (Caokifen et al, 2003). 2 pairs of SSR primers distributed on 13 # and 16 # chromosomes of the apple are selected from an apple high-density microsatellite genetic map constructed by a HiDRAS website and Okada and the like, and the HIDRAS markers of the linkage group of the regenerated plant are shown in Table 1; the primers were synthesized by Biotechnology engineering (Shanghai) Inc.
Table 1:
2. PCR amplification
The total volume of the PCR reaction system is 15 mu L, 10 × PCR Buffer 1.5 mu × 0, 2.5 mM dNTPsmixture 1.2 mu × 1, 10 ng/mu L Primers F1.5 mu L, 10 ng/mu L Primers R1.5 mu L, 5U Taq polymerase 0.15 mu L, 100 ng/mu L DNA template 0.75 mu L are contained in the PCR reaction system, deionized water is added to 15 mu L, and the amplification program is pre-denaturation at 94 ℃ for 2 min30 s, denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 40s, 35 cycles, and preservation at 72 ℃ for 10 min and 4 ℃ for later use.
3. PCR product detection
And (3) performing 8% non-denaturing polyacrylamide electrophoresis, adding 1/2 non-denaturing L oadingBuffer into each reaction product of the PCR, uniformly mixing, keeping the constant voltage at 150V for about 150min, fixing the mixture by glacial acetic acid, and performing AgNO3 staining and photographing.
The method comprises the steps of screening bands obtained by 8% of non-denatured polyacrylamide electrophoresis, analyzing markers for generating polymorphism in Gala and anther culture plants thereof, counting, selecting a primer for amplifying bands at two alleles/sites in Gala apples and only one allele/site in the Gala apple anther culture plants, amplifying products of the Gala apples with specific bands of 140 bp and 170 bp in a primer GD147 (L G13), amplifying products with specific bands of 235 bp and 241 bp in a primer Hi22f06 (L G16), and screening 2 pairs of SSR molecular markers distributed on 13 th and 16 th chromosomes to amplify the allele sites in the Gala apples and anther culture plants thereof.
And thirdly, chromosome ploidy analysis of the regeneration strain, namely cutting the regenerated plant leaves in 500 mu L lysate by using a blade, standing for 2 min, filtering in an EP tube, dyeing PI, analyzing the DNA content in the leaves by using a BD Accuri C5 flow cytometer, and taking heterozygous diploid Gala test-tube seedling leaves obtained by stem tip culture as a reference standard for ploidy identification.
And (3) carrying out chromosome ploidy identification on the survival and regeneration strain by adopting a flow cytometer. The heterozygous diploid Gala donor is taken as a control, and the result shows that: besides haploid Gala17, Gala16, Gala18, Gala19, Gala20 regenerating lines are all diploid.
Genotyping of the regenerating lines:
extraction of genomic DNA: after total DNA of leaves is extracted by adopting an improved CTAB method, the concentration of the DNA is detected by a nucleic acid protein instrument (Bio-Rad), and the concentration and the quality of the DNA are detected by 1 percent agarose gel electrophoresis.
The PCR reaction system is 25 mu L, and contains dNTP mix (10 mM) 0.5 mu L, 10 × PCR Buffer 2.5 mu L and 25 mM MgCl22.0 mu L, rTaq enzyme (5U/. mu. L) 0.2 mu. L, DNA template (100 ng/. mu. L) 1 mu. L, deionized water 17.8 mu. L. the PCR reaction program comprises the first step of pre-denaturation at 95 ℃ for 3min, the second step of denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 30 s, 10 cycles, the third step of denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 30 s, 20 cycles, and the fourth step of sufficient extension at 72 ℃ for 6 min, and storage at 4 ℃.
The results of SSR identification of the Gala regenerated plant genotype are shown in Table 2, and the results show that 2 SSR markers on the linkage group show that a heterozygote is two peaks, while a regenerated strain only has one peak. It was demonstrated that Gala16, Gala18, Gala19 and Gala20 are all homozygous genotype plants except haploid Gala 17.
