An InDel marker primer for identifying the authenticity of F1 generation of interspecific hybridization of sisal and its application

Abstract

The present invention discloses an InDel marker primer for identifying the authenticity of the F1 generation of interspecific hybridization of sisal hemp and its application. According to the resequencing data of sisal parental varieties, the present invention develops an InDel marker primer for identifying the authenticity of the F1 generation of hybridization of sisal hemp '292' and 'Moyin 7', and performs identification at the seedling stage of the hybrid F1 generation. The results show that the offspring that can simultaneously amplify the characteristic bands of the father and the mother are true hybrids, and the lack of any one of the bands or the lack of two bands are regarded as false hybrids, which improves the detection purity of the true hybrids of the F1 generation and also advances the identification time. At the same time, a method for identifying the authenticity of the F1 hybrids of interspecific hybridization of sisal hemp varieties is provided. Compared with the traditional hybrid identification method, it is fast, convenient, accurate and reliable, has a short breeding cycle and low breeding cost, and can also screen the breeding target offspring early, effectively improve the identification efficiency of interspecific hybrids of sisal hemp, and provide a more effective method for quickly identifying the authenticity of the offspring of interspecific hybridization of sisal hemp.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding, and more specifically, relates to an InDel marker for identifying the authenticity of the hybrid F1 generation of sisal '292' and 'Moyin 7' varieties and its application. Background Art

[0002] Sisal is a general term for plants of the subfamily Agavoideae in the family Asparagaceae. The subfamily Agave includes 9 genera and nearly 300 species. At present, fibers are often extracted from the leaves of Agave plants. Due to its high tensile strength, strong toughness and corrosion resistance, it is widely used in the inner core of ship cables and steel ropes of industrial equipment. Sisal originated from the Mexican plateau and is a typical tropical crop. It is now mainly distributed in tropical and subtropical regions around the world. More than 100 resources of Agave have been collected. These varieties are precious materials for the creation of excellent sisal germplasm and the cultivation of new varieties.

[0003] At present, the main method of sisal breeding is sexual hybrid breeding. The existing sisal hybrid breeding has the following problems: (1) In the process of hybrid seed production, there are problems such as exogenous pollen contamination. Sisal is monoecious and can be self-pollinated and heterozygous. Sisal pollen and stamens are collected manually. The sisal rachis is 5-10 meters high and can be pollinated by bees, moths, bats, birds and wind. There are cases of animal and wind pollination before artificial pollination, resulting in the hybrid F1 generation may not be the real hybrid F1, but pollinated by other plants of the father, thus affecting the accuracy and genetic diversity of the genetic offspring. (2) The use of phenotypes to identify hybrid offspring is easily affected by the environment. Before this, hybrid offspring were mainly identified by phenotype, but the sisal juvenile period is long, usually 2-3 years, and many traits are not manifested in the juvenile period, such as sisal leaf thorns. There are no leaf thorns in the young age, but leaf thorns grow after adulthood. This makes it impossible to identify hybrid offspring by phenotype during the sisal juvenile period. In addition, environmental factors can also affect the phenotype of sisal. For example, in harsh conditions such as high temperature and drought, or in an environment with good water and fertilizer, sisal will bloom early and the size of the plant will also vary. (3) Sisal has a long juvenile period and management is time-consuming and labor-intensive. Sisal has a long juvenile period and needs to be planted densely and sparsely for the phenotype to stabilize, which usually takes 3 years. During this period, a lot of cultivation and management is required, which consumes time and manpower.

[0004] Purple leaf curl is the main disease that restricts the sisal industry. At present, there is an urgent need to cultivate more high-yield sisal varieties that are resistant to purple leaf curl. The sisal variety Mexican Introduction 7 (M7, referred to as "Mexican Introduction 7") is a sisal material introduced from Mexico. Its Latin name is unknown. It has long leaves, thorns on the leaf edges, and is resistant to purple leaf curl. The sisal variety "292" is a hybrid strain with high yield and no thorns on the leaf edges. After hybridization with "292" as the male parent and "Mexican Introduction 7" as the female parent, phenotypic observations revealed that the F1 generation has rich genetic variation. It not only has the characteristics of the male and female parents, but also has individuals with characteristics different from the parents. The identification of hybrid F1 through traditional hybrid breeding has problems such as long cycle, poor reliability, low accuracy, and poor efficiency in identifying the authenticity of the hybrid F1 generation. It is impossible to accurately identify whether it is a true hybrid offspring through phenotype.

