An InDel molecular marker for genetic diversity analysis of yellow peel and its application

Abstract

The present invention provides a high-throughput specific InDel molecular marker for the whole genome of yellow peel and its application. The present invention mines InDel sites to develop molecular markers based on the results of whole genome resequencing analysis of 94 yellow peel germplasm resources, and selects 138 pairs of polymorphic marker primers through verification. 28 InDel markers were randomly selected from them to carry out cluster analysis on 38 yellow peel germplasm resources, revealing their genetic diversity. The results showed that the effective allele number ranged from 1.7304 to 2, the gene polymorphism index was between 0.333 and 0.375, and the Shannon diversity index was between 0.613 and 0.693. The cluster analysis results showed that at the genetic similarity coefficient of 0.57, the 38 yellow peel resources were divided into 2 subgroups. The results of this study laid a foundation for the subsequent breeding of yellow peel.

Description

Technical Field

[0001] The invention relates to the field of genetic breeding, and in particular to an InDel molecular marker for yellow peel genetic diversity analysis and an application thereof. Background Art

[0002] Clausena lansium (Lour.) Skeels is a tropical and subtropical evergreen fruit tree belonging to the genus Clausena Burm.f. of the subfamily Aurantioideae of the family Rutaceae. There are about 30 species of Clausena genus in the world, distributed in Asia, Africa and Oceania, and there are about 10 species and 2 variants in my country. Clausena lansium is a cultivated species of this genus, and currently only large-scale commercial cultivation is carried out in China. Clausena lansium has sweet and sour flesh, unique flavor and rich nutrition. Because of its antioxidant and anti-cancer effects, it is a dual-purpose fruit for medicine and food, and is deeply loved by consumers. In recent years, the market for fresh Clausena fruit has been in short supply, and the price of Clausena has risen sharply, bringing higher economic benefits to fruit farmers. Therefore, the cultivation area of ​​Clausena lansium has continued to grow, becoming a highlight of rural economic growth in the production area.

[0003] my country is the origin of yellow peel, with a long history of cultivation. Long-term natural evolution and artificial selection have formed rich germplasm resources. Due to the lack of systematic research and collation, the genetic background and kinship between different germplasms are unclear; at the same time, many homonyms and homonyms have appeared in the process of germplasm exchange between different regions, which is very unfavorable for germplasm resource research and breeding. Molecular markers have the advantages of genetic stability and are not easily affected by the environment and materials. They are valuable tools for germplasm resource analysis and identification and evaluation. Recently, the use of random amplified polymorphic DNA (RAPD) marker pairs, simple sequence repeat interval (ISSR) markers, SSR markers, etc. to analyze the genetic diversity of yellow peel germplasm has been reported. InDel markers (insertion-deletion) refer to individual variations caused by the insertion and deletion of nucleotide fragments in the allele point sequences of different individuals between populations. Their stability and separation effect are better than SSR. In addition, InDel markers have the advantages of low development cost and wide distribution in the genome, so they are widely developed and utilized in the fields of crops and fruit trees. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an InDel molecular marker primer set for yellow peel genetic diversity analysis.

[0005] Another object of the present invention is to provide an application of the above primer set.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] An InDel molecular marker primer set for yellow peel genetic diversity analysis includes the following primer pairs:

[0008] The primer pairs used to identify the InDel molecular markers InDel-29, InDel-27, InDel-30, InDel-39, InDel-53, InDel-74, InDel-82, InDel-84, InDel-102, InDel-108, InDel-114, InDel-127, InDel-130, InDel-135, InDel-147, InDel-170, InDel-171, InDel-181, InDel-187, InDel-202, InDel-208, InDel-212, InDel-214, InDel-223, InDel-224, InDel-255, InDel-252, and InDel-270, whose nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.56.

[0009] An InDel molecular marker for yellow peel genetic diversity analysis is obtained by amplifying the yellow peel genome using the primer set.

[0010] The application of the above primer set or molecular markers in constructing a fingerprint map of yellow peel.

[0011] Application of the above primer sets or molecular markers in the analysis of genetic diversity of yellow peel.

[0012] Compared with the prior art, the present invention has the following advantages and effects:

[0013] The present invention mines InDel sites and develops molecular markers based on the results of whole genome resequencing analysis of 94 yellow peel germplasm resources. By verifying the effectiveness of 270 pairs of primers, 138 pairs of primers with polymorphism were selected, and the polymorphism rate reached 50.55%. The present invention conducted a cluster analysis on 38 yellow peel germplasm resources based on 28 InDel markers with the best amplification effect, revealing its genetic diversity. The results showed that the effective allele number ranged from 1.7304 to 2, the gene polymorphism index was between 0.333 and 0.375, and the Shannon diversity index was between 0.613 and 0.693. The cluster analysis results showed that at the genetic similarity coefficient of 0.57, 38 yellow peel resources were divided into 2 subgroups. The results of this study laid the foundation for the subsequent breeding of yellow peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the electrophoresis diagram of the amplification products of the 28 pairs of primers shown in Table 3 in 38 yellow peel materials.