And (3) carrying out plant observation on a regeneration line: and (5) carrying out the phytology characteristic survey on the test-tube plantlet of the regenerated plant. The plant observation results of the regenerated plants are shown in table 3, and the results in table 3 show that the gala heterozygous donor plant height is 5.67 cm (n = 1) and the average plant height of the homozygous diploid is 2.96 cm ± 0.44 cm (n = 28). We also observed that homozygous diploid vigour was weaker relative to gala heterozygous donors. The different diploid homozygous plants also have different vegetative characteristics, namely smaller leaves and more leaves of Gala 18.
Table 3:
fourth, results and analysis
Haploid breeding is one of the most efficient methods to obtain dominant parent donor material. The anther wall of anther is heterozygote cell, and it is possible to induce the regeneration plant of heterozygote diploid, so the obtained diploid plant is not necessarily homozygote. Heterozygous diploids and homozygous diploids of anther-cultured regenerated plants can be distinguished by identifying alleles of the regenerated plants. Previous techniques including isozyme markers, S alleles and SSR molecular markers have all been applied to the homozygosity identification of anther regenerated plants. The invention also adopts SSR identification method. Firstly, the SSR marker (from HIDRAS database (http:// www.hidras.unimi.it /)) selected is used for carrying out PCR amplification on all regeneration plants, and the SSR marker which can distinguish the regeneration plants as homozygous is screened out. To further differentiate the genotypes of the regenerated plants, we screened SSR markers distributed in linkage groups on apple chromosomes 13 and 16. The SSR marker can clearly mark different plants of the Gala apple, and provides a technical index for the genotype identification of the Gala apple in the future.
The alleles implied by the parents are detectable in anther-cultured plants, but only one allele/locus is amplified in anther-cultured plants. This indicates that the plants grown from anthers are homozygous for their genotype, i.e. haploid from the parent.
Fifth, conclusion
Homozygote plants with rich ploidy can be obtained through anther culture, which provides rich test materials for developing ploidy genetic breeding research of apples in future.
The regeneration strain and the SSR identification system obtained by the invention have important significance for analyzing and identifying the Gala excellent character gene research, field grafting and crossbreeding and phenotype-genotype correlation analysis.
And the next step is combined with the whole genome sequencing result of the Gala apples, and the parents and the anther culture plants thereof are thoroughly analyzed and compared in the genome range so as to analyze the molecular mechanism of important characters (characteristics) of the Gala apples.
Sequence listing
110 center for researching biotechnology of Shanxi province academy of agricultural sciences, institute of fruit tree of Shanxi province academy of agricultural sciences, institute of agricultural resources and economy
SSR molecular marker VI for identifying progeny plants of Gala apples and application thereof
〈160〉4
〈210〉1
〈211〉17
〈212〉DNA
Artificial sequence of < 213 >
〈220〉
Upstream primer of GD147 marked with < 223 > L G13
〈400〉1
TCCCGCCATTTCTCTGC
〈210〉2
〈211〉22
〈212〉DNA
Artificial sequence of < 213 >
〈220〉
Downstream primer of < 223 > L G13 marker GD147
〈400〉2
GTTTAAACCGCTGCTGCTGAAC
〈210〉3
〈211〉20
〈212〉DNA
Artificial sequence of < 213 >
〈220〉
Upstream primer of Hi22f06 labeled with < 223 > L G16
〈400〉3
CAATGGCGTCTGTGTCACTC
〈210〉4
〈211〉24
〈212〉DNA
Artificial sequence of < 213 >
〈220〉
Downstream primer of Hi22f06 marked by < 223 > L G16
〈400〉4
GTTTACGACGGGTAAGGTGATGTC
Claims (1)
