CN112226533A - Molecular marker related to cotton leaf rolling character and application thereof - Google Patents
Molecular marker related to cotton leaf rolling character and application thereof Download PDFInfo
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Abstract
本发明涉及一种与棉花卷叶性状相关的分子标记及其应用，该分子标记由2‑5个特定的InDel/SNP分子标记组成，所述InDel/SNP分子标记均位于棉花第11号染色体上。与现有技术相比，SNP/InDel分子标记直接以DNA的形式表现，在棉花的各个组织和发育阶段均可检测，不受环境和季节限制，不存在表达与否等问题影响。棉花叶片的适当卷曲可使叶片维持直立，从而减少群体冠层荫蔽，提高群体光能利用率。与棉花卷叶性状紧密连锁SNP/InDel分子标记，可用于棉花卷叶性状的鉴定和棉花卷叶相关的株型辅助选择育种，为棉花高光效育种提供可用标记和基因资源。The present invention relates to a molecular marker related to cotton leaf rolling traits and its application. The molecular marker is composed of 2-5 specific InDel/SNP molecular markers, and the InDel/SNP molecular markers are all located on the 11th chromosome of cotton . Compared with the prior art, the SNP/InDel molecular marker is directly expressed in the form of DNA, which can be detected in all tissues and developmental stages of cotton, and is not restricted by the environment and season, and is not affected by problems such as expression or not. The proper curling of cotton leaves can keep the leaves upright, thereby reducing the shade of the group canopy and improving the utilization rate of light energy in the group. SNP/InDel molecular markers closely linked to cotton leaf rolling traits can be used for the identification of cotton leaf rolling traits and plant type-assisted selection breeding related to cotton leaf rolling, providing available markers and gene resources for cotton high light efficiency breeding.
Description
Technical Field
The invention belongs to the field of plant molecular biology, particularly relates to the field of molecular detection related to cotton leaf rolling traits, and particularly relates to a molecular marker related to cotton leaf rolling traits and application thereof.
Background
The cotton is used as a raw material of cotton textile industry, is closely related to the daily life of people, is a global important economic crop, and is also an important economic and strategic material in China. The cotton is used as the largest commodity cotton production base in China and has a vital role in the economic development of Xinjiang. However, because the canopy of cotton is shaded due to the growth characteristics of cotton, the light energy utilization efficiency of the population is directly influenced, and the cotton breeding with high light efficiency is slower than that of other model plants.
The plant type of the crops is the morphological structure of the crops for adapting to the environment, and directly determines the quality and the yield of cotton. The leaves are the main place of photosynthesis, are the main source organs of plant growth and development, and bear 90% of organic matters required by plant growth and development. The shape of the leaf has important influence on the growth and development of cotton, canopy shading is easily caused due to the growth habit of the cotton, the utilization rate of the light energy of cotton groups is low, the high-light-efficiency breeding of the cotton is slower than that of other mode crops, the moderately curled leaf can maintain the upright leaf and reduce the shadow area of plants, so that the light saturation point is improved under the condition of not influencing the light compensation point, the utilization rate of the light energy of the cotton groups is improved, and the aims of improving the accumulation of dry matters and the yield of the crops are fulfilled. In addition, the wax content of the cotton curling leaves is higher than that of common broad leaves, which is beneficial to reducing plant transpiration under drought conditions, reducing the damage of light radiation to the leaves and simultaneously has very important significance for resisting insects.
The leaves are one of important plant type characters of crops, have important significance for constructing the ideal plant type of cotton, and no related report about the molecular marker gene closely linked with the cotton rolling leaves exists at present. Can provide gene resources and germplasm resources for high-light-efficiency breeding of cotton, and has important significance for exploring a molecular mechanism for forming cotton leaf shapes.
Disclosure of Invention
The invention aims to provide a molecular marker related to cotton leaf rolling traits and application thereof, so as to provide a novel SNP/InDel molecular marker closely linked with the cotton leaf rolling traits and provide a novel method for detecting the cotton leaf rolling traits.
