CN110791588A - CAPS molecular marker capable of identifying elongated, elliptical and round watermelon fruits simultaneously, primers and application - Google Patents

CAPS molecular marker capable of identifying elongated, elliptical and round watermelon fruits simultaneously, primers and application Download PDF

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CN110791588A
CN110791588A CN201911261079.2A CN201911261079A CN110791588A CN 110791588 A CN110791588 A CN 110791588A CN 201911261079 A CN201911261079 A CN 201911261079A CN 110791588 A CN110791588 A CN 110791588A
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primer
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CN110791588B (en
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高美玲
张艳玲
梁晓雪
郭宇
高越
刘继秀
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Qiqihar Guokang Seed Industry Co.,Ltd.
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Abstract

A CAPS molecular marker, a primer and an application capable of identifying long, oval and round watermelon fruits simultaneously belong to the technical field of biology. The molecular marker is SUN-24, and the nucleotide sequence is shown in SEQ ID NO. 1. The nucleotide sequence of the forward primer in the identifying primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3. It is applied to identification and auxiliary screening of the fruit shape of the watermelon in seedling stage. According to the invention, the molecule is marked SUN-24, and long melon, oval melon and round melon are simultaneously distinguished through PCR products and enzyme digestion products; the method is used for judging the shape of the watermelon fruit in the seedling stage, rapidly screening required individuals and accelerating the breeding process of the shape of the watermelon fruit. The method is suitable for identifying the watermelon type.