[0005] Since the key to obtaining hybrid breeding offspring with excellent traits lies in the identification of the authenticity of the hybrid F1 generation, it is very necessary to detect the authenticity of the hybrid F1 generation. By developing molecular markers to identify the purity of hybrids, it is possible to accurately identify hybrid offspring in the early growth stage without being affected by the environment. It has the advantages of being simple, convenient, efficient, stable, time-saving and labor-saving. At present, there are no reports on the use of molecular markers to identify the purity of hybrids in sisal. In order to cultivate more new varieties with high yield and resistance to purple leaf curl, it is urgent to develop methods or technologies that can quickly, accurately and efficiently identify sisal hybrid offspring, which is of great significance for the identification of sisal ('292'×'Moyin 7') hybrid offspring and the cultivation of more high-yield sisal varieties with resistance to purple leaf curl. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the problems existing in the traditional identification of offspring of sisal hybrid breeding, such as long period, poor reliability, low accuracy, poor efficiency in identifying the authenticity of hybrid F1 generation, and the shortcomings of the authenticity identification method of sisal hybrid F1 generation, and provide an InDel marker primer for identifying the authenticity of sisal interspecific hybrid F1 generation and its application.

[0007] The first object of the present invention is to provide an InDel labeled primer.

[0008] The second object of the present invention is to provide the application of InDel labeled primers.

[0009] The third object of the present invention is to provide a kit.

[0010] The fourth object of the present invention is to provide the use of the kit.

[0011] The fifth object of the present invention is to provide a method for identifying the authenticity of F1 hybrids of interspecific hybridization of sisal varieties using InDel markers.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The invention provides an InDel labeling primer, comprising an upstream primer: 5'-AGGGGGAGACGATAGCACA-3', and a downstream primer: 5'-TCCCCACCAACCCTAGGC-3'.

[0014] According to the resequencing data of sisal parent varieties '292' and 'Moyin 7', the present invention develops InDel marker primer C03_463164855 for identifying the authenticity of the F1 generation of interspecific hybridization of sisal from 645 InDel markers. The primer can be used for identification in the seedling stage of the hybrid F1 generation. The offspring that can simultaneously amplify the characteristic bands of the father and the mother are true hybrids, and the lack of any one of the bands or the lack of two bands are regarded as false hybrids. The use of the molecular marker primer for identification greatly advances the identification time, and can quickly, accurately and efficiently identify the authenticity of the offspring of interspecific hybridization of sisal. Compared with the traditional hybrid identification method, it is not only accurate and reliable, but also can shorten the breeding cycle, reduce breeding costs, and can screen the breeding target offspring early, effectively improve the identification efficiency of interspecific hybrids of sisal, and provide a more effective method for quickly identifying the authenticity of the offspring of interspecific hybridization of sisal.

[0015] Therefore, the present invention provides the use of InDel marker primers in identifying the authenticity of hybrids of sisal varieties in the F1 generation, or in screening sisal hybrids in the F1 generation.

[0016] The invention provides application of InDel labeled primers in a kit for preparing authenticity test of F1 hybrids of sisal varieties.

[0017] The invention provides a kit comprising InDel labeled primers.

[0018] Preferably, the kit contains reagents for extracting sample DNA.

[0019] Preferably, the kit also contains reagents required for PCR amplification.

[0020] The present invention provides application of the kit in identifying the authenticity of hybrids of sisal varieties and F1 generation, or in screening sisal hybrids and F1 generation.

[0021] The present invention provides a method for identifying the authenticity of F1 hybrids of interspecific hybridization of sisal varieties by using InDel markers, comprising the following steps:

[0022] S1. Extract genomic DNA from sisal parents and interspecific hybrid F1 hybrids respectively;

[0023] S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification was performed using InDel labeled primers, and then agarose gel electrophoresis was performed;

[0024] S3. Identification of the authenticity of F1 hybrids: Analyze the S2 test results. The offspring that can simultaneously amplify the characteristic bands of the father and the mother are true hybrids. The lack of any one of the bands or the lack of two bands are considered false hybrids.

[0025] Furthermore, the sisal varieties are sisal '292' and 'Moyin 7'.

[0026] Preferably, the PCR amplification system is: 2 μL of DNA template, 0.2 μL of upstream and downstream primers, 10 μL of Mix, and a total volume of 20 μL made up with sterile water; PCR amplification program: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing and extension at 65°C for 20 s, 40 cycles; extension at 72°C for 5 min, and storage at 25°C.

