CN111088388B - InDel marker and primer pair for identifying flesh red/non-red character of peach fruit and application of InDel marker and primer pair - Google Patents
InDel marker and primer pair for identifying flesh red/non-red character of peach fruit and application of InDel marker and primer pair Download PDFInfo
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Abstract
The invention discloses an InDel marker, a primer pair and application thereof for identifying peach flesh red/non-red traits, wherein the InDel marker is positioned between 707474bp and 707475bp of a 5 th chromosome of a peach genome, the sequence of a corresponding upstream primer BLproF is shown as SEQ ID No.2, and the sequence of a corresponding downstream primer BLproR is shown as SEQ ID No. 3. The invention utilizes the upstream primer of the primer pair on the known reference sequence and the downstream primer on the insertion sequence, the amplification product is 1041bp, and the accuracy of the molecular marker genotype is verified on 455 single plants to be detected in total of 3 hybridization groups. The result shows that all the single plants with the amplified band of about 1041bp are red flesh peaches, and the single plants without the amplified band are non-red flesh peaches, and the verification accuracy rate reaches 100%. The InDel marker disclosed by the invention is used for identifying the color of peach pulp, has the advantages of rapidness, convenience, low cost and the like, and can be applied in large scale in production.
Description
Technical Field
The invention relates to an InDel marker and a primer pair for identifying the color (red/non-red) character of peach pulp and application thereof, belonging to the technical field of biology.
Background
The pulp color is used as the main agronomic character of the peach, and plays an important sensory role in evaluating the quality of the peach fruit. The peach pulp is divided into white meat, yellow meat and red meat according to the content of anthocyanin, wherein the red meat is rich in anthocyanin, so that the peach pulp not only has strong visual impact, but also has the effects of removing free radicals in vivo, resisting tumors and preventing cardiovascular sclerosis; in addition, anthocyanin in the red meat is used as a pigment additive, and has the advantages of being natural, healthy and the like. Therefore, the red-flesh peaches are very popular among consumers. However, most of red-meat peaches planted at present are local varieties, and have defects in appearance, flavor and hardness, and breeding strength needs to be increased to obtain a red-meat peach variety with good commodity. However, for fruits, the color of the fruits can be observed only after the fruits are planted, and at least 3 years are needed from the field planting of seedlings to the fruit setting of the peach trees, so that the selection time is later; in addition, peach trees are tall and large, occupy a lot of land, have long growing seasons, and a large amount of seedling management needs high cost in a few years before the observation of the color of the pulp. These are not beneficial to the breeding effect, so that the development of molecular markers closely linked with the color character eliminates plants irrelevant to the red meat character in the seedling stage, can reduce the selective population, reduce the breeding cost and improve the breeding efficiency, and has important significance for promoting the breeding process of the red meat peaches.
The research on the red meat character and the molecular marker positioning is related to scholars at home and abroad. Quilot et al mapped the red meat trait to chromosome 3 using 'pruneus davidiana clone P1908' and 'Summergrand', and the molecular markers linked to this trait were CFF14, UDP 96-008. Gillen et al mapped the red meat trait on chromosome 4 and the linked molecular marker was C41H, which is about 10.3cM genetic distance from the red meat trait. Shenshijun et al also mapped the red meat trait on chromosome 4, and the linkage markers were SNP-IGA-386619 and SNP-IGA-378198. In addition, the authors mapped another red meat trait to the top of chromosome 5, and the linked molecular markers were AMPPGl44, AMPPGl52, AMPPGl57, AMPPGl78 and CPPCT040, and genotyping indicated that 80 individual mapping populations had only one marker recombined with the trait. The red meat trait is positioned between the BPPCT016 and M7/E6-280 markers by utilizing the populations of 'Daidao' and 'Tianjin honey' F1 in autumn peace and the like, and the genetic distance between the two markers is about 0-20 cM. Zhou Hui et al used the ` Dahongpao ` and ` eosin ` F2 populations as materials, and also located the red flesh trait at the top of the 5 th chromosome, the linked molecular markers were WPS21, WPS22 and WPS23, wherein WPS23 was heterozygous in Dahongpao and belonged to the ` ef. eg ` type marker, and when the genotype of the filial generation was ee or eg, the flesh was red and when ef or fg, the flesh was non-red.
