CN107058601B - SNP (Single nucleotide polymorphism) marker for identifying low stone cell content of pear pulp based on high-resolution dissolution curve and application of SNP marker - Google Patents
SNP (Single nucleotide polymorphism) marker for identifying low stone cell content of pear pulp based on high-resolution dissolution curve and application of SNP marker Download PDFInfo
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Abstract
The invention discloses SNP molecular marker primers closely related to the content of stone cells in pear pulp and application thereof. SNP markers Psc397-7 and Psc397-12 closely related to pear stone cell content, primers: SEQ ID No. 1; SEQ ID No.2; SEQ ID No. 3; SEQ ID NO. 4. The specific primer can be used for well typing the low stone cell content of the pulp based on a high-resolution dissolution curve. The molecular marker primer for the pear pulp stone cell content can be used for molecular marker-assisted selective breeding of pear pulp with low stone cell content, and has important theoretical and practical guiding significance for accelerating the genetic improvement process of pear varieties and improving the breeding selection efficiency.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and relates to an SNP (single nucleotide polymorphism) marker for identifying the content of pear pulp low stone cells based on a high-resolution dissolution curve and application thereof.
Background
The pear has a long cultivation history in China and is an important deciduous fruit tree in the world. The pear has high nutritive value, crisp meat and juicy fruit, and is popular with people. The edible quality of the pear is always a concern of people and is an important factor for determining the economic value of the pear, so that the edible quality of the pear is improved, and the edible quality of the pear is of great significance. The eating quality of pear fruits is influenced by a plurality of factors, wherein stone cells are one of the important factors influencing the quality of the pear fruits. Stone cells are a specific type of sclerenchyma cells in pear fruits, which not only affect the eating quality of the pear fruits, but also affect the processing quality of the pear fruits. The content of stone cells in the pear fruits is reduced, and the method is important for improving the quality of pears. In recent years, the Dangshan pear as the main planting variety of Chinese pears has the disadvantages of increasing the content of stone cells, thickening the meat quality and generating more residues in the mouth feel due to the degeneration of the variety or the poor management, and the quality and the economic value of the Dangshan pear are seriously influenced. Therefore, the breeding of the high-quality low stone cell content pears not only becomes one of the main targets of the international pear breeding in the twenty-first century, but also becomes a hotspot and a remarkable trend for improving the quality of the pear varieties in China in recent years.
The breeding of new pear varieties is mainly hybrid breeding, the hybrid breeding is usually carried out through phenotype, most genes controlling economic traits of pear fruits are Quantitative Trait (QTL) genes, the juvenile period of pears is long, and the breeding of new varieties by using the traditional breeding technology needs a long time, so that the breeding of new varieties with excellent comprehensive traits and good quality, disease resistance, insect resistance, high yield and storage resistance is very difficult. The breeding technology of molecular Marker Assisted Selection (MAS) can improve the selection efficiency and accelerate the breeding pace. The SNP (single nucleotide polymorphism) molecular marker is taken as a representative of the third-generation molecular marker, has the characteristics of rich variation, high stability, easy realization of high-throughput automatic analysis and the like, and is incomparable with the traditional molecular marker. In recent years, SNP molecular markers are highly concerned by the fields of molecular breeding and phylogenetic research [1,2], are widely applied to the aspects of population inheritance and phylogenetic analysis [3], genetic map construction [4], species diversity analysis [5] and the like, and become an important tool in the field of crop research.
At present, QTL positioning research related to quantitative traits of pears still belongs to a starting stage, the existing research mainly aims at some diseases or growth traits, and the research on development and application of SNP molecular markers with low stone cell content in pear pulp is not reported. Therefore, development of SNP molecular markers with low stone cell content in pear pulp is carried out, and a natural population auxiliary selection technical system is established, which is very important for improving breeding efficiency and saving production cost.
Disclosure of Invention
The invention aims to develop primers of SNP specific markers Psc397-7 and Psc397-12 for identifying the low stone cell content of pear pulp based on a high-resolution melting curve by utilizing the re-sequencing data information of pears. The molecular marker primer can be used for predicting the pear varieties with low content of the pulp stone cells, and provides technical support of molecular marker assisted breeding for early identification and screening of the content of the pulp stone cells of the pears.
A SNP marker closely related to the content of pear pulp stone cells, selected from the position Psc397-7 or Psc397-12 at base 26607192 or base 26607490 of the pear genomic sequence Chr5 of AJSU 00000000; the Psc397-7 locus is A in the low stone cell content variety and is G in the high stone cell content variety; the Psc397-12 locus is G in low stone cell content varieties and A in high stone cell content varieties.
The SNP marker disclosed by the invention is applied to identification of the content of high or low stone cells in pear pulp.
The SNP marker disclosed by the invention is applied to molecular breeding for early identification and screening of the content of the stone cells in the pear pulp.
The SNP marker primer pair closely related to the content of the stone cells of the pear pulp is characterized by being selected from any one of the following pairs: psc 397-7-F: SEQ ID NO.1, Psc 397-7-R: SEQ ID No.2; or Psc 397-12-F: SEQ ID NO.3, Psc 397-12-R: SEQ ID NO. 4.
The SNP marker primer pair disclosed by the invention is applied to identification of the content of high or low stone cells in pear pulp.
The SNP marker primer pair disclosed by the invention is applied to molecular breeding for early identification and screening of the content of pear pulp stone cells.
The invention discovers that positions Psc397-7 and Psc397-12 related to the low stone cell content of pear pulp exist at 26607192 and 26607490 bases of a pear genome sequence Chr5 of AJSU 00000000. The Psc397-7 locus is A in the low stone cell content variety and is G in the high stone cell content variety; the Psc397-12 locus is G in low stone cell content varieties and A in high stone cell content varieties. The content of stone cells in pear pulp can be predicted by detecting the genotype of the site, so that the early identification and screening of the content of the stone cells in pulp with low content can be realized. Wherein the SNP marker primer pair of Psc397-7 is Psc 397-7-F: SEQ ID NO.1, Psc 397-7-R: SEQ ID No.2; the primer pair at the Psc397-12 site is Psc 397-12-F: SEQ ID NO.3, Psc 397-12-R: SEQ ID NO. 4.
A method for identifying the content of high or low stone cells in pear pulp based on a high resolution melting curve is characterized in that any pair of SNP marker primers is adopted to carry out high resolution melting curve analysis on a control pear variety with high content of stone cells in pulp and a control pear variety with low content of stone cells in pulp and a pear variety with the content of stone cells to be identified, and whether the content of stone cells in pulp of the pear variety is high or low is identified by comparing the high resolution melting curve of the pear variety to be identified with the high resolution melting curves of the two control pear varieties; wherein the control pear species having a high content of pulp stone cells is selected from the group consisting of: dangshan pear; the control pear varieties with low pulp stone cell content are selected from: and is crisp and green.
Wherein, the High Resolution dissolution curve reaction system is carried out according to the instructions in a LightCycler480 High Resolution Messing Master kit, and the HRM analysis is carried out on a LightCycler480 II fluorescent quantitative PCR instrument.
The high resolution dissolution curve reaction system is 20 μ L: contains 30ng of pear genome DNA template, 1 × Master Mix, 2.5mmol/L MgCl2200nM of the SNP marker primer set of the invention; the amplification procedure used a touchdown PCR: pre-denaturation at 95 deg.C for 10min, denaturation at 95 deg.C for 10s, annealing at 65-55 deg.C for 10s, and performing 45 cycles at 72 deg.C for 10 s; and melting after the PCR cycle is finished, wherein the program comprises the following steps: heating to 95 deg.C for 1min, 40 deg.C for 1min, and 65 deg.C for 1s, continuously heating from 70 deg.C to 95 deg.C, increasing per liter by 0.04 deg.C, and collecting fluorescence for 1 time, and collecting fluorescence for the mostThen cooling to 40 ℃; finally, the melting curve of the amplification product was automatically generated in the Gene Scanning software of LightCycler480 II.
Advantageous effects
The invention discovers that 26607192 and 26607490 base positions of the pear genome sequence Chr5 of AJSU00000000 respectively have a site Psc397-7 and Psc397-12 related to the content of pear pulp stone-poor cells, and the content of the pear pulp stone-poor cells can be predicted by detecting the genotype of the site, so that the early identification and screening of the content of the pulp stone-poor cells are realized. Based on the above findings, the applicants developed SNP marker primers for the above two sites. The two pairs of developed SNP marker primers are utilized to carry out differential genotype detection on 56 natural pear groups, and the method can be well used for identifying the group with low content of pulp stone cells and is consistent with the actual content of the pulp stone cells of the fruits. Group tests show that the developed SNP marker specific primers can well type natural group varieties with low content of pulp stone cells, so that the SNP marker specific primers have good application value and can realize the pre-selection and assisted breeding of the varieties with low content of pulp stone cells.
Drawings
In the figure 1, HRM specific marker primers developed by SNP markers Psc397-7 and Psc397-12 and normalized translational lysis curves detected in 56 pear natural population individuals can be well typed.
(a) Normalized translational lysis curve for detection of Psc397-7 labeled primer.
(b) And (3) detecting the arrangement of individuals, wherein A1-C6 is a high stone cell content population, and E1-G2 is a low stone cell content population.
(c) Normalized translational lysis curve for detection of Psc397-12 labeled primer.
(d) And (3) detecting the arrangement of individuals, wherein A1-C6 is a high stone cell content population, and E1-G2 is a low stone cell content population.
Detailed Description
The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Example 1 determination of the content of Stone cells
Isolation and weighing of stone cells were performed according to the method of Tao et al (2009) [6 ]. Peeling and removing core of fruit, pulping 100g of fruit pulp with distilled water, diluting with distilled water to obtain homogenate, stirring, standing at room temperature for 30min, discarding the floating substance, resuspending the precipitate in 0.5mol/L hydrochloric acid for 30min, discarding the floating substance, and washing with distilled water. Repeating the steps until the stone cells are separated from other cell fragments, drying and weighing. The pulp stone cell content 35 days after the flower of the 56 pear natural groups is shown in table 1.
35 days after flowering of 156 natural pear groups in Table, the pulp stone cell content
Note: the serial numbers 1-30 are varieties with high stone cell content, and 31-56 are varieties with low stone cell content.
EXAMPLE 2 development of HRM-specific primers Using SNP marker loci Psc397-7 and Psc397-12
The DNA sequences of SNP markers Psc397-7 and Psc397-12 on the 5 th chromosome were searched from the Dangshan pear genome database, sequences of 300bp around the site were selected, and SNP marker primers were developed and designed according to the primer design principle (Table 2). The designed SNP marker primers are used for carrying out PCR amplification on the genomic DNA of the Dangshan pear, the primers are all normally amplified, and the PCR product conforms to the predicted size.
TABLE 2 SNP marker primers
Example 3 typing of Pear pulp Stone cell content Using HRM (high-resolution melting) technique
The HRM reaction was performed according to the instructions in the LightCycler480 High Resolution Messing Master kit, and the HRM analysis was performed on a LightCycler480 II fluorescent quantitative PCR instrument. 20 μ L reaction: contains 30ng of pear genome DNA template, 1 × Master Mix, 2.5mmol/L MgCl2Primers described in 200nM (Psc 397-7-F: SEQ ID NO.1/Psc 397-7-R: SEQ ID NO.2 or Psc 397-12-F: SEQ ID NO.3,/Psc 397-12-R: SEQ ID NO. 4); the amplification procedure used was touchdown pcr: pre-denaturation at 95 ℃ for 10min, followed by denaturation at 95 ℃ for 10s, annealing at 65-55 ℃ (0.5 ℃ per cycle) for 10s, and extension at 72 ℃ for 10s for 45 cycles. And melting after the PCR cycle is finished, wherein the program comprises the following steps: 95 ℃ for 1min, 40 ℃ for 1min, 65 ℃ for 1s, continuously heating from 70 ℃ to 95 ℃, raising the temperature by 0.04 ℃ per liter, collecting fluorescence for 1 time, and finally cooling to 40 ℃. Finally, the melting curve of the amplification product was automatically generated in the Gene Scanning software of LightCycler480 II.