[0027] The present invention has the following beneficial effects:

[0028] The present invention provides InDel marker primers that can be used to identify the authenticity of the hybrid F1 generation of sisal '292' and 'Moyin 7' varieties, and can be used for identification at the seedling stage of the hybrid F1 generation. The results show that the offspring that can simultaneously amplify the characteristic bands of the father and the mother are true hybrids, and the lack of any one of the bands or the lack of two bands are regarded as false hybrids. The use of the primers for identification greatly advances the identification time, and can quickly, accurately and efficiently identify the authenticity of the offspring of sisal interspecific hybrids. Compared with the traditional hybrid identification method, it is not only accurate and reliable, but also can shorten the breeding cycle, reduce breeding costs, and can screen breeding target offspring early, effectively improve the identification efficiency of sisal interspecific hybrids, and provide a more effective product for quickly identifying the authenticity of sisal interspecific hybrid offspring.

[0029] At the same time, the present invention provides a method for identifying the authenticity of interspecific hybrid F1 hybrids of sisal varieties using InDel markers. The method can simply, conveniently, efficiently, stably and accurately identify the authenticity of the hybrid F1 generation, without having to wait until the F1 generation grows into plants to make a judgment, and can accurately, conveniently and quickly identify the genetic traits of the hybrid offspring. The present invention fills the gap in identifying the authenticity of sisal hybrid offspring using InDel markers, and the provided method can quickly identify the authenticity of interspecific hybrids of sisal, which is beneficial to the genetic improvement of sisal varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 2 shows the SNP and InDel analysis results of the resequencing of ‘Moyin7’ and ‘292’ ( Figure 1 A in the figure indicates the distribution of SNP and InDel markers on the sisal genome; Figure 1 B in the figure is the variation type of the effective SNP marker; Figure 1 The C in the figure is the length distribution frequency of the effective InDel markers (1-11 means 1-11 bp in length, and the same applies to 12-84 and 85-276).

[0031] Figure 2 This is the result of InDel primer screening (in the figure, M: DNA 2000 marker; 292: male material '292'; M7: female material 'Moyin 7'; 11648: reference gene variety) Agave hybrid NO.114648).

[0032] Figure 3 This is the result of F1 generation identification (in the figure, M: DNA 2000 Marker; 292: paternal material '292'; M7: maternal material 'Moyin 7'). DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The materials used in this example are: sisal parent materials '292', 'Moyin 7' and their hybrid F1 generation, where '292' and 'Moyin 7' are the male and female materials respectively, and are also the resequencing materials of this experiment; 'Moyin 7' was introduced from Mexico in 2005, and '292' was obtained by hybridization in 1995 (common sisal (female parent) × H.11648 (♂) (male parent)). A hybrid population of '292' (male parent) and 'Moyin 7' (female parent) was created through artificial pollination, and 7261 seeds were obtained, which were sown in the sisal germplasm resource garden of the South Asian Crop Research Institute of China Tropical Agricultural College, and 3147 hybrid offspring were obtained.

[0036] Example 1 Whole genome resequencing and InDel site analysis

[0037] 1. DNA extraction and detection

[0038] The method for extracting DNA from sisal '292' (male parent) and 'Moyin 7' (female parent) was based on the prior art Yang et al. (Ziping Yang, Qian Yang, Qi Liu, Xiaolong Li, Luli Wang, Yanmei Zhang, ZhiKe, Zhiwei Lu, Huibang Shen, Junfeng Li and Wenzhao Zhou. A chromosome-level genome assembly of Agave hybrid NO.11648 provides insights into the CAMphotosynthesis. Horticulture Research, 2024, 11(2): uhad269.). The DNA concentration of each tube was then measured using a photophotometer, and 1% agarose gel electrophoresis was used for detection, and the integrity of the DNA was analyzed using a gel imaging system. DNA samples with qualified concentration and quality were stored at -80°C for later use.

[0039] 2. Whole genome resequencing

[0040] The DNA of the paternal and maternal samples was sent to Lianchuan Bio for whole-genome resequencing to obtain the original data. After removing the adapter sequence and data quality control from the original data, the valid data was obtained. The valid data was compared with the existing sisal H.11648 genome data (Yang et al., 2024) using BWA software to obtain a BAM format file. Then, the SAMtools software was used to sort the comparison results, and the Picard software was used to mark and delete the duplicate sequences; the GATK software was then used to re-align, calibrate and readjust the read quality. Later, the vcftools software was used to calculate the InDel site distribution density on the whole genome based on the vcf file, and the analysis results were composed and displayed using the R language circlize package, RColorBrewer package and ggsci package.

[0041] 3. Results Analysis

[0042] Using the existing sisal H.11648 genome data, DNA resequencing analysis was performed on the hybrid population parents 'Moyin 7' and '292'. The resequencing data analysis results are as follows Figure 1 As shown in A, 55,046,628 SNPs and 21,976,197 InDel sites were searched between the two materials.