At present, some of the existing linkage markers are far away from the red meat character, and the accuracy rate in early identification of filial generations is low; although some molecular markers are closely linked with red meat characters and have high identification accuracy in filial generations, the molecular markers are not main factors causing the red meat, are SSR type markers and have complicated identification steps, so that the development of the molecular markers with high accuracy and convenient operation is necessary.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an InDel marker for identifying a peach pulp color (red/non-red) character, a primer pair and application thereof, wherein the marker has high accuracy in identifying the peach pulp color (red/non-red) character.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an InDel marker for identifying the flesh red/non-red character of peach fruits is positioned between 707474bp and 707475bp of the 5 th chromosome of a peach genome.
An InDel marker for identifying peach flesh red/non-red traits is disclosed, wherein a nucleotide sequence of a corresponding upstream primer BLproF is shown as SEQ ID No.2, and a nucleotide sequence of a corresponding downstream primer BLproR is shown as SEQ ID No. 3.
A PCR amplification primer pair for detecting an InDel marker is disclosed, wherein an upstream primer is designed according to an upstream sequence of an insertion deletion site, and a downstream primer is designed according to a sequence on the insertion site.
The primer pair consists of two single-stranded DNAs shown by an upstream primer BLproF and a downstream primer BLproR.
The nucleotide sequence of the upstream primer BLproF is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer BLproR is shown in SEQ ID NO. 3.
A kit for identifying the peach flesh red/non-red character is characterized by comprising the primer pair.
An application of the InDel marker in the aspect of peach fruit color character molecular marker-assisted selective breeding.
An application of the InDel marker in identifying the color (red/non-red) of peach pulp.
A method for identifying peach pulp color (red/non-red) character by using an InDel marker, comprising the following steps:
(1) and (3) extracting DNA: collecting leaves, extracting genome DNA as a template for PCR amplification;
(2) and (3) PCR amplification: performing PCR amplification by using the extracted DNA as a template and a PCR primer pair to obtain a corresponding PCR amplification product;
(3) electrophoresis: detecting the size of the PCR amplification product band obtained in the step (2) by using agarose gel electrophoresis;
(4) genotyping: and (4) observing the molecular weight of the PCR amplification product, and carrying out genotyping according to the size.
The invention has the beneficial effects that:
the invention utilizes the upstream primer of the primer pair on the known reference sequence and the downstream primer on the insertion sequence, the amplification product is 1041bp, and the accuracy of the molecular marker genotype is verified on 455 single plants to be tested which are counted by 3 hybridization groups. The result shows that all the single plants with the amplified band of about 1041bp are red flesh peaches, and the single plants without the amplified band are non-red flesh peaches, and the verification accuracy rate reaches 100%. The method for identifying the color of peach pulp by using the nucleotide insertion deletion (InDel) marker has the advantages of rapidness, convenience, low cost and the like, and can be applied to large scale in production.
Drawings
FIG. 1 is a partial electrophoretogram of a population of hybrids 07-4-28 (red meat) x Yangzhou 431 (white meat).
FIG. 2 is a partial electrophoretogram of a 09-1 West-28 (red meat) x morning glory (white meat) hybrid population.
FIG. 3 is a partial electrophoretogram of a 07-4-28 (red meat). times.medium oil No. 13 (yellow meat) hybrid population.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 obtaining of an insertion deletion marker (InDel)
The method takes two germplasms of peach germplasm resources of Zhengzhou fruit tree research institute of China academy of agricultural sciences, 'Tianjin honey (red meat)' and 'Baifeng (white meat)' as samples, extracts sample DNA by adopting a CTAB improvement method, and performs re-sequencing on the two germplasms through third-generation sequencing to obtain 18Gb data, wherein the average sequencing depth is about 30 multiplied. From sequencing of 10kb-100kb reads, aligned with peach reference genome v.2.0(ftp:// ftp. biooinfo. wsu. edu/species/Prunus _ Persica-genome. v2.0.a1/), 12692 large nucleotide indels were identified, where at the BL gene promoter position (between 707474bp and 707475bp of the 5 th chromosome of peach genome), "Tianjin Honey" has an insertion of 6647 bp. While 'white phoenix' does not. Then, an upstream primer is designed according to an upstream reference sequence of the insertion site, and a downstream primer is designed according to a 5' end sequence of the insertion fragment and is used as a molecular marker for identifying the color (red/non-red) of the pulp.
Example 2 method for identifying peach pulp color using nucleotide insertion (InDel) labeling
1. DNA extraction
Extracting the DNA of a sample tissue (leaf) to be detected by adopting an improved CTAB method, wherein the volume of the DNA sample is not less than 30 mu l. Measuring OD values of DNA sample at 260nm and 280nm with ultraviolet spectrophotometer, and calculating DNA content and OD260/280The ratio of (a) to (b). DNA sample purity OD260/280The value should be between 1.8-2.0, and the concentration should be diluted to about 10 ng/. mu.l.