HRM analysis was performed on 56 natural pear populations using the SNP marker primers obtained in example 2 (FIG. 1).
Psc397-7 primer typing results:
line 1-red curve, 21 individuals with similar line type, of which 20 individuals with high pulpal cell content; line 2-blue curve, 31 individuals with similar line pattern, of which 25 individuals with low pulp stone cell content; line 3-green curve, 4 individuals are similar in line, with 4 individuals being high pulp stone cell content. Statistical analysis shows that 56 individuals are typed into three genotypes, and the separation ratio is 21: 31: 4. according to the Psc397-7 primer typing result, the physiological data of the content of the pulp stone cells of 56 natural populations are subjected to Fisher's exact test (p-value is 3E-9).
Psc397-12 primer typing results:
line 1-red curve, similar line for 21 individuals, where 21 individuals are high pulp stone cell content; line 2-blue curve, 31 individuals with similar line pattern, of which 25 individuals with low pulp stone cell content; line 3-green curve, 4 individuals are similar in line, with 3 individuals being high pulp stone cell content. Statistical analysis shows that 56 individuals are typed into three genotypes, and the separation ratio is 21: 31: 4. according to the Psc397-12 primer typing result, the physiological data of the content of the pulp stone cells of 56 natural populations are subjected to Fisher's exact test (p-value is 5.83E-10).
The typing results of the two pairs of primers are combined, and the group identification accuracy rate of pear pulp with low stone cell content reaches 92.3%. Therefore, through the comparative analysis of the physiological character determination result of the pulp stone cell content 35 days after pear blossom and the HRM typing result, the genotype of the Psc397-7 and Psc397-12 markers is proved to be related to the pulp stone cell content, and the two-component results are combined, so that the method can be better used for detecting the individual with low pulp stone cell content.
Primary references
1.Mcnally KL,Childs KL,Bohnert R,Davidson RM,Zhao K,Ulat VJ,Zeller G,Clark RM,Hoen DR,Bureau TE:Genomewide SNP variation reveals relationships among landraces and modern varieties of rice.Proceedings of the National Academy of Sciences 2009,106(30):12273-12278.
2.Rafalski A:Applications of single nucleotide polymorphisms in crop genetics.Current Opinion in Plant Biology 2002,5(2):94-100.
3.Monk D,Arnaud P,Apostolidou S,Hills FA,Kelsey G,Stanier P,Feil R,Moore GE:SNPs in ecology,evolution and conservation.Trends in Ecology&Evolution 2004,19(4):208-216.
4.Tsang S,Sun Z,Luke B,Stewart C,Lum N,Gregory M,Wu X,Subleski M,Jenkins NA,Copeland NG:A comprehensive SNP-based genetic analysis of inbred mouse strains.Mammalian Genome 2005,16(7):476.
5.Sattarzadeh A,Achenbach U,Lübeck J,Strahwald J:Single nucleotide polymorphism(SNP)genotyping as basis for developing a PCR-based marker highly diagnostic for potato varieties with high resistance to Globodera pallida pathotype Pa2/3.Molecular Breeding 2006,18(4):301-312.
6.Tao S,Khanizadeh S,Zhang H,Zhang S:Anatomy,ultrastructure and lignin distribution of stone cells in two Pyrus species.Plant Science 2009,176(3):413-419.
