CN112941219B

CN112941219B - Development and application of NBS-SSR specific markers of sweet potatoes and kindred wild species thereof

Info

Publication number: CN112941219B
Application number: CN202110154149.5A
Authority: CN
Inventors: 王连军; 杨新笋; 王崇; 柴沙沙; 雷剑; 宋峥; 刘意; 焦春海
Original assignee: Hubei Academy Of Agricultural Sciences Institute Of Food Crops
Current assignee: Hubei Academy Of Agricultural Sciences Institute Of Food Crops
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2022-05-17
Anticipated expiration: 2041-02-04
Also published as: CN112941219A

Abstract

The invention relates to development and application of NBS-SSR specific markers of sweet potatoes and kindred wild species thereof, and discloses a primer composition for identifying resistance to sweet potato vine cutting diseases. The SSR marker has the advantages of high polymorphism, good repeatability, stable heredity, codominance and the like; closely linked with the candidate gene for resisting the vine cutting disease, greatly improves the efficiency of molecular marker-assisted selection in the breeding process, and has important significance in breeding of the sweet potato for resisting the vine cutting disease.

Description

Development and application of NBS-SSR specific markers of sweet potatoes and kindred wild species thereof

Technical Field

The invention belongs to the technical field of agriculture, and particularly relates to NBS-SSR specific marker development and application of sweet potato and its kindred wild species Ipomoea trifida whole genome.

Background

Sweet potatoes (Ipomoea batatas (L.) Lam.) are important grain, feed, industrial raw materials and novel root crops for energy, China is the biggest sweet potato producing country in the world, and the perennial planting area is 550 ten thousand hm²The yield of fresh potatoes is about 1.2 multiplied by 10⁸t, respectively accounting for 60% and 85% of the total area and the total yield of the sweet potato planting in the world. The disease in south and north is mixed due to the non-standard introduction, and the disease tends to spread and gradually aggravate. The breeding of disease-resistant varieties is the most economic and effective prevention and treatment means, the traditional breeding mainly depends on the phenotypic selection of plants, and various factors such as environmental conditions, gene interaction, genotype and environmental interaction influence the phenotypic selection efficiency, for example, the identification of disease resistance is influenced by disease conditions, plant physiological conditions, evaluation standards and the like.

The molecular marker-assisted selection can be utilized to remarkably improve the selection efficiency and accuracy and accelerate the breeding process. The SSR loci of NBS gene sequences of 353 sweet potato cultivars and 280 sweet potato wild species are diagnosed by utilizing a sweet potato genome and transcriptome database, SSR molecular markers are developed, specific markers are screened out, and the developed markers are verified by utilizing sweet potato anti-vine-cutting-disease and vine-cutting-disease-susceptible materials. The molecular marker, especially the marker developed by functional gene, can obviously improve the accuracy of target character, greatly shorten the breeding period and improve the breeding efficiency.

Disclosure of Invention

In order to solve the problems in the prior art, the research provides a method for developing and applying NBS-SSR specific markers of the whole genome of Ipomoea trifida and a related wild species thereof.

The invention provides a primer composition for identifying sweet potato vine cutting disease resistance, which consists of at least one of the following 6 pairs of specific primer pairs:

A1) the primer pair NBS2 consists of a primer NBS2-F and a primer NBS 2-R;

the primer NBS2-F is (a1) or (a 2);

(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;

the primer NBS2-R is (a3) or (a 4);

(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;

A2) the primer pair NBS4 consists of a primer NBS4-F and a primer NBS 4-R;

the primer NBS4-F is (b1) or (b 2);

(b1) a single-stranded DNA molecule shown in sequence 3 of the sequence table;

(b2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;

the primer NBS4-R is (b3) or (b 4);

(b3) a single-stranded DNA molecule shown in a sequence 4 of a sequence table;

(b4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;

A3) the primer pair NBS8 consists of a primer NBS8-F and a primer NBS 8-R;

the primer NBS8-F is (c1) or (c 2);

(c1) a single-stranded DNA molecule shown in sequence 5 of the sequence table;

(c2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;

the primer NBS8-R is (c3) or (c 4);

(c3) a single-stranded DNA molecule shown in sequence 6 of the sequence table;

(c4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same functions as the sequence 6;

A4) the primer pair NBS11 consists of a primer NBS11-F and a primer NBS 11-R;

the primer NBS11-F is (d1) or (d 2);

(d1) a single-stranded DNA molecule shown in sequence 7 of the sequence table;

(d2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and having the same functions as the sequence 7;

the primer NBS11-R is (d3) or (d 4);

(d3) a single-stranded DNA molecule shown in sequence 8 of the sequence table;

(d4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 8 and have the same functions as the sequence 8;

A5) the primer pair NBS14 consists of a primer NBS14-F and a primer NBS 14-R;

the primer NBS14-F is (e1) or (e 2);

(e1) a single-stranded DNA molecule shown in sequence 9 of the sequence table;

(e2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 9 and has the same function as the sequence 9;

the primer NBS14-R is (e3) or (e 4);

(e3) a single-stranded DNA molecule shown in sequence 10 of the sequence table;

(e4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 10 and has the same function as the sequence 10;

A6) the primer pair NBS17 consists of a primer NBS17-F and a primer NBS 17-R;

the primer NBS17-F is (F1) or (F2);

(f1) a single-stranded DNA molecule shown in sequence 11 of the sequence table;

(f2) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 11 and having the same functions as the sequence 11;

the primer NBS17-R is (f3) or (f 4);

(f3) a single-stranded DNA molecule shown in sequence 12 of the sequence table;

(f4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 12 and has the same function as the sequence 12.