2. Primer design
Primers were designed based on the reference sequence upstream of the insertion site and the 5' end sequence of the insert (total of about 1680bp), as shown in Table 1 below.
TABLE 1 insertion site two-terminal sequence information
Note: the nucleotide sequence of the grey part is the reference sequence upstream of the insertion site, and the nucleotide sequence of the rest part is the sequence on the insertion fragment.
Primers were synthesized by Biotech and then applied ddH2O was dissolved and diluted to 10. mu.M. The primer sequences are shown in Table 2.
TABLE 2 primer sequences
3. PCR amplification and banding pattern analysis
(1) PCR amplification system, see table 3:
TABLE 3 PCR amplification System
The prepared reagent mixture is transferred to a PCR tube or plate and put into a PCR instrument for PCR amplification, and the amplification procedures are shown in Table 4.
TABLE 4 PCR amplification procedure
Preparing 1% agarose gel, carrying out electrophoresis on the amplified product at 150V for 15-20min, and observing whether the band exists or not. When the amplified bands are at the 1041bp position of the Marker, the corresponding single plants are all red meat, and when the bands are not amplified, the corresponding single plants are all non-red meat.
Example 3, 3 Cross-over populations were verified by blind testing for performance using the InDel marker of the invention
1. Selection of test materials
Conventional peach varieties planted in resource gardens of Zhengzhou fruit tree research institutes are taken as experimental materials, 3 red flesh peach hybrid groups with investigated phenotypic characters are selected from the experimental materials, and 455 individual plants are selected in total, and the specific table 5 shows.
TABLE 5 name of test Cross population and population size information (Unit: Strain)
Note: 07-4-28 and 09-1 west-28 are red meat excellent lines, Yangzhou 431 is a white meat local variety, morning glory is a white meat breeding variety, and Medium oil No. 13 is a yellow meat breeding variety.
2. Identification method using InDel marker of the invention
The InDel marker is utilized to carry out blind test identification on the color of 455 individual pulps of 3 red flesh peach crossbred groups, and the specific identification method refers to the method in the embodiment 2. Meanwhile, SSR markers reported in China are used as references (Zhouhui, research on a regulation and control mechanism of anthocyanin coloration and procyanidine synthesis, institute of Chinese academy of sciences (Wuhan plant Garden)), and are compared and analyzed with the InDel markers disclosed by the invention.
3. Accuracy of typing results in hybrid populations to table prediction
As can be seen from the identification results in Table 6, when the InDel marker of the invention is used for performing phenotype prediction on a hybrid population, the identification accuracy of the red meat/non-red meat character of the fruit reaches 100 percent, and is consistent with the identification result of the SSR marker, and partial electrophoresis charts are shown in figures 1-3. Therefore, the InDel marker disclosed by the invention is high in identification accuracy and can be used as a molecular marker to be applied to red meat/non-red meat peach assisted breeding. In addition, the marker has strong operability and simple operation steps, and the identification efficiency is higher than that of an SSR marker which is a molecular marker.
TABLE 6 typing results (unit: strain) of InDel markers in the Red meat hybrid population
Sequence listing
<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences
<120> InDel marker and primer pair for identifying peach flesh red/non-red character and application thereof
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<213> peach (Amygdalus persica L.)
