CN115976251B - A kind of InDel marker and its application for cassava genetic diversity analysis - Google Patents

Abstract

The invention provides a primer set developed based on the InDel marker of the whole cassava genome and its application. Based on the whole genome resequencing analysis results of 22 cassava germplasm resources, the present invention excavates InDel sites to develop molecular markers, and verifies the effectiveness of 64 pairs of primers. As a result, 20 pairs of primers have polymorphism, and the polymorphism rate is 31.25%. . Based on the cluster analysis results of 18 InDel markers with the best amplification effect, the genetic diversity of 72 cassava germplasm resources was revealed. The gene diversity index of each marker was between 0.21 and 0.50, and the Shannon diversity index was between 0.36 and 0.70 between. The results of cluster analysis showed that 72 cassava materials were divided into two groups at the genetic similarity coefficient of 0.62. The results of this study will lay a certain foundation for the subsequent development of cassava breeding.

Description

A kind of InDel marker and its application for cassava genetic diversity analysis

technical field

The invention relates to the field of biotechnology, in particular to an InDel marker for cassava genetic diversity analysis and its application.

Background technique

Cassava (Manihot esculenta Crantz) belongs to Euphorbiaceae and is one of the sixth most important crops in the world. Originating in Brazil, it originated in America at first, and was later introduced and promoted by more than 100 countries in Africa and Asia. It was first introduced into my country's Guangdong Province in the 1820s, and is now mainly distributed in the southeast coastal areas of my country, such as Guangxi, Hainan and other provinces and regions. In addition, it is also planted in Fujian, Jiangxi, Hunan and Guizhou. Because it is rich in a large amount of starch, it is widely used in the production of industrial raw materials, and can also be used as a source of calories needed by the human body, so it has become the staple food of nearly 1 billion people in the world. As a key food crop and an important industrial raw material, it plays an important role in international animal husbandry, chemical industry, food, environmental protection and other industries.

It has been nearly 200 years since cassava was introduced into my country. At present, Chinese researchers have screened a series of main plant varieties from the introduced germplasm. Due to its complex genetic background and diverse germplasm resources, the progress of collection, preservation and breeding of its germplasm materials in my country is relatively slow. However, the breakthrough of breeding work depends on the degree of genetic diversity of resources among varieties, so the analysis of genetic diversity among populations is of great significance for species breeding. According to previous reports, molecular markers have the advantages of stable genetics, not easily affected by the environment and materials. At present, it has been proved to be a valuable tool for germplasm resource analysis and identification and evaluation, because it has the advantages of genetic stability, not easily affected by the environment and material selection. At present, scholars have used SRAP (Sequence-related amplified polymorphism), SSR (Simple Sequence Repeats), AFLP (Amplified Fragment Length Polymorphism), RAPD (random amplified polymorphic DNA) and other markers to analyze the genetic diversity of cassava germplasm. InDel marker (insertion-deletion) refers to the individual variation caused by the insertion and deletion of nucleotide fragments in different individual allele point sequences between populations, that is, a gap is found after comparing homologous sequences. Compared with SNP, the application of InDel is faster, and the electrophoresis technology platform can be used for quick detection, and the requirements for facilities and methods are simpler. Some scholars compared it with the amplification results of SSR, and found that its stability and separation effect were better than those of SSR. In addition, InDel markers also have the advantages of low development cost and wide distribution in the genome, so they have gradually become a research hotspot in the field of crops, such as pepper, rapeseed, tomato, upland cotton, kale, etc. However, there are still few research reports on the application of InDel markers to the analysis of cassava germplasm resources.

In this study, the whole genome resequencing results of 22 cassava germplasm resources were analyzed, and InDel markers suitable for electrophoresis detection were excavated and developed, and 5 cassava germplasm resources were selected for molecular marker effectiveness identification. Finally, 18 pairs of InDel markers with polymorphism and clear bands were screened out to analyze the genetic diversity and population structure of 72 cassava germplasm resources, laying a certain foundation for the subsequent development of cassava breeding.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, and provide a primer set developed based on the InDel marker of the whole cassava genome and its application.