[0015] Figure 2 This is the dendrogram of UPMGA cluster analysis of 38 yellow peel germplasms.

[0016] Figure 3 This is the genetic distance map of 38 yellow peel germplasm resources. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0018] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0019] Example 1 Wampee materials and field trials

[0020] 94 core resources of Huangpi (see Table 1) were selected for whole genome resequencing. 12 Huangpi germplasm materials were randomly selected for InDel marker validity identification, and 38 Huangpi resources (see Table 2) were used for cluster analysis. The above experimental materials were planted in the Guangzhou Huangpi resource nursery of the Ministry of Agriculture and Rural Affairs (geographic location 113°22′E, 23°09′N, altitude 20m) and breeding nursery. The germplasm resources are all over 15 years old, and the soil and fertilizer management is consistent.

[0021] Table 1 Basic information of 94 Wampee germplasm resources used for resequencing

[0022]

[0023]

[0024] Table 2 Basic information of 38 yellow peel resources used for cluster analysis

[0025]

[0026] Example 2 Whole-genome high-throughput sequencing and InDel site mining

[0027] (1) The young leaves of the yellow-peeled material were placed in liquid phase and the total genomic DNA was extracted using the CTAB method. The concentration, purity and integrity were tested, and the qualified samples were used for subsequent library construction and sequencing. The above work was completed by Beijing Novogene Biotechnology Co., Ltd. The raw reads were quality evaluated and filtered to obtain Clean Reads, which were then compared with the reference genome sequence using BWA, and InDel detection and annotation were performed based on the comparison results.

[0028] (2) The raw data obtained by Illumina sequencing of 94 Huangpi germplasm resources were filtered to obtain Cleandata. The Q20 of the Clean data of each sample was above 95%, and the GC content was between 34.25% and 35.15%. After comparing the reads with the reference genome, the contrast ratio was between 98.51% and 99.58%. In summary, the sequencing quality, GC content, and data volume of the 94 Huangpi germplasm resources were qualified and suitable for the development of InDel markers. After alignment with the Huangpi reference genome sequence, high-quality InDels were obtained through the following filtering and screening. The filtering conditions were: the average sequencing depth of the variant site was >10; the minimum allele frequency MAF>0.05; the proportion of missing genotypes in all resequencing samples was <10%. After filtering, a total of 219,120 high-quality InDel sites were obtained, of which 12,568 were located in the coding region. Based on the above InDel results, the polymorphic information content (PIC) was calculated, and InDel sites with PIC>0.45 were screened as polymorphic sites, and a total of 65,326 polymorphic InDel sites were obtained.

[0029] Example 3 InDel primer design and marker validity identification

[0030] Sequences of 200 bp upstream and downstream of the polymorphic InDel site were extracted, and primers were designed for InDel using primer3 (a software for designing primers under Linux or Unix system), with the following conditions: primer length between 18 and 27 bp, annealing temperature between 60 and 65 °C, and product length between 200 and 500 bp. 60,955 polymorphic InDel marker primers were successfully designed, with a primer design success rate of 93.91%. 30 markers were randomly selected on each chromosome, and a total of 270 pairs of InDel primers were finally selected (Table 3).

[0031] The 270 pairs of InDel primers were used for labeling validity identification, and PCR amplification was performed using DNA from 12 yellow peel germplasm materials with significant phenotype differences as templates. PCR reaction system (20u1): DNA template (20-50ng / ul) 1u1, 2×Taq PCRStarMix (GenStar) 10ul, upstream primer (10uM) 0.4u1, downstream primer (10uM) 0.4u1, ddH 2 O 8.2u1, amplification procedure: 94℃ pre-denaturation: 2min; 94℃ denaturation 30s, 55-65℃ annealing 30s, 72℃ extension 15s, a total of 32 cycles; 72℃ extension 5min, 4℃ hold. The amplified product was tested by 3% agarose gel electrophoresis, and then the gel running results were photographed and recorded using a gel performance system to count the band types. In the test results, 270 pairs of primers amplified the target bands, and 0 pairs of primers did not amplify the bands, with an efficiency of 100%. Statistics of the agarose gel electrophoresis test results showed that 138 pairs of primers out of 270 pairs of primers were polymorphic in 12 yellow peels, and the polymorphism rate was 50.55%.