Description

CAPS molecular marker capable of identifying elongated, elliptical and round watermelon fruits simultaneously, primers and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a CAPS molecular marker capable of identifying long, oval and round watermelon fruits simultaneously, a primer and application thereof.
Background
Watermelon (Citrullus lanatus) is an important annual crop in cucurbitaceae, an important economic horticultural crop in the world and occupies 7 percent of the vegetable production area in the world. The genetic diversity of the watermelon is rich, and the different germplasm resources have more character differences, so that the analysis of the characters on the molecular level is favorable for the research of the genetic basis of the watermelon.
In recent years, molecular assisted selective breeding technology is widely developed and applied, and can rapidly screen required individuals on a molecular level, greatly shorten breeding time and greatly promote breeding work. Whether a marker closely linked with the character can be found or not and whether the marker can be used in different materials is a sign for the success of molecular marker-assisted selective breeding.
The fruit shape is one of important appearance quality traits of watermelons and is also an important index for classifying and evaluating watermelon varieties, and researches on other crops show that the fruit shape is controlled by genes such as SUN, OVATE, m/CsACS (Liu et al, 2002, van der Knaap et al, 2014, Tan et al, 2015) and the like. In the watermelon study, Weetman (1937) was first thought to be controlled by a single gene that was not fully dominant; the Wangmji (2013) locates the QTL (Fsi3.1) of the fruit shape index on the third linkage group by constructing a map; chenyao (2015) detected 11 QTL sites associated with fruit shape on linkage group No.3 and No. 11, respectively; lupropylu (2016) locates 1 major QTL site related to fruit shape index, and is positioned on linkage group No. 3; the deletion of 159bp on Cla011257 gene found by Guiling et al (2018) results in the formation of watermelon-long melon. The above studies can only distinguish the long melon from other shapes, and fail to find the molecular markers closely linked to the long melon, the oval melon, and the round melon.
Disclosure of Invention
The invention aims to provide a CAPS molecular marker, a primer and application capable of identifying long, oval and round watermelon fruits simultaneously, which are used for judging the shape of the watermelon fruits at the seedling stage and accelerating the breeding process of the shape of the watermelon fruits.
A CAPS molecular marker capable of identifying long, oval and round watermelon fruits simultaneously is a molecular marker SUN-24, and the nucleotide sequence of the CAPS molecular marker is shown in SEQ ID NO. 1.
The identification primer of CAPS molecular marker capable of identifying long, oval and round watermelon fruits simultaneously has the nucleotide sequence shown in SEQ ID No.2 as the forward primer and the nucleotide sequence shown in SEQ ID No.3 as the reverse primer.
A CAPS molecular marker capable of identifying long, oval and round watermelon fruits simultaneously is applied to identification and auxiliary screening of fruit shapes of watermelons in seedling stage.
The invention provides a CAPS molecular marker capable of identifying elongated, elliptical and round watermelon fruits simultaneously, which is a molecular marker SUN-24 and is used for distinguishing elongated melons, elliptical melons and round melons simultaneously through PCR products and enzyme digestion products; the method is used for judging the shape of the watermelon fruit in the seedling stage, rapidly screening required individuals and accelerating the breeding process of the shape of the watermelon fruit.
The method is suitable for identifying the watermelon type.
Drawings
FIG. 1 is a diagram showing the actual shape of the watermelon fruit of the example, wherein A represents a long watermelon, B represents an oval watermelon, and C represents a round watermelon;
FIG. 2 is an electrophoretogram for detection of amplification products and enzyme-cleaved products in the examples, wherein region A represents a long watermelon, region B represents an oval watermelon, and region C represents a round watermelon; lane 1 is DM2000marker, lanes 2-7 are all elongated watermelons; lanes 8-13 are all oval watermelons; lanes 14-19 are all round watermelons.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the CAPS molecular marker capable of identifying the long, oval and round watermelon fruits simultaneously is a molecular marker SUN-24, and the nucleotide sequence of the CAPS molecular marker is shown as SEQ ID No. 1.
In the embodiment, the molecular marker SUN-24 is tightly linked with the watermelon fruit type, the parental sequences are respectively positioned from the 26846850 th nucleotide to the 26847281 th nucleotide on the Cla011257 gene of the No.3 chromosome of the watermelon genome 97103V1 and from the 29790628 th nucleotide to the 29791059 th nucleotide on the Cla97 gene of the No.3 chromosome of the watermelon genome 97103V2, the sizes of the nucleotides are 432bp, the parental sequences are compared, the existence of non-synonymous mutation on the parental is determined, the mutation of G-A is generated on the 26,847,041 site of the Cla011257 gene, and the aspartic acid is mutated into the asparagine.
Acquisition of molecular marker SUN-24 in the present embodiment:
utilizing the parent watermelon K with larger fruit shape difference2And L1Hybridizing to obtain a separated population F2Binding to F by parental Whole genome Re-sequencing2Establishing high-density genetic map by GBS (Genotyping-by-sequencing) typing technology, obtaining closely linked QTL (quantitative trait locus) on No.3 chromosome of watermelon genome, and utilizing amplified F2The population maps finely to chromosome 3, 26,801,978-26,864,789 segments. 6 candidate genes exist in the segment, and a SNP site is found on the Cla011257 gene by aligning the parental sequences, the site is located on the 26,847,041 site of the No.3 chromosome of the watermelon genome, and the base group is G-A. According to the mutation point, molecular markers are developed and named as SUN-24, an upstream primer SUN-24L and a downstream primer SUN-24R. The restriction endonuclease is TaqI.
Partial watermelon natural population:
ChangGua (Changgrey; Zhengkang No. 1-5; Zhengzi No. 017; Zhengzi No. 20; Mangu watermelon-1; Jiubeli);
oval melon (K)2(ii) a Red Xingcheng; disease-resistant watermelon-1; zhengzhu No. 05; PI 255137; PI 596666);
round melon (L)1(ii) a Small seed number one; early martial arts; xigai true Chepu; spain; short vines).
The second embodiment is as follows: in the embodiment of the identifying primer of the CAPS molecular marker capable of identifying the long, oval and round watermelon fruits simultaneously, the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3.
The third concrete implementation mode: the CAPS molecular marker capable of identifying the long, oval and round watermelon fruits is applied to identification and auxiliary screening of the shapes of the watermelon fruits at the seedling stage.
Example (b):
the method for identifying the shape of the watermelon fruit by using the molecular marker SUN-24 comprises the following specific steps:
firstly, extracting the DNA of a watermelon natural population by using an improved CTAB method:
1. when the watermelon plant has three leaves and one heart, taking young leaves into a 2ml centrifugal tube, taking the young leaves back by using an ice bag, performing pumping drying by using a vacuum freezing and pumping machine, and grinding the young leaves into powder by using a grinding instrument after the pumping drying;
2. adding 1000ul of 2% CTAB preheated for 1h in a 65 ℃ water bath into a centrifuge tube, shaking to make the powder fully contact with the liquid, and putting into a 65 ℃ water bath for 1h, wherein the mixing is carried out once every 10 min;
3. cooling to room temperature after water bath, and centrifuging for 15min (centrifugation: 4 ℃, 13000 r/min);
4. centrifuging, sucking out supernatant, transferring into a new 2ml centrifuge tube, adding 900ul chloroform/isoamylol (24:1) solution, shaking thoroughly, centrifuging for 15min (centrifuging at 4 deg.C, 13000 r/min); repeating the step for 2 times;
5. sucking out 600 plus 700ul of supernatant and 1.5ml of centrifuge tube, adding 700ul of anhydrous ethanol precooled at 4 ℃, fully shaking up, and precipitating for 1h at 4 ℃;
6. taking out the centrifuge tube, centrifuging for 15min (at 4 deg.C, 13000r/min), sucking out supernatant, and retaining precipitate;
7. washing with 75% ethanol for 2-3 times, air drying at room temperature, adding 50ul ddH2O, can be permanently stored in a refrigerator at minus 80 ℃;
secondly, using the DNA of the natural population as a template, and carrying out PCR amplification by using an identifying primer to obtain an amplification product:
the reaction system for PCR amplification is a 10-microliter reaction system, and consists of the following components:
Figure BDA0002311596370000031
Figure BDA0002311596370000041
and (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 30s, for 25 cycles, extension at 72 deg.C for 7min, and preserving at 4 deg.C;
thirdly, detecting the amplification product obtained in the second step by using 1.5 percent agarose gel electrophoresis and determining the elongated watermelon:
the detection result is shown in figures 1 and 2, the amplified band of the oblong watermelon is about 282bp, and the amplified band of the round watermelon and the oval watermelon is 432 bp;
fourthly, carrying out enzyme digestion on the PCR product and determining the oval watermelon and the round watermelon:
the enzyme digestion system is as follows:
Figure BDA0002311596370000042
the enzyme digestion conditions are as follows: enzyme digestion is carried out for 4h at 65 ℃;
the enzyme digestion product was detected by 1.5% agarose gel electrophoresis, and the detection results are shown in FIGS. 1 and 2, in which the oval melon had a band at 432bp, and the round melon was cut into two bands of 240bp and 188bp, respectively.
The above results were the same as expected experimental results.
Sequence listing
<110> university of ziqi hall
<120> CAPS molecular marker capable of identifying elongated, elliptical and round watermelon fruits simultaneously, primer and application
<160>3
<210>1
<211>432
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence of molecular marker SUN-24.
<400>1
gacttgaaat ccgagccaga aaatcaatgg tgaacactaa gaaactttat ttacaatttg 60
tcattactgt tttcgtttgc tgaattgaaa ttaaaccccc aaataaaatt ttcaatgcag 120
gagagatttg atgacacaaa aagcgaacac acagcttcgg tacacagcag aagactctct 180
gcttcattag ataacacagc attcacagca atggaagaaa gccccaaaat cgtggaaatc 240
aacaccggcc gaccaaaatc ctggtcacgc cggacgaaca cgtcggcgtc ggaattaagc 300
gacgacctat ttcaccaaac tctctcgtct ccactccctt gccggactcc gtcgcgtttg 360
caaatcccag attgccggaa tttccatgac tctgactggt gcggcggcga cgactggcgt 420
ttaatctcga cg 432
<210>2
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> identifying the nucleotide sequence of the forward primer in the primer.
<400>2
cgtcgagatt aaacgccagt 20
<210>3
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> identifying the nucleotide sequence of the reverse primer in the primer.
<400>3
tctggctcgg atttcaagtc 20