The first aspect of the present invention is to provide a primer set developed based on the InDel markers of the whole cassava genome, the primer set includes 18 pairs of InDel primers whose nucleotide sequences are shown in SEQ ID NO.1-36.

Preferably, the primer set further includes 2 pairs of InDel primers whose nucleotide sequences are shown in SEQ ID NO.37-40.

The second aspect of the present invention is to provide a kit comprising the primer set described in the first aspect of the present invention.

Preferably, the kit also includes reagents required for PCR reaction and/or electrophoresis.

The third aspect of the present invention is to provide the application of the primer set described in the first aspect of the present invention or the kit described in the second aspect of the present invention in cassava genetic diversity analysis.

The fourth aspect of the present invention is to provide a method for analyzing the genetic diversity of cassava, which is carried out by using the primer set described in the first aspect of the present invention or the kit described in the second aspect of the present invention.

Preferably, the analytical method comprises the steps of:

(1) extract the genomic DNA of cassava to be analyzed;

(2) using step (1) DNA to be tested as a template, applying the primer set described in the first aspect of the present invention or the kit described in the second aspect of the present invention to carry out PCR amplification;

(3) Analyze the genetic diversity of cassava to be analyzed according to the amplified genomic DNA polymorphic bands.

Genetic diversity analysis includes but is not limited to analyzing genetic parameters such as effective allele number, gene diversity index, and Shannon diversity index, and constructing UPMGA clustering dendrograms, etc.

The fifth aspect of the present invention is to provide the application of the primer set described in the first aspect of the present invention or the kit described in the second aspect of the present invention in distinguishing cassava germplasm.

Based on the whole genome resequencing analysis results of 22 cassava germplasm resources, the present invention excavates InDel sites to develop molecular markers, and verifies the effectiveness of 64 pairs of primers. As a result, 20 pairs of primers have polymorphism, and the polymorphism rate is 31.25%. . Based on the cluster analysis results of 18 InDel markers with the best amplification effect, the genetic diversity of 72 cassava germplasm resources was revealed. The gene diversity index of each marker was between 0.21 and 0.50, and the Shannon diversity index was between 0.36 and 0.70 between. The results of cluster analysis showed that 72 cassava materials were divided into two groups at the genetic similarity coefficient of 0.62. The results of this study will lay a certain foundation for the subsequent development of cassava breeding.

Description of drawings

Figures 1 to 18 are the electrophoresis diagrams of the amplification products of 18 pairs of primers shown in Table 3 in 72 cassava materials.

Figure 19 is the electrophoresis diagram of the amplification products of the primer pair IDC49 in 72 cassava materials.

Figure 20 is the UPMGA clustering dendrogram of 72 cassava diversity.

Detailed ways

Referring to the accompanying drawings, the present invention will be further described in conjunction with specific embodiments, so as to better understand the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

1 Cassava materials and field tests

22 genetically stable cassava materials (see Table 1 for the basic information of the 22 cassava materials) were selected for whole genome resequencing. Five cassava germplasm materials were randomly selected for the identification of InDel marker validity, and 72 cassava materials (the basic information of the 72 cassava germplasm resources are shown in Table 4) were used for cluster analysis. The above materials were all planted in the National Cassava Germplasm Resource Garden of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences in March 2021.

Table 1 Basic information of 22 cassava germplasm resources

serial number material name Germplasm type source serial number material name Germplasm type source C4 SC201 Breeding varieties China C37 GR024-8 Breeding varieties China C6 Yunnan 7 local breeds China C40 CMR37-14-9 Introduced strains Thailand C9 Switzerland W4 Introduced strains Switzerland C46 COL739 Introduced strains Colombia C12 ZM8625 strain China C47 Switzerland P11 Introduced strains Switzerland C15 CM4040-1 Introduced strains Colombia C49 Indonesian species Introduced strains Indonesia C17 R5 Introduced strains Thailand C57 Swiss Q10 Introduced strains Switzerland C22 KM98-6 Introduced strains Vietnam C58 COL2626 Introduced strains Colombia C27 Swiss H19 Introduced strains Switzerland C60 R80 Introduced strains Thailand C31 Brazil 7 Introduced strains Brazil C69 Guire No. 5 Breeding varieties China C35 SC6 Breeding varieties China C71 SC12 Breeding varieties China