[0032] Example 4 Application of InDel primers in analyzing and constructing the fingerprint of yellow peel germplasm resources

[0033] InDel markers with clear bands and polymorphism were selected, and 38 yellow peel germplasm resources genomic DNA were used as templates for PCR amplification and agarose gel electrophoresis detection. The amplification results were photographed and counted in the same way as above. Finally, 28 pairs of primers with the best amplification effect were selected (Table 3) for PCR amplification of 38 yellow peel varieties. The amplified products were separated by 3% agarose gel, the electrophoresis buffer was 0.5X TBE, and the electrophoresis was separated by 130V constant voltage electrophoresis for 30-50min. After electrophoresis, the band characteristics were observed by imaging with a gel imaging system (Bio-Rad, lercules, CA, USA), and the genotyping results were counted. Band statistical method: "1" is recorded for the presence of bands at the allele, "0" is recorded for the absence of bands, and "9" is recorded for the missing ones. After generating the band matrix, NTSYS-pc was used to calculate the genetic distance of the germplasm resources ( Figure 3 ), and the UPGMA algorithm was used to perform cluster analysis on 38 yellow peel germplasm resources ( Figure 2 Except for the samples of germplasm resources No. 29 and No. 89, which were the same germplasm resource or the candidates were the same germplasm resource, 28 pairs of primers could completely separate the remaining 37 resources, and each material had a unique fingerprint (Table 4).

[0034] Table 3 28 pairs of primers with the best amplification effect

[0035] InDel Marker Chromosome Position (bp) Forward Primer (5'-3') Reverse Primer (5'-3') InDel-27 Chr1 34131371 AGCGCAAGGTAGTTTTTCTCAGTC TGAAGGTTAAGGTTTTTCCACAATG InDel-29 Chr1 38881569 GGGCTTGATCTTCAGATGTGTCTT CGCAGGCGATTTAAATTTTGTTAT InDel-30 Chr1 40592449 AACACGATTTTCTCATGTTGGGTT CGTAAATAGTCCAAATTCGCATCC InDel-39 Chr2 3323403 TCCAGTTAAAGTATGAAATTGCTTCAA GGGAGTCTCAAAATATCAAGCTAAAAA InDel-53 Chr2 25309582 TCCTACACCCACTACATGAAACCA TTCTCCTCCCATATCACACTAAACG InDel-74 Chr3 12142947 TTGCTCTCCCTAAATCCAACCTTT ATTTAATTTTTGGTGCCCAAATCC InDel-82 Chr3 22958689 TATCCTCCGTCAGCTTTCAGAATC ATCCCTGCTGGTGAGATATCAGTC InDel-84 Chr3 25073164 AATAATCCGGGAAGGACCTTATGA TGAGTTTGCTTGATTGATGGATTG InDel-102 Chr4 6037468 TGGTTCAATGGCATTTTAGGATCT ATCCTGCATGTTGGCCTTAAGATA InDel-108 Chr4 14589279 TGGGGATTAGAATTGATTGCTTGT ATCTGGTTTTATCCAAATTGGCCT InDel-114 Chr4 23946496 ACATGGAGGGGGAATTATCAGAGT ACATCAACCGAAATGAAGTGTCCT InDel-127 Chr5 2089732 TTGTCAAAGAAGGAAATACACGGG AATAACTTCAGAGGGGGAGCATTG InDel-130 Chr5 4303332 ATTTGGTGGTAATTGGAGTTTTGA TGCTCCATTTAGTATGATTGTCTAGC InDel-135 Chr5 11603497 AGAGGAACCCCATACAAAGTGTGA GCTTACGTGGCTGGTTTTTATTTG InDel-147 Chr5 25261146 AGATTGGGTGAATTTGTGCTTGTT TTTTAATTTCGGAATCCATAGCCC InDel-170 Chr6 20020950 GGCAGCATTGGAAGACACTTTAAC AAAAGAATCGCATTATGCCAGAAA InDel-171 Chr6 21043011 AAATTAATCCTGATCCCTACCGTAAAA ATTGACACGTGGGATTAGAAAGGA InDel-181 Chr7 613083 CCATGTGCATTTACAATTTTGCC ATCCTTTCCCTTCGACGAGTATTG InDel-187 Chr7 4255862 TGAAGAGAAGAAGGCCTAAACGTG GCAGCATTTCAAGTTTTAACCCAC InDel-202 Chr7 20340035 TACTGCAGAGCACAGAAGCACTCT TTACAAGCACAAACTCTCGCTTTG InDel-208 Chr7 25060664 GCCAAGCATGAAGGTTAACAGAAG GCCAATGAGGGGATCGTTAAAAT InDel-212 Chr8 1030265 AAAGCGAGGAGATTCAAAGTGATG CCATCCTTCAAATTTATGCAAAGC InDel-214 Chr8 2004780 CAATTGATTTCAACGTCACCAACT AAACTGAATTGGACGGAATTAGCA InDel-223 Chr8 10019616 TTAGAGTCGAGCACTGATGTGTCC TACATTGGTGAAGCTCAAGCAAAC InDel-224 Chr8 11039163 CAAATTCATTGCCTCTAAAGCACC AAAAATTCTTCAAGACCTGTCCCC InDel-255 Chr9 9616545 AAAGTTTTGGGTTTGAATCTCGC TGGTAATAACCAGAATTGTCAACCAA InDel-258 Chr9 12362073 TGAGGTCAATTTTCGCAACTTTTT TCTCAAATTAATCCCTTTCCGTCA InDel-269 Chr9 22620899 AATGGATCGAAAGGTTTGCAGATA TAATGCTGGGGACTGTTTATCGTT