1. The application of an SSR molecular marker VI for identifying the progeny plants of the Gala apples is characterized by comprising the following steps:
(1) apple genome DNA is used as a PCR amplification template, two pairs of Primers of SSR molecular marker VI are respectively used for PCR amplification, and the reaction system is 15 mu L, wherein the reaction system contains 10 × PCR Buffer 1.5 mu × 0, 2.5 mM dNTPs mix 1.2 mu × 1, 10 ng/mu L Primers F1.5 mu L, 10 ng/mu L Primers R1.5 mu L, 5U Taq polymerase 0.15 mu L, 100 ng/mu L DNA template 0.75 mu L, and deionized water is supplemented to 15 mu L;
(2) the amplification procedure was: pre-denaturation at 94 deg.C for 2 min and 30 s, denaturation at 94 deg.C for 30 s, annealing at 60 deg.C for 30 s, extension at 72 deg.C for 40s, and storing at 72 deg.C for 10 min and 4 deg.C for 35 cycles;
(3) detecting PCR product, performing 8% non-denaturing polyacrylamide electrophoresis, adding each reaction product of PCR into 1/2 non-denaturing L loading Buffer, mixing, keeping constant voltage at 150V for 150min, fixing with glacial acetic acid, and AgNO3Dyeing and photographing;
(4) when the SSR molecular marker VI is used for identifying anther culture plants, the apple to be detected can amplify a corresponding single specific strip in the two pairs of primers of the SSR molecular marker VI, so that the apple plant to be detected is a homozygous material, and if two strips appear, the apple plant to be detected is not a homozygous material;
the SSR molecular marker VI consists of L G13 marked GD147 and L G16 marked Hi22f06, two pairs of primers of the SSR molecular marker VI are used simultaneously in the detection process, and the sequences of the primers are as follows:
l G13 labeled GD 147:
upstream: 5，- TCCCGCCATTTCTCTGC -3，
Downstream: 5，- GTTTAAACCGCTGCTGCTGAAC -3，
L G16 marker Hi22f 06:
upstream: 5，- CAATGGCGTCTGTGTCACTC -3，
Downstream: 5，- GTTTACGACGGGTAAGGTGATGTC -3，。
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| EP1962212A1 (en) * | 2007-01-17 | 2008-08-27 | Syngeta Participations AG | Process for selecting individuals and designing a breeding program |
| CN102653790A (en) * | 2012-03-31 | 2012-09-05 | 中国农业科学院果树研究所 | Improved TP-M13-SSR molecular arking method of apple germplasm resource |
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2017
- 2017-01-20 CN CN201710040798.6A patent/CN106755483B/en not_active Expired - Fee Related
- 2017-01-20 CN CN201710040784.4A patent/CN106755481B/en not_active Expired - Fee Related
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- 2017-01-20 CN CN201710040796.7A patent/CN106755482B/en not_active Expired - Fee Related
- 2017-01-20 CN CN201710040768.5A patent/CN106755480B/en not_active Expired - Fee Related
- 2017-01-20 CN CN201710040753.9A patent/CN106755479B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104531844A (en) * | 2014-12-05 | 2015-04-22 | 中国农业科学院郑州果树研究所 | SSR genotype-based fruit tree variety distinguishing and characteristic fingerprint displaying method |
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| Microsatellite markers spanning the apple (Malus x domestica Borkh.) genome;E. Silfverberg-Dilworth等;《Tree Genetics & Genomes》;20060809;第2卷(第4期);第202-224页 * |
| Molecular identification of commercial apple cultivars with microsatellite markers;Zsolt Galli等;《HortScience》;20051231;第40卷(第7期);第1974-1977页 * |
| 苹果花药培养再生植株的倍性鉴定及SSR分析;张春芬等;《园艺学报》;20151231;第2580页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755479A (en) | 2017-05-31 |
| CN106755478A (en) | 2017-05-31 |
| CN106755483A (en) | 2017-05-31 |
| CN106755479B (en) | 2020-07-17 |
| CN106755478B (en) | 2020-07-17 |
| CN106755483B (en) | 2020-07-17 |
| CN106755480B (en) | 2020-07-17 |
| CN106755481A (en) | 2017-05-31 |
| CN106755482B (en) | 2020-07-17 |
| CN106755482A (en) | 2017-05-31 |
| CN106755480A (en) | 2017-05-31 |
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