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aattaattac gtttagggaa gtaaaattga caaaaggacc aactccctca acagaaaatc 180
tgatgatcaa caaccccacc acctactagt agtagtaggc agtagagaga gagaagttct 240
tcctcacagg gagaggtggg gtccacaact gagctacact acactacaca cacctactgg 300
gtatacaata ggagtagggg tgtccgtgtc cctcacaaat ttacatcatg catgcatgcg 360
actaatagtt taataggttt tattctgtag taaaattaac tccattgaat tatattgagc 420
atgttatatg taaatgttat accacctgat ctggatggat gctcaaacct gttaggaaat 480
ctttttattt ttggcctttt tggtgatatt attattatca tctgtttgtg ttaaaagtta 540
aaagttgagc aaacagatag atatatatat tgttgtaaag aagaaacatg tggagcccac 600
atttatgggg cccacaaaga aaattaagca aagtatttgg ctgcacactc acggatgtgt 660
attgtagaaa cacgggtgcc atttaacatg tctataaata gagaaagaag aagagagaaa 720
aaaaagagga aaagaagaag agagaaagag ataggaaacg gggagaagaa aaggaaagga 780
ggaaagagag aagaaaggga aaggaggaaa gagaagagga aagggaaagg aggaaagaaa 840
agaggaatga gagtcaggca gagaagagga agagaaggaa gaaataggtg agatatatat 900
aaaattcagc cgaaatttta catatcagaa ctctcagcca tactctatta tttcataaat 960
gaaaaagtta ctaccgccgc ttctctccca tgtgcatagc caacctcaag taaatatcaa 1020
atggttcatc tactctttac gccaaaattt caatattcgc gacatatctc cgtacatttt 1080
ataacacgct atcaagacga gaagcctttt ataatttttt tgtgctgcac tattatttat 1140
actactaacc attatgttga tgtaaagaag aaaagccaac gcatctagct ttccactaat 1200
atattatata tatgaaaagt tgatggaaat ataatatact atatttatct cgcttttcta 1260
ttactaacca ttaacaaaaa tggacccttg gtaatactaa acatattatc ggtgggagac 1320
cgttactaat actaacactt tccttatctt gttcgctggt ggtttttatt cccacattta 1380
ttttatttac tgtatggtgt aggtttatta cccatacaac aagatttatt taaaagggtg 1440
gtgcggttta tcgcccacgc tttgctttta tttaaatata tatataatta atattttata 1500
caagactttt actttgtatt tctccaaatt tacttttact taaagtatta tttatggtat 1560
ggtgtaggtt tattacccat acggcaatat ttatttaagg gtgaatgctt gagtttataa 1620
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<210> 2
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<213> Artificial sequence ()
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ggaccaactc cctcaacaga 20
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<212> DNA
<213> Artificial sequence ()
<400> 3
gtggaaagct agatgcgttg g 21
Claims (4)
1. A primer pair for identifying peach flesh red/non-red characters is characterized in that the nucleotide sequence of an upstream primer BLproF of the primer pair is shown as SEQ ID No.2, the nucleotide sequence of a downstream primer BLproR is shown as SEQ ID No.3, when an 1041bp amplification band appears, peach flesh is judged to be red characters, and when the amplification band does not appear, the peach flesh is judged to be non-red characters.
2. The use of the primer pair of claim 1 for identifying the red/non-red trait of peach pulp, wherein peach pulp is judged to be in the red trait when 1041bp amplification band occurs, and peach pulp is judged to be in the non-red trait when no amplification band exists.
3. A method for identifying a peach flesh red/non-red trait using the primer pair of claim 1, comprising the steps of:
(1) and (3) extracting DNA: collecting leaves, extracting genome DNA as a template for PCR amplification;
(2) and (3) PCR amplification: performing PCR amplification by using the DNA extracted in the step (1) as a template and using the primer pair of claim 1;
(3) electrophoresis: detecting the size of the PCR amplification product band obtained in the step (2) by using agarose gel electrophoresis;
(4) genotyping: and (3) observing the molecular weight of the PCR amplification product, judging that the peach pulp is red when 1041bp of amplification band appears, and judging that the peach pulp is not red when the amplification band does not appear.
4. A kit for identifying the red/non-red character of peach fruit flesh is characterized by comprising the primer pair of claim 1, wherein when 1041bp amplification band appears, the peach fruit flesh is judged to be the red character, and when the 1041bp amplification band does not appear, the peach fruit flesh is judged to be the non-red character.
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| CN112831593B (en) * | 2021-03-16 | 2022-08-30 | 安徽省农业科学院园艺研究所 | SNP molecular marker primer for variety identification of red peach trees in early summer and identification and application method thereof |
| CN113265411A (en) * | 2021-05-28 | 2021-08-17 | 中国农业科学院郑州果树研究所 | Gene for controlling peach blossom/fruit peel color and application thereof |
| CN114480716B (en) * | 2022-03-04 | 2022-11-11 | 广东省科学院南繁种业研究所 | Molecular marker related to corn kernel color and application thereof |
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| ES2386131B1 (en) * | 2010-09-08 | 2013-05-08 | Instituto Valenciano De Investigaciones Agrarias (Ivia) | DIRECT PROCEDURE FOR SPECIFIC DETECTION OF VIROID PEACH LATENT MOSAIC VIROID THROUGH IMMOBILIZED TARGETS AND REAL-TIME RT-PCR AND KIT FOR DETECTION. |
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