Wherein the primer composition consists of a primer pair NBS4 and a primer pair NBS 14.

The application of the primer composition in identification or auxiliary identification of sweet potato vine cutting disease resistance also belongs to the protection scope of the invention.

The application of the primer composition in the breeding of the sweet potatoes with high resistance to the vine cutting disease also belongs to the protection scope of the invention.

The invention provides a product for identifying or assisting in identifying sweet potato vine cutting disease resistance, which contains the primer composition.

The application of the product in identification or auxiliary identification of sweet potato vine cutting disease resistance also falls into the protection scope of the invention.

The application of the product in the breeding of the sweet potato with high resistance to the vine cutting disease also falls within the protection scope of the invention.

The invention provides a method for identifying or assisting in identifying sweet potato vine cutting disease resistance, which comprises the following steps: 1) carrying out PCR amplification by using the primer composition by using the genomic DNA of the sweet potato as a template to obtain a PCR amplification product;

2) carrying out electrophoresis on the PCR amplification product obtained in the step 1) to obtain a cluster analysis map of the sweet potato, and analyzing the resistance of the sweet potato to the vine cutting disease according to the cluster analysis map.

The application of the method in identification or auxiliary identification of sweet potato vine cutting disease resistance also belongs to the protection scope of the invention.

The application of the method in the breeding of the sweet potatoes with high resistance to the vine cutting disease also belongs to the protection range of the invention.

The invention has the following beneficial effects: 1. SSR markers have the advantages of high polymorphism, good repeatability, stable heredity, codominance and the like; 2. closely linked with the candidate gene for resisting the vine cutting disease, greatly improves the efficiency of molecular marker-assisted selection in the breeding process, and has important significance in breeding of the sweet potato for resisting the vine cutting disease.

Drawings

FIG. 1NBS-SSR site verification (primers: NBS2, NBS4, NBS8, NBS11, NBS14, NBS 17; R: Ipomoea batatas Lam. Var. deltoides 11, S: Ipomoea batatas Lam 13);

FIG. 2 primer NBS4 amplification results;

FIG. 3 shows the result of amplification with primer NBS 14;

FIG. 4 clustering analysis results.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail.

1.1 plant Material

The tested material is a sweet potato variety Hubei potato 11 with high resistance to vine cutting and root rot; sweet potato variety Hubei purple potato 13 with the symptoms of vine cutting and high root rot. Provided by the research institute of food crops of agricultural science institute of Hubei province. Purple sweet potato 13 national identification number: sweet potato 2016020 for national reference; sweet potato 11, purple sweet potato 3, commercial sweet potato 9, sweet potato 8, sweet potato 87, Wansu 178, Szechwan vegetable potato 211, sweet potato green 1, quan potato 830, Fucai potato 18, Fuxu potato 6, sweet osmanthus powder 3 and Ningcai potato 1: reference documents: wanglijun, Leishang, a beautiful gem, etc. the national appraisal situation of the sweet potato variety in 2015.2005-2014 is analyzed, Hubei agricultural science, 54(12), 2844 and 2849; the public is available from the institute of food crops, academy of agricultural sciences, Hubei province. Yushu 27 reference: xixiu plum, old bamboo, plum cloud, etc. 2019, screening and evaluating edible sweet potato fine varieties, cultivating and cultivating 39(2) 48-51; zhenghong 23 reference: yangyfeng, Li Junxia, generation of winter, etc. 2015, breeding and cultivating of Zhenghong 23, a new variety of sweet potato, Henan agricultural science 44(9) 22-24; sweet potato 29 reference: jiazhao Dong, horse earwear, Xiaofeng, etc. 2018, selection of high-starch multi-resistance sweet potato variety, sweet potato 29, southern agriculture newspaper 49(5) 848-; nicotiana 29 reference: the method comprises the following steps of 1, breeding 29 high-quality high-starch sweet potato varieties of Yanshu potato, breeding quality characteristics and cultivation key points, 37(3) of Jiangsu university (Nature science edition), 48-50; sweet potato 24 reference: jiazhao Dong, masage, Bian Xiaofeng, etc. 2016. research on breeding of high-yield and high-quality starch type sweet potato variety, sweet potato No. 24. Chinese agricultural report 32(12) 42-48; ji potato 982, disclosed in https:// baike.baidu.com/% E5% 86% 80% E8% 96% AF982/22690707fr ═ aladdin; sweet potato 16 reference: xiyizhi, Guo Xiao Ding, Jia Zhao Dong, etc. 2012, breeding and cultivating technology of edible new variety of sweet potato, Sushu 16, Jiangsu agricultural horse euyong science 40(7) 104-; guangdong potato No. 2 reference: fuyufan, Yangchunxian, Zhaoyite, etc. 2010. different leaf-vegetable type sweet potato varieties have chlorogenic acid content in stem tