CN113736902B - Molecular marker related to pear fruit ethylene jump, detection method and application - Google Patents
Molecular marker related to pear fruit ethylene jump, detection method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and discloses a molecular marker related to pear ethylene jump, a detection method and application thereof, wherein the molecular marker is expressed as an insertion/deletion length polymorphism of a nucleotide sequence shown as SEQ ID NO. 1. The molecular marker is closely related to the ethylene jump character of pear fruits, is used for storing and managing the pear fruits, and can avoid resource waste and misjudgment of the fruits.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker related to pear fruit ethylene jump, a detection method and application thereof.
Background
Pear (Latin name: pyrus spp, english name: pear), a plant of genus Pyrus of family Rosaceae, is usually a deciduous arbor or shrub, few varieties are evergreen, leaves are often oval, and the sizes are different from variety to variety.
The flower is white or yellowish and pink, and has five petals. The fruit has a round shape and also has a thinner base and a thicker tail, which is commonly called a pear shape. The peel colors of different varieties are different, and yellow, green, yellow with yellow, yellow brown, green brown, red brown and brown are respectively provided, and each variety also has mauve. The fruit diameter of the wild pear is smaller and is between 1 and 4 cm, and the fruit diameter of the artificially cultivated variety can reach 8 cm and the length can reach 18 cm.
The pear fruit is usually eaten, has a delicious juice, is sweet and sour, is rich in nutrition, contains multiple vitamins and cellulose, and has completely different tastes and textures. The pears can be eaten raw or steamed. In medical efficacy, the pears can relax constipation, promote digestion and have the benefit to cardiovascular diseases.
The ethylene content of the jump-type fruit increases sharply at the beginning of ripening, according to which the harvest time can be determined by measuring the ethylene content of the fruit. At present, a portable ethylene tester is researched by michigan state university in the united states, so that the determination of the fruit maturity is more scientific and reliable, and when the pear is picked and stored, the fruit maturity is different to a certain extent, so that the fruit is excessively ripe to cause putrefaction, and the pear is difficult to distinguish whether the pear is stored or needs to be consumed as soon as possible only by appearance, thereby wasting resources.
Most of fruit ripening still depends on appearance judgment in the breeding process, and the result is subjective and lacks a quantitative detection method, and the pear fruit breeding process also lacks a molecular marker capable of directly detecting ethylene jump.
Disclosure of Invention
The invention provides a molecular marker related to pear fruit ethylene jump, a detection method and application thereof, wherein the molecular marker is closely related to pear fruit ethylene jump character and is used for breeding research of pear fruits.
The invention provides a molecular marker related to pear fruit ethylene jump, which is expressed as an insertion/deletion length polymorphism of a nucleotide sequence shown as SEQ ID NO. 1.
Further, an individual having the nucleotide sequence shown in SEQ ID NO.1 and homozygous at the molecular marker locus exhibits a non-ethylene jump;
individuals lacking the nucleotide sequence shown in SEQ ID NO.1 and homozygous at the molecular marker locus show a vinylic transition;
individuals who lack the nucleotide sequence shown in SEQ ID NO.1 and are heterozygous at the molecular marker locus are shown as ethylene jumps.
The invention also provides a primer pair for detecting the molecular marker, which comprises a forward primer F with a nucleotide sequence shown as SEQ ID NO.2 and a reverse primer R with a nucleotide sequence shown as SEQ ID NO. 3.
Further, the primer pair is utilized to carry out PCR amplification on the genomic DNA of the pear and detect the length of an amplified fragment, when the amplified fragment is 146bp, the pear to be detected has the insertion of a nucleotide sequence shown as SEQ ID NO.1 and is expressed as non-ethylene jump;
when the amplified fragment is 121bp, the pear to be detected lacks the nucleotide sequence shown as SEQ ID NO.1 and shows ethylene jump;
and when the amplified fragments are 146bp and 121bp, the molecular marker loci of the pear to be detected are heterozygous, and the pear to be detected shows ethylene jump.
The invention also provides application of the primer pair in pear breeding.
The invention also provides a kit comprising the primer pair, and the kit is used for detecting the molecular marker.
The invention also provides application of the kit in pear breeding.
The invention also provides a method for detecting pear fruit ethylene jump character by using the molecular marker, which comprises the following steps:
and carrying out PCR amplification on the genome DNA of the pear to be detected by using the primer pair or the kit, detecting the length of an amplified fragment, wherein the amplified fragment shows non-ethylene jump when the amplified fragment is 146bp, shows ethylene jump when the amplified fragment is 121bp, and shows ethylene jump when the amplified fragment is 146bp and 121 bp.
Further, the amplified fragment length was detected by agarose gel electrophoresis.
The invention also provides application of the molecular marker in pear storage management.
Compared with the prior art, the molecular marker related to pear fruit ethylene jump provided by the invention has the following beneficial effects:
1. the molecular marker provided by the invention is closely related to the pear fruit ethylene jump character, and whether the ethylene jump occurs or not is judged through the length of the gene segment obtained by PCR amplification, so that the mature condition of the pear fruit can be rapidly and accurately judged, and the molecular marker can be used for pear breeding.
2. The molecular marker is closely related to the ethylene jump character of pear fruits, is used for storing and managing the pear fruits, and can avoid resource waste and misjudgment of the fruits.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that, the drawings in the following description are only examples of embodiments of the present invention and other drawings may be made from these drawings by those of ordinary skill in the art without undue burden.