[0043] Subsequently, these markers were filtered according to the following process: (1) non-binary mutation sites were screened out; (2) heterozygous mutation sites were removed; and (3) sites with no deletions in the parents and different genotypes were obtained.

[0044] Results A total of 9,524,689 SNPs and 1,659,292 InDel markers were obtained, and the number of effective polymorphic markers on chromosomes is shown in Table 1.

[0045] Table 1 Number of effective polymorphic markers on chromosomes

[0046]

[0047] By typing the effective SNP markers, the results are as follows Figure 1 As shown in B, C>T (C mutates to T, the same below) and A>G are the main mutation types. For InDel markers, the number of inserted / deleted bases is used as the statistical object, and the results are as follows: Figure 1 As shown in Figure C, the vast majority of InDels are mainly mutations of several bases (1-11 bp), and the number of markers decreases with the increase in the number of mutated bases. In addition, there are 645 InDel markers larger than 100 bp, which can be used for the design and screening of subsequent InDel site primers.

[0048] Example 2 InDel Primer Screening

[0049] 1. InDel primer design

[0050] According to the InDel sites obtained by resequencing analysis of the parents, sites with variable InDel>50 bp between the parents were selected, and primers were designed in the conserved region 200 bp upstream and downstream of the variable site using Primer Premier 5.0 software. The primer design parameters were: GC content 40%~50%, annealing temperature 50℃~60℃, and primer fragment length 18 bp~22 bp. Ten pairs of primers were randomly selected and designed from 645 InDel markers, as shown in Table 2. Primers were synthesized by Guangzhou Aiji Biotechnology Co., Ltd.

[0051] Table 2 InDel primer list

[0052]

[0053] 2. PCR system and electrophoresis

[0054] Using the DNA of the parents as templates, PCR amplification was performed using the primers in Table 2. The primers that can amplify the characteristic bands of the father and mother are used to identify the authenticity of the hybrid offspring. Agave hybridThe reference gene NO.114648 was used as a control. PCR system: 2 μL DNA template, 0.2 μL upstream and downstream primers, 10 μL Mix, and a total volume of 20 μL made up with sterile water. PCR amplification program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 65℃ annealing and extension for 20 s, 40 cycles; 72℃ extension for 5 min, and storage at 25℃. 2% agarose gel was used for electrophoresis at 120 V for 15 minutes. Images were acquired using the BIO-RAD gel imaging system.

[0055] The amplification results are as follows Figure 2 As shown, C28_35176484 can amplify multiple bands for three materials, C04_175683614 can amplify multiple bands for DNA of 292 and M7, but no band for 11648; C05_275624710 can amplify 2 bands for DNA of 292 and M7, but no band for 11648; C18_76724499 amplifies a single band for three templates, but the difference in band size is not obvious; C03_416856662 can amplify 1 band for DNA of 292 and M7, but no 2 bands for 11648; C03_463164855 amplifies a single band for three templates, and the size of the DNA amplified band for 292 is about 250 bp, and the size of the DNA amplified band for M7 is about 500 bp. bp, the amplified product size difference is obvious, which can clearly distinguish 292 and M7; the other 4 primer pairs cannot amplify bands. In summary, C03_463164855 can be used to distinguish 292 and M7.

[0056] Example 3 Authenticity Identification of Hybrid F1 Generation

[0057] The C03_463164855 primer was used to identify 298 randomly selected F1 generations, and the leaf tissue DNA of 298 hybrid materials was extracted: the samples were placed in 2 mL centrifuge tubes, and 2 steel beads with a diameter of 5.2 mm and 200 μL of DNA extraction solution were placed in each 2 mL centrifuge tube containing leaf samples. The samples were ground on a fast grinder with the following parameters: running time 40 s, interruption time 5.0 s, grinding times 2, and frequency 60.00 Hz. After the samples were ground, 200 μL of DNA extraction solution was added and mixed evenly, and then the genomic DNA was extracted according to the method of the Tiangen kit.

[0058] The DNA of the F1 generation plant was used as a template for PCR amplification, and the amplification method and conditions were the same as those in Example 2, followed by gel electrophoresis detection. The offspring that can simultaneously amplify the characteristic bands of the male and female parents are true hybrids, and the lack of any one of the bands or the lack of two bands are considered false hybrids. At the same time, the purity of the authenticity of the hybrid F1 generation is calculated, and the specific calculation formula is:

[0059] Purity = (total amount detected - number of false hybrids) / total amount detected × 100.

[0060] Test results such as Figure 3 As shown, 292 F1 plants were able to amplify a band of about 250 bp for the male parent and about 500 bp for the female parent. Four F1 plants were able to amplify only one band, and two F1 plants did not amplify any bands. Based on the above results, the 292 F1 plants tested were true hybrids with a purity of 97.98%.

[0061] At the same time, the phenotypic situation of leaf thorns of 292 F1 hybrid offspring was investigated in the field, showing that all materials contained leaf thorns, and the phenotype tended to be the mother parent 'Moyin 7'. However, the 6 F1 hybrid offspring that did not amplify the bands had no thorns on the leaf edges. Therefore, the structure identified by primer C03_463164855 was consistent with the phenotypic observation results.

[0062] Example 4 A method for identifying the authenticity of the F1 hybrid of sisal '292' and 'Moyin 7'

[0063] This embodiment provides a method for identifying the authenticity of F1 hybrids of interspecific hybridization between sisal '292' and 'Moyin 7' varieties using InDel marker primers, comprising the following steps:

[0064] S1. Extract genomic DNA from parental sisal and interspecific hybrid F1 hybrids respectively;

[0065] S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification was performed using InDel-labeled primers C03_463164855: 5'-AGGGGGAGACGATAGCACA-3', 5'-TCCCCACCAACCCTAGGC-3', and then detected by agarose gel electrophoresis;

[0066] S3. Identification of the authenticity of F1 hybrids: Analyze the S2 test results. The offspring that can simultaneously amplify the characteristic bands of the father and the mother are true hybrids. The lack of any one of the bands or the lack of two bands are considered false hybrids.

[0067] At the same time, a kit for identifying the authenticity of the F1 generation of the hybrid of sisal '292' and 'Moyin 7' varieties is also provided, which contains InDel labeled primer C03_463164855: 5'-AGGGGGAGACGATAGCACA-3', 5'-TCCCCACCAACCCTAGGC-3', as well as reagents for extracting sample DNA and reagents required for PCR amplification.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An application of an InDel marker primer in identifying the authenticity of the F1 hybrid of sisal '292' and 'Moyin 7' varieties, or in screening the F1 hybrid of sisal '292' and 'Moyin 7' varieties, characterized in that: The InDel marker primers include an upstream primer: 5'-AGGGGGAGACGATAGCACA-3', and a downstream primer: 5'-TCCCCACCAACCCTAGGC-3'.

2. The use of InDel labeled primers in the preparation of a kit for identifying the authenticity of F1 hybrids of sisal '292' and 'Moyin 7' varieties, characterized in that: The InDel marker primers include an upstream primer: 5'-AGGGGGAGACGATAGCACA-3', and a downstream primer: 5'-TCCCCACCAACCCTAGGC-3'.

3. The application according to claim 2, characterized in that: The kit contains reagents for extracting sample DNA.

4. The application according to claim 2, characterized in that: The kit also contains reagents required for PCR amplification.

5. Use of the kit for identifying the authenticity of the hybrid F1 generation of sisal '292' and 'Moyin 7' varieties, or for screening the hybrid F1 generation of sisal '292' and 'Moyin 7' varieties, characterized in that: The kit contains InDel labeled primers; the InDel labeled primers include an upstream primer: 5'-AGGGGGAGACGATAGCACA-3', and a downstream primer: 5'-TCCCCACCAACCCTAGGC-3'.

6. A method for identifying the authenticity of F1 hybrids of interspecific hybridization between sisal '292' and 'Moyin 7' varieties using InDel marker primers, characterized in that: The following steps are involved: S1. Extract genomic DNA from the parents of sisal '292' and 'Moyin 7' and the interspecific hybrid F1 generation; S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification was performed using InDel labeled primers, and then agarose gel electrophoresis was performed; S3. Identification of the authenticity of F1 hybrids: Analyze the results of S2 test. The offspring that can simultaneously amplify the characteristic bands of the father and the mother are true hybrids. The lack of any one of the bands or the lack of two bands are considered false hybrids. The InDel marker primers include an upstream primer: 5'-AGGGGGAGACGATAGCACA-3', and a downstream primer: 5'-TCCCCACCAACCCTAGGC-3'.

7. The method according to claim 6, characterized in that: The PCR amplification system is as follows: 2 μL of DNA template, 0.2 μL of upstream and downstream primers, 10 μL of Mix, and a total volume of 20 μL made up with sterile water; PCR amplification program: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 65°C annealing and extension for 20 s, 40 cycles; 72°C extension for 5 min, and storage at 25°C.

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