Claims (3)

1. A CAPS molecular marker capable of identifying long, oval and round watermelon fruits simultaneously is characterized in that the CAPS molecular marker is a molecular marker SUN-24, and the nucleotide sequence of the CAPS molecular marker is shown as SEQ ID NO. 1.
2. An identifying primer of CAPS molecular markers capable of identifying long, oval and round watermelon fruits simultaneously is characterized in that the nucleotide sequence of a forward primer in the identifying primer is shown as SEQ ID NO.2, and the nucleotide sequence of a reverse primer is shown as SEQID NO. 3.
3. The use of the CAPS molecular marker for simultaneously identifying elongated, oval and round watermelon fruits according to claim 1 for identifying and assisting in the screening of fruit shape of watermelon in seedling stage.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115820907A (en) * 2022-10-25 2023-03-21 东北农业大学 Application of primer pair in identification of melon leaf shape

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107619875A (en) * 2017-10-11 2018-01-23 中国农业科学院郑州果树研究所 A kind of insertion and deletion marker site, primer and application for being used to identify Watermelon Fruit shape

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107619875A (en) * 2017-10-11 2018-01-23 中国农业科学院郑州果树研究所 A kind of insertion and deletion marker site, primer and application for being used to identify Watermelon Fruit shape

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DOU等: "Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.)", 《THEORETICAL AND APPLIED GENETICS》 *
豆峻岭等: "西瓜果实及株型相关性状的遗传及分子机理研究", 《中国博士学位论文全文数据库 农业科技辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115820907A (en) * 2022-10-25 2023-03-21 东北农业大学 Application of primer pair in identification of melon leaf shape
CN115820907B (en) * 2022-10-25 2023-09-08 东北农业大学 Application of primer pair in identification of melon leaf shape

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