2 Whole genome high-throughput sequencing and InDel site mining

The young leaves of cassava material were placed in liquid nitrogen to extract the total genomic DNA by CTAB method, and its concentration, purity and integrity were tested, and the qualified samples were used for subsequent library construction and sequencing respectively. The above work was completed by Beijing Baimaike Biotechnology Co., Ltd. The obtained raw reads were evaluated for quality and filtered to obtain CleanReads, which were compared with the reference genome sequence using BWA, and InDel detection and annotation were performed based on the comparison results.

The raw data obtained by Illumina sequencing of 22 cassava germplasm resources were filtered to obtain 671.57G Clean Data. The Q20 of the clean data of each sample is above 85%, and the GC content is between 36.9% and 45.04%. After comparing the reads with the reference genome, the comparison ratio is between 84.93% and 98.60%. In summary, the sequencing quality, GC content, and data volume of 22 cassava germplasm resources are all qualified, which can be applied to the development of InDel markers. After comparing with the cassava reference genome sequence, a total of 1,737,846 InDels were detected in the whole genomes of 22 cassava germplasm resources, including 15,499 InDels located in the coding region.

3InDel primer design and marker validity identification

Screen the InDel sites identified by the sequencing results, and the screening conditions are: Insertion/deletion base number 13bp≥5bp, sequencing depth>10. Based on the position of the InDel site on the reference genome (Mesculenta_v7.0), 250 bp upstream and downstream of the InDel site were taken, and primers were designed using Primer5.0. The design ranges of the upstream and downstream primers are 1-225bp and 270-501bp respectively, the primer length is between 20-25bp, and the annealing temperature is between 47-65°C. Random selection was performed according to the InDel markers detected in the whole genome, and a total of 64 InDel sites were finally selected (Table 2).

Sixty-four pairs of InDel primers were used for the identification of marker validity, and PCR amplification was carried out by randomly selecting the DNA of 5 cassava germplasm materials as templates. PCR reaction system (20ul): DNA template (50～100ng/ml) 3ul, 2×Rapid Tap MasterMix (Vazyme) 10ul, upstream primer (20ng/ul) 0.3ul, downstream primer (20ng/ul) 0.3ul, ddH2O 6.4 ul. Amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 15 s, annealing at 52-60°C for 15 s, extension at 72°C for 15 s, a total of 32 cycles; extension at 72°C for 5 min, hold at 4°C. The amplified products were detected by 3% agarose gel electrophoresis, and then the gel results were taken and recorded by the gel performance system, and the band types were counted. In the test results, 62 pairs of primers amplified the target bands, and 2 pairs of primers did not amplify the bands, with an effective rate of 96.88%. According to the results of agarose gel electrophoresis, 20 pairs of primers among 64 pairs of primers were polymorphic in 5 cassava samples, and the polymorphic rate was 31.25%.

Table 2 64 InDel tag information

4 Application of InDel primers to identify specific germplasm resources

Using 20 pairs of primers with polymorphisms to carry out population gene analysis on 72 germplasm resource materials, it was found that among the amplified bands of IDC46, most of the materials only amplified polymorphic bands at 184bp and 198bp. In the cassava materials, polymorphic bands were amplified at 184bp and 170bp. According to the analysis of band amplification results, it was found that IDC46 could directly identify specific cassava materials Brazil No. 14 (C38) and COL1395 (C70). After the two specific germplasms are identified, they can be combined with other polymorphic Indel primer electrophoresis bands for germplasm differentiation. As shown in the amplification results of IDC06 in Brazil No. 14, the InDel primers only amplify the band at 152bp band, while in COL1395, bands were amplified at 152bp and 173bp. In addition, IDC03, IDC06, IDC07, IDC17, IDC21, IDC28, IDC43, IDC50, IDC51, IDC53, IDC56, IDC49 can be used for subsequent identification of two cassava materials (Table 3). Therefore, the use of IDC46 for germplasm identification, combined with the statistics of the band amplification results of the above primers, can directly identify and distinguish cassava materials Brazil 14 and COL1395.

If multiple specific InDel primers that can identify specific cassava materials are selected and combined, it can provide an effective tool for the identification of cassava germplasm resources. The above findings also fully demonstrate that InDel marking is expected to be used in the construction of cassava fingerprints and molecular ID cards in the later stage.

Table 3 Other polymorphic Indel primers for the amplification results in Brazil 14 and COL1395

5 Genetic diversity analysis of cassava germplasm resources

Select InDel markers with clear bands and polymorphisms, and use 72 cassava germplasm resources as templates for PCR amplification and agarose gel electrophoresis detection, the same as the above method. Finally, the amplification results were photographed and counted. Finally, 18 pairs of primers with the best amplification effects (Table 2) were selected for PCR amplification of 72 cassava varieties. After PCR amplification detection, the 18 pairs of primers screened out can amplify the target bands, and the types of bands amplified by the 18 pairs of primers are counted. The band statistics method: the band at the allele is marked as "1", Mark "0" if there is no strip, and "9" if there is missing. After the strip matrix was generated, Popgene32 was used to calculate the equivalent value of primers Nei and Shannon, and the UPGMA algorithm in NTSYS-pc was used to perform cluster analysis on 72 cassava germplasm resources. The results are shown in Table 4-5 and Figure 4.

It can be seen from Table 4 that among the 72 cassava germplasms, the effective allele number ranged from 1.2712 to 1.9862, the alleles amplified at the IDC53 locus were the least, and the alleles amplified at the IDC15 locus were the most. The variation range of the index is 0.2134-0.4965, and the variation range of the Shannon diversity index is 0.3698-0.6897. The number of effective alleles, the gene diversity index, and the Shannon diversity index show similar trends, and the maximum values all appear at the IDC15 locus , the minimum value is at the site IDC53.

It can be seen from Table 5 and Figure 4 that the cluster analysis of 72 cassava germplasm resources using 18 InDel markers with good polymorphism found that the 72 cassava germplasm resources were divided into 2 groups at the genetic similarity coefficient of 0.62. Among them, the largest group is the 1# group, and its germplasm sources and types are relatively extensive. Most of the local varieties and selected varieties from my country are divided into the 1# subgroup, and the remaining 1# subgroup materials have no geographical distribution rules, and the 2# subgroup Group materials mainly come from Colombia and China. According to the results of cluster analysis, the minimum genetic similarity coefficient of 72 cassava germplasm resource populations is 0.61, and the genetic similarity coefficient of 7 cassava materials reaches 100%, indicating that the genetic basis of cassava germplasm is relatively narrow, which is similar to previous research results . According to the research results of scholars at home and abroad, the level of genetic variation among existing cassava germplasm is low, and the genetic basis is very narrow, which is difficult to meet the needs of cassava breeding.