[0036] Table 4 DNA fingerprints of 37 yellow peel germplasm resources

[0037]

[0038]

[0039] Example 5 Application of InDel primers in analyzing genetic diversity of yellow peel germplasm resources

[0040] 28 pairs of polymorphic InDel primers were selected for PCR amplification of 38 yellow peel varieties. The amplified products were separated by 3% agarose gel, the electrophoresis buffer was 0.5X TBE, and the electrophoresis was separated by 130V constant voltage electrophoresis for 30-50 minutes. After electrophoresis, the band characteristics were observed by gel imaging system (Bio-Rad, lercules, CA, USA), and the genotyping results were statistically analyzed. Band statistical method: "1" is recorded for the presence of bands at the allele, "0" is recorded for the absence of bands, and "9" is recorded for the absence. After generating the band matrix, Popgene32 was used to calculate the primer Nei, Shannon and other values, and the results are shown in Table 5.

[0041] Table 5 Genetic diversity of 28 pairs of InDel primers in 38 yellow peel germplasms

[0042]

[0043] As shown in Table 5, among the 38 yellow peel germplasms, the effective allele number ranged from 1.7304 to 2, the gene polymorphism index was between 0.333 and 0.375, and the Shannon diversity index was between 0.613 and 0.693. The effective allele number, gene diversity index, and Shannon diversity index showed similar trends, with the maximum values ​​appearing at InDel-138 and InDel-205, and the minimum values ​​at InDel-54 and InDel-83.

[0044] From Table 5 and Figure 3 It can be seen that cluster analysis of 38 yellow peel germplasm resources using 28 InDel markers with good polymorphism found that at the genetic similarity coefficient of 0.57, the 38 yellow peels were divided into two groups. The largest group is group 1#, whose germplasm comes from a wide range of sources, from the main cultivation areas of yellow peel in my country, including Guangdong, Fujian, Hainan and a small number of varieties in Guangxi Zhuang Autonomous Region; subgroup 2# materials mainly come from Guangxi Zhuang Autonomous Region. At the genetic similarity coefficient of 0.592, group 1# can be divided into two subgroups. The larger subgroup 1#A is mainly varieties from Guangdong and Fujian, while the yellow peel varieties in Hainan are concentrated in subgroup 1#B. There are 2 yellow peel materials with a genetic similarity of 100%, and the primer amplification bands of the two materials are exactly the same, which determines that the two varieties are or candidates for the same variety.

[0045] The above results indicate that the 270 InDel markers are suitable for constructing fingerprints of yellow peel germplasm resources, identifying the specificity of germplasm resources, and analyzing the genetic diversity of germplasm resources.

[0046] 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 InDel molecular marker primer set for analyzing the genetic diversity of yellow peel, characterized in that The following primer pairs are included: The primer pairs used to identify the InDel molecular markers InDel-29, InDel-27, InDel-30, InDel-39, InDel-53, InDel-74, InDel-82, InDel-84, InDel-102, InDel-108, InDel-114, InDel-127, InDel-130, InDel-135, InDel-147, InDel-170, InDel-171, InDel-181, InDel-187, InDel-202, InDel-208, InDel-212, InDel-214, InDel-223, InDel-224, InDel-255, InDel-252, and InDel-270, whose nucleotide sequences are shown in SEQ ID NO.1 to SEQ ID NO.

56.

2. Application of the primer set described in claim 1 in constructing a fingerprint map of yellow peel.

3. Use of the primer set described in claim 1 in the analysis of genetic diversity of yellow peel.

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