The invention realizes the purpose through the following technical scheme:
the invention provides a molecular marker related to cotton leaf rolling traits, which consists of one InDel molecular marker and one or more InDel/SNP molecular markers in 4 SNP molecular markers, is named as InDelNo.1 and SNPIDNo.1-4, is positioned on No. 11 chromosome of cotton, wherein, InDelNo.1 has a nucleotide sequence consisting of 89027119 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.1 has a nucleotide sequence consisting of 89074679 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.2 has a nucleotide sequence consisting of 89074986 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.3 has a nucleotide sequence consisting of 89621017 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.4 has a nucleotide sequence consisting of 89736957 th base and upstream and downstream bases of cotton 11 th chromosome, and the mutation forms of the molecular markers are shown in the following table:
as a further optimization scheme of the invention, the molecular marker consists of 2-5 InDel/SNP molecular markers.
The invention also provides application of the molecular marker in detecting cotton leaf rolling characters or cotton molecular marker assisted breeding.
The invention also provides a specific primer sequence for detecting the molecular marker, which is shown in the following table:
the invention also provides a kit for detecting the cotton leaf rolling character, and the kit comprises one or more pairs of the 5 pairs of specific primer sequences.
The invention also provides a method for detecting the leaf rolling character of cotton by using the molecular marker, which comprises the following steps: extracting cotton tissue DNA to be detected, designing a specific primer for PCR reaction and single base extension according to the information of the molecular marker locus by utilizing MASSARRAY technology, carrying out PCR amplification, SAP digestion and extension by utilizing the designed specific primer by taking the cotton tissue DNA as a template, detecting the genotype of the SNP molecular marker locus, and judging according to the genotypes shown in the following table:
if the detection result of 1 of the molecular markers is the genotype of the broad leaf or the leaf roll, the cotton variety/line to be detected is generally considered to be the broad leaf or the leaf roll phenotype. If the detection results of 2 molecular markers are inconsistent, other molecular markers or combined identification can be reused to ensure the accuracy of the detection results. The more and consistent the number of labels used for detection, the more reliable the detection result. The invention has the beneficial effects that:
the invention provides a SNP/InDel molecular marker related to cotton leaf rolling traits and application thereof, the marker is used for identification of the cotton leaf rolling traits and cotton auxiliary selective breeding, can solve the problems that canopy shading is easily caused by cotton growth habit, the utilization rate of group light energy is low and the like, properly rolled leaves enable the leaves to be kept upright, and reduce the shadow area of cotton group, thereby improving the utilization rate of the group light energy and laying a foundation for high-light efficiency cotton breeding.
The SNP/InDel molecular marker associated with cotton rolling leaves provided by the invention is directly expressed in a DNA form, can be detected in each tissue and development stage of cotton, is not limited by environment and season, and is not influenced by the problems of expression and the like. Through extracting cotton leaf DNA to carry out PCR amplification and comparing with a reference sequence, typing is carried out on different detected single plants, so that different SNP/InDel sites and different genotypes are effectively distinguished, different cotton leaf samples are screened, and the aim of molecular marker assisted breeding is fulfilled.
Detailed Description
The present application is described in further detail below, and it should be noted that the following detailed description is provided for illustrative purposes only, and is not intended to limit the scope of the present application, which is defined by the appended claims.
Example 1
1. Material
And crossing to obtain F1 with Xinluzao 45 and M091 as parents, and selfing to form F2 as a mapping population.
2. Leaf shape survey of F2 population
Leaf shape character investigation was performed on each individual plant of the F2 population, and the findings met the mendelian segregation law with a segregation ratio of 3: 1, indicating that the leaf rolling character is controlled by a pair of recessive genes.
3. BSA high throughput sequencing
Based on the results, two extreme DNA mixing pools with the characteristics are constructed to carry out BSA high-throughput sequencing. And comparing the sequencing result with a reference genome, eliminating false positives in the variation result, and correcting the SNP and InDel false marks by using the GATK software to ensure that the detection result is correct and reliable. Based on the results of genotyping, markers that are homozygously different between the two parents were screened, and a total of 11774 polymorphic markers were selected. And then calculating the SNP-index of the mixed pool sample according to the detected SNP locus, and calculating the frequency difference of the two extreme character mixed pools. Selecting a 95% confidence level as a screening threshold, selecting SNP sites with obvious SNP-index difference of two filial generations, namely selecting sites with the SNP-index of the filial generation 2 (extreme character B) close to 1 and the SNP-index of the filial generation 1 (extreme character A) close to 0, screening to obtain 22 polymorphic marker sites, and further selecting 9 mutation sites causing stop loss or stop gain or nonsynonymous mutation or alternative splicing as potential mutation sites of the candidate gene.