Table 4 Genetic diversity of 18 pairs of Indel primers in 72 cassava germplasms

Table 5 Basic information of 72 cassava germplasm resources

serial number material name Germplasm type source Subgroup serial number material name Germplasm type source Subgroup C1 petioleless wild species 1# C37 GR024-8 Breeding varieties China 1# C2 KM94 Introduced strains Vietnam 1# C38 Brazil 14 Introduced strains Brazil 1# C3 SC14 Breeding varieties China 1# C39 CM965-3 Introduced strains Colombia 1# C4 SC201 Breeding varieties China 1# C40 CMR37-14-9 Introduced strains Thailand 1# C5 Yunnan 8 local breeds China 1# C41 Switzerland T7 Introduced strains Switzerland 1# C6 Yunnan 7 local breeds China 1# C42 ZM9242 strain China 1# C7 SC7 Breeding varieties China 1# C43 ZM99250 strain China 1# C8 Yunnan 2 local breeds China 1# C44 COL629-4 Introduced strains Colombia 1# C9 Switzerland W4 Introduced strains Switzerland 1# C45 CM4031-2 Introduced strains Colombia 1# C10 ZM93236 strain China 1# C46 COL739 Introduced strains Colombia 1# C11 SC205 Breeding varieties China 1# C47 Switzerland P11 Introduced strains Switzerland 1# C12 ZM8625 strain China 1# C48 Brazil 15 Introduced strains Brazil 1# C13 Swiss R9 Introduced strains Switzerland 1# C49 Indonesian species Introduced strains Indonesia 1# C14 ZM99247 strain China 1# C50 Swiss S8 Introduced strains Switzerland 1# C15 CM4040-1 Introduced strains Colombia 1# C51 COL244 Introduced strains Colombia 1# C16 SC8002 Breeding varieties China 1# C52 R90 Introduced strains Thailand 1# C17 R5 Introduced strains Thailand 1# C53 Indonesian Fine Leaf Introduced strains Indonesia 1# C18 R72 Introduced strains Thailand 1# C54 CM7595 Introduced strains Colombia 1# C19 COL198 Introduced strains Colombia 1# C55 R7 Introduced strains Thailand 1# C20 sticky wood species Introduced strains Indonesia 1# C56 CM1568-2 Introduced strains Colombia 1# C21 R60 Introduced strains Thailand 1# C57 Swiss Q10 Introduced strains Switzerland 1# C22 KM98-6 Introduced strains Vietnam 1# C58 COL2626 Introduced strains Colombia 1# C23 CM3993-9 Introduced strains Colombia 1# C59 KM98-7 Introduced strains Vietnam 1# C24 Switzerland N13 Introduced strains Switzerland 1# C60 R80 Introduced strains Thailand 1# C25 GR911 Breeding varieties China 1# C61 GR024-3 Breeding varieties China 1# C26 Brazil 14 Introduced strains Brazil 1# C62 COL1061 Introduced strains Colombia 1# C27 Swiss H19 Introduced strains Switzerland 1# C63 Switzerland U6 Introduced strains Switzerland 1# C28 Brazil No. 9 Introduced strains Brazil 1# C64 CM483-2 Introduced strains Colombia 1# C29 Switzerland B25 Introduced strains Switzerland 1# C65 COL777 Introduced strains Colombia 1# C30 CMR36-40-9 Introduced strains Thailand 1# C66 SC11 Breeding varieties China 2# C31 Brazil 7 Introduced strains Brazil 1# C67 SC8 Breeding varieties China 2# C32 COL1049-1 Introduced strains Colombia 1# C68 CM901 Introduced strains Colombia 2# C33 ZM7901 strain China 1# C69 Guire No. 5 Breeding varieties China 2# C34 ZM8229 strain China 1# C70 COL1395 Introduced strains Colombia 2# C35 SC6 Breeding varieties China 1# C71 SC12 Breeding varieties China 2# C36 SC13 Breeding varieties China 1# C72 CM3327-4 Introduced strains Colombia 2#

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this practice are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims (8)

1. A primer set based on the InDel marker development of cassava whole genome, characterized in that, said primer set includes 18 pairs of InDel primers with nucleotide sequences as shown in SEQ ID NO.1-36. 2 . The primer set according to claim 1 , further comprising 2 pairs of InDel primers whose nucleotide sequences are shown in SEQ ID NO.37-40. 3. A kit comprising the primer set of claim 1 or 2. 4. The kit according to claim 3, characterized in that it further comprises reagents required for PCR reaction and/or electrophoresis. 5. application of the primer set as claimed in claim 1 or 2 or the test kit as claimed in claim 3 or 4 in cassava genetic diversity analysis. 6. An analysis method of cassava genetic diversity, characterized in that, the primer set according to claim 1 or 2 or the kit according to claim 3 or 4 are used. 7. analysis method according to claim 6, is characterized in that, comprises the steps: (1) extract the genomic DNA of cassava to be analyzed; (2) using step (1) sample DNA as a template, applying the primer set described in claim 1 or 2 or the kit described in claim 3 or 4 to carry out PCR amplification; (3) Analyze the genetic diversity of cassava to be analyzed according to the amplified genomic DNA polymorphic bands. 8. Use of the primer set as claimed in claim 1 or 2 or the test kit as claimed in claim 3 or 4 in distinguishing cassava germplasm.

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