FIG. 1 is a graph showing the change trend of ethylene production amount of pear fruit samples 1 to 12 of example 1 according to the present invention on different days;
wherein, the graphs (1) to (12) represent the ethylene production amount change trend graphs of different days of the samples 1 to 12 respectively;
FIG. 2 is a graph showing the variation trend of ethylene production on different days of pear fruit samples 13 to 24 in example 1 of the present invention;
wherein, the graphs (13) - (24) represent the ethylene production amount change trend graphs of different days of the samples 13-24 respectively;
FIG. 3 shows the results of electrophoresis of the detection of pear samples 1 to 24 in example 1 of the present invention.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1
1. Collecting mature pear sample, and detecting ethylene production
Collecting mature pears as samples to be detected, wherein 24 samples are taken, the serial numbers of the samples are respectively 1,2,3,4 and … and 24, all the samples are detected for ethylene production, and whether the samples have ethylene jump is judged by utilizing the ethylene production change trend of the samples on different days;
the results show that: as can be seen from the results of the ethylene production test of samples 1-12 in FIG. 1, the ethylene production in samples 1-12 is stable according to the trend of the ethylene production change of different samples in FIG. 1, and no ethylene jump occurs, i.e. samples 1-12 are all judged as non-ethylene jumps;
FIG. 2 shows the results of the ethylene production measurements of samples 13-24, and it can be seen from the trend of the ethylene production changes of the different samples in FIG. 2 that the ethylene production of samples 13-24 is changed, i.e., samples 13-24 are all judged to be ethylene changes.
2. Molecular marker verification related to pear fruit ethylene jump
The invention provides a molecular marker related to pear ethylene jump, which is expressed as an insertion/deletion length polymorphism of a nucleotide sequence shown as SEQ ID NO. 1;
SEQ ID NO.1：ACGGTAGTACCATTCAACTTTTTTG。
the steps for detecting the pear fruit ethylene jump character by using the molecular marker are as follows:
1. extracting the pear genome DNA of the 24 samples;
2. designing a forward primer F and a reverse primer R
The nucleotide sequence of the forward primer F is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer R is shown as SEQ ID NO. 3;
SEQ ID NO.2：TAGTCACTGACCATTTGATCTTAATTC
SEQ ID NO.3：GAGTCCCTGTTCTCGTTAACTCTAA
carrying out PCR amplification on all the genome DNA samples of the pears to be detected by using primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3, and detecting the length of amplified fragments, wherein an amplification system is as follows: DNA template 0.5 u L, forward primer F0.5 u L, reverse primer R0.5 u L, 2X ExTaqTM 0.5 u L, RNase Free water 0.5 u L;
the amplification procedure was: 95.0 ℃ for 3min;95.0 ℃,30s,50.0 ℃,30s,72.0 ℃,30s,34cycles;72.0 ℃ for 5min;4.0 ℃, plus;
3. result judgment
As shown in FIG. 3, the amplified fragment showed non-ethylene jump when 146bp, ethylene jump when 121bp, and heterozygous molecular marker locus of the pear to be detected when 146bp and 121bp were amplified.
The molecular marker related to pear ethylene transition provided by the invention can accurately judge the occurrence of pear ethylene transition by detecting the molecular marker, can rapidly and accurately judge the maturity of pear fruit, is used for storing and managing pear fruit, and can avoid resource waste and misjudgment of fruit.
It should be noted that the preferred embodiments of the present invention have been described in order to prevent redundancy, but that additional variations and modifications can be made to these embodiments once the basic inventive concepts are known to those skilled in the art. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
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Claims (7)
1. The primer pair for detecting the molecular marker related to pear ethylene jump is characterized by comprising a forward primer F with a nucleotide sequence shown as SEQ ID NO.2 and a reverse primer R with a nucleotide sequence shown as SEQ ID NO. 3.
2. The primer pair of claim 1, wherein the primer pair is used for carrying out PCR amplification on the genomic DNA of the pear and detecting the length of an amplified fragment, and when the amplified fragment is 146bp, the pear to be detected has the insertion of a nucleotide sequence shown as SEQ ID NO.1 and shows non-ethylene jump;
when the amplified fragment is 121bp, the pear to be detected lacks the nucleotide sequence shown as SEQ ID NO.1 and shows ethylene jump;
and when the amplified fragments are 146bp and 121bp, the molecular marker loci of the pear to be detected are heterozygous, and the pear to be detected shows ethylene jump.
3. Use of the primer pair of claim 1 in pear breeding.
4. A kit comprising the primer pair of claim 1.
5. Use of the kit of claim 4 in pear breeding.
6. A method for detecting pear fruit ethylene jump character by using the primer pair of claim 1, which is characterized by comprising the following steps:
carrying out PCR amplification on the genomic DNA of the pear to be detected and detecting the length of an amplified fragment, wherein the amplified fragment is shown as non-ethylene jump when 146bp, and the amplified fragment is 121bp, the amplified fragments are 146bp and 121bp, and the molecular marker loci of the pear to be detected are heterozygous and show ethylene jump.
7. The method for detecting the ethylene jump character of pear fruit according to claim 6, characterized in that the amplified fragment length is detected by agarose gel electrophoresis.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690046A (en) * | 2020-07-03 | 2020-09-22 | 南京农业大学 | Pear transcription factor PbHB.G7.2 and application thereof in promoting fruit ripening |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690046A (en) * | 2020-07-03 | 2020-09-22 | 南京农业大学 | Pear transcription factor PbHB.G7.2 and application thereof in promoting fruit ripening |
Non-Patent Citations (2)
| Title |
|---|
| Validation of molecular markers associated with fruit ripening day of Japanese pear (Pyrus pyrifolia Nakai) using variance components;Sogo Nishio等;Scientia Horticulturae;第199卷;9-14 * |
| 基于CAPS标记的沙梨果实乙烯释放量水平的快速鉴定;张靖国等;中国南方果树;第47卷;58-60 * |
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