4. SNP/InDel detection
The accuracy and reliability of the SNP/InDel obtained by the screening are verified by utilizing MASSARRAY technology. In combination with the F2 population individual phenotype, 5 of the SNP/InDel markers located on chromosome A11 of cotton (see Table below) were found to be closely linked or co-segregating with the cotton leaf roll trait.
The steps of SNP/InDel detection are as follows:
firstly, designing a specific extension primer according to the SNP/InDel locus, as follows:
designing specific primers for PCR reaction and single-base extension according to SNP/InDel site information, carrying out DNA extraction and PCR amplification on leaves of 200 individuals in an F2 population, carrying out single-base extension by treating PCR products with alkaline phosphatase, diluting reaction products (9 mu l in total) by 3 times, desalting by using resin, spotting samples subjected to desalting treatment on sample targets, naturally crystallizing, and finally carrying out mass spectrum detection on a machine.
Specific primers for each SNP/InDel molecular marker are shown in the following table:
the PCR reaction process comprises the following steps:
reaction system for PCR (384-well PCR plate + 38% reagent loss):
PCR reaction procedure
The SAP digestion reaction process includes:
SAP digestion reaction system
| Reagent | Concentration | Volume(μL) |
| Water | NA | 810.9 |
| SAP Buffer | 10x | 90.1 |
| SAP | 1.7U/μL | 159.0 |
| Total |
SAP reaction procedure
| Temperature(℃) | Time(second) | Cycle |
| 37 | 40 | 1 |
| 85 | 5 | 1 |
| 4 | ∞ | 1 |
The extension reaction process comprises the following steps:
extension reaction System (384-well PCR plate + 38% reagent loss)
Extension reaction procedure
The results show that the 5 SNP/InDel loci are closely linked with the cotton leaf rolling traits, and can be used as molecular markers which are closely linked with the cotton leaf rolling traits and used for identifying the cotton leaf rolling traits. The leaf is used as one of important characters of the cotton plant type, the invention has important significance for constructing the ideal plant type of the cotton, can improve the problems of canopy shading, low utilization rate of group light energy and the like caused by the growth habit of the cotton to a certain extent, and lays a foundation for high-light efficiency breeding of the cotton.
5. Conclusion
The invention utilizes the hybridization of Xinluzao 45 and M091 to obtain F1Selfing to form F2And (4) screening 5 SNP/InDel sites which are closely linked with the cotton rolling leaves. The SNP/InDel sites are obtained by screening through a BSA high-throughput technology, in order to ensure the correctness and reliability of results, the invention utilizes MASSARRAY technology to detect the relativity of the 5 SNP/InDel sites and cotton rolling leaves, and the results show that the 5 SNP/InDel sites are tightly linked with the cotton rolling leaves. The 5 SNP/InDel sites can be used for rapidly and effectively identifying the leaf rolling characters of cotton. It is known that leaves are the main site for photosynthesis of plants, and are responsible for 90% of the nutrients in cotton throughout its growth period. However, because the cotton grows habit, canopy shading is easily caused, so that the light energy utilization rate of cotton groups is low, and the high-light-efficiency breeding of the cotton is slower than that of other mode crops. The blade which is properly curled can keep the blade upright and reduce the shadow area so as to improve the optical saturation point under the condition of not influencing the optical compensation point, thereby improving the utilization rate of the group light energy. The leaves are one of important plant type traits of crops, and have important significance for constructing an ideal plant type of cotton.
Example 2
The embodiment provides a method for detecting cotton leaf rolling traits by using molecular markers related to the cotton leaf rolling traits, wherein the molecular markers comprise any two or more of InDelNo.1 and SNPIDNo.1-4, and the sites and mutation forms of the molecular markers are shown in the following table:
the method for detecting the leaf rolling character of the cotton by using the molecular marker comprises the following steps:
(1) extracting the DNA of the cotton tissue to be detected by using a conventional plant tissue DNA extraction method;
(2) using MASSARRAY technique, designing specific primers based on the SNP molecular markers, the sequences are as follows:
(3) the PCR reaction solution was obtained by using cotton tissue DNA as a template and using a designed specific primer for PCR amplification, and referring to example 1 for the amplification system and procedure.
(4) The PCR reaction solution was subjected to SAP digestion, digestion system and procedure with reference to example 1, to obtain an SAP reaction solution.
(5) Extension reaction was performed on the SAP reaction solution, and the extension reaction system and procedure were as in example 1, to complete the obtaining of the detection solution.
(6) Analyzing by using an MASSARRAY nucleic acid mass spectrometry system, judging the base type of the target site, and judging the leaf type of the cotton variety to be detected according to the following table:
if the detection result of 1 of the molecular markers is the genotype of the broad leaf or the leaf roll, the cotton variety/line to be detected is generally considered to be the broad leaf or the leaf roll phenotype. If the detection results of 2 molecular markers are inconsistent, other molecular markers or combined identification can be reused to ensure the accuracy of the detection results. The more and consistent the number of labels used for detection, the more reliable the detection result. The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (6)
1. A molecular marker related to cotton leaf rolling traits is characterized in that the molecular marker consists of one InDel molecular marker and any one or more InDel/SNP molecular markers in 4 SNP molecular markers, which are named as InDelNo.1 and SNPIDNo.1-4 and are positioned on No. 11 chromosome of cotton, wherein, InDelNo.1 has a nucleotide sequence consisting of 89027119 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.1 has a nucleotide sequence consisting of 89074679 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.2 has a nucleotide sequence consisting of 89074986 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.3 has a nucleotide sequence consisting of 89621017 th base and upstream and downstream bases of cotton 11 th chromosome, SNPIDNO.4 has a nucleotide sequence consisting of 89736957 th base and upstream and downstream bases of cotton 11 th chromosome, and the mutation forms of the molecular markers are shown in the following table:
2. the molecular marker related to the cotton leaf rolling trait of claim 1, wherein the molecular marker consists of 2-5 InDel/SNP molecular markers.
3. Use of a molecular marker associated with a cotton leaf curl trait of any one of claims 1-2 for detecting a cotton leaf curl trait or for assisting in breeding with a cotton molecular marker.
4. A specific primer sequence for detecting a molecular marker related to a cotton leaf rolling trait according to any one of claims 1-2, wherein the specific primer sequence is shown in the following table:
5. a kit for detecting cotton leaf curl characteristics, comprising one or more pairs of the specific primer sequences of claim 3.
6. A method for detecting a cotton leaf curl trait using a molecular marker associated with the cotton leaf curl trait of any one of claims 1 to 2, comprising the steps of: extracting cotton tissue DNA to be detected, designing a specific primer for PCR reaction and single base extension according to the information of the molecular marker locus by utilizing MASSARRAY technology, carrying out PCR amplification, SAP digestion and extension by utilizing the designed specific primer by taking the cotton tissue DNA as a template, detecting the genotype of the SNP molecular marker locus, and carrying out the judgment of the leaf rolling character according to the genotype shown in the following table:
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| CN116656864A (en) * | 2023-06-19 | 2023-08-29 | 石河子大学 | Molecular marker for identifying cotton leaf cracking character, method and application |
| CN117126962B (en) * | 2023-10-26 | 2024-02-02 | 中国农业科学院生物技术研究所 | Cotton leaf wrinkling control gene GhZY, linkage SNP locus and application thereof |
| CN118272574A (en) * | 2024-05-28 | 2024-07-02 | 三亚中国农业科学院国家南繁研究院 | Cotton GaIAA gene mutation site and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116656864A (en) * | 2023-06-19 | 2023-08-29 | 石河子大学 | Molecular marker for identifying cotton leaf cracking character, method and application |
| CN117126962B (en) * | 2023-10-26 | 2024-02-02 | 中国农业科学院生物技术研究所 | Cotton leaf wrinkling control gene GhZY, linkage SNP locus and application thereof |
| CN118272574A (en) * | 2024-05-28 | 2024-07-02 | 三亚中国农业科学院国家南繁研究院 | Cotton GaIAA gene mutation site and application |
| CN118272574B (en) * | 2024-05-28 | 2024-08-16 | 三亚中国农业科学院国家南繁研究院 | Cotton GaIAA gene mutation site and application |
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