<110> Nanjing university of agriculture
<120> SNP (single nucleotide polymorphism) marker for identifying pear pulp low stone cell content based on high-resolution dissolution curve and application thereof
<160> 2
<210> 1
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<212> DNA
<213> Artificial sequence
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<223> Psc397-7-F
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tacatgcaaa gctggttcat aact 24
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<212> DNA
<213> Artificial sequence
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<223> Psc397-7-R
<400> 2
tccgcagttt tgattgatgc 20
<210> 3
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<213> Artificial sequence
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<223> Psc397-12-F
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caactataat tcttccaaat ggcc 24
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ggtattccct cgtaatcctg tg 22
Claims (6)
1. SNP-tagged primer pairs closely related to the content of stone cells of the pear flesh, characterized by being selected from any one of the following pairs: psc 397-7-F: SEQ ID NO.1, Psc 397-7-R: SEQ ID NO.2, or Psc 397-12-F: SEQ ID NO.3, Psc 397-12-R: SEQ ID NO. 4.
2. The SNP marker primer pair of claim 1, wherein the SNP marker primer pair is used for identifying the content of stone cells in pear pulp.
3. The SNP marker primer pair of claim 1, wherein the SNP marker primer pair is applied to molecular breeding for early identification and screening of the content of the pyritum cells.
4. A method for identifying the content of stone cells in pear pulp based on a high-resolution melting curve is characterized in that the SNP marker primer of claim 1 is used for carrying out high-resolution melting curve analysis on a control pear variety with high content of stone cells in pulp and a control pear variety with low content of stone cells in pulp and a pear variety with the content of stone cells in pulp to be identified, and the content of stone cells in pulp of the pear variety is identified to be high or low by comparing the high-resolution melting curve of the pear variety to be identified with the high-resolution melting curves of the two control pear varieties; wherein the control pear species having a high content of pulp stone cells is selected from the group consisting of: dangshan pear; the control pear varieties with low pulp stone cell content are selected from: and is crisp and green.
5. The method of claim 4, wherein the High Resolution Melting curve reaction system is performed according to the instructions of the LightCycler480 High Resolution Melting Master kit, and the HRM analysis is performed on a LightCycler480 II fluorescence quantitative PCR instrument.
6. The method of claim 5, wherein the high resolution dissolution profile reaction system is 20 μ L: contains 30ng of pear genome DNA template, 1 × Master Mix, 2.5mmol/L MgCl2200nM of the SNP marker primer set of claim 1; amplification procedureUsing a falling-down PCR: pre-denaturation at 95 deg.C for 10min, denaturation at 95 deg.C for 10s, annealing at 65-55 deg.C for 10s, and performing 45 cycles at 72 deg.C for 10 s; and melting after the PCR cycle is finished, wherein the program comprises the following steps: continuously heating to 95 deg.C from 70 deg.C for 1min, 40 deg.C for 1min, and 65 deg.C for 1s, increasing the temperature per liter by 0.04 deg.C, collecting fluorescence for 1 time, and cooling to 40 deg.C; finally, the melting curve of the amplification product was automatically generated in the Gene Scanning software of LightCycler480 II.
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| CN102071194A (en) * | 2010-11-19 | 2011-05-25 | 南京农业大学 | Molecular marker of major quantitative trait locus (QTL) of fruit stone cell content of Dangshan pear |
| CN102071195A (en) * | 2010-11-19 | 2011-05-25 | 南京农业大学 | Molecular marker for content of sclereid in pyrus communis fruits |
| CN105400887A (en) * | 2015-12-16 | 2016-03-16 | 安徽农业大学 | Method for screening pollination pear varieties suitable for Dangshan pears |
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| CN102071194A (en) * | 2010-11-19 | 2011-05-25 | 南京农业大学 | Molecular marker of major quantitative trait locus (QTL) of fruit stone cell content of Dangshan pear |
| CN102071195A (en) * | 2010-11-19 | 2011-05-25 | 南京农业大学 | Molecular marker for content of sclereid in pyrus communis fruits |
| CN105400887A (en) * | 2015-12-16 | 2016-03-16 | 安徽农业大学 | Method for screening pollination pear varieties suitable for Dangshan pears |
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| Title |
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| The genome of the pear (Pyrus bretschneideri Rehd.);Jun Wu. et al.;《Genome Research》;20130228;第23卷(第2期);摘要，第397页右栏第2-3段，第402页左栏最后一段-右栏第1段，第407页左栏第2段，补充图5，补充图7，补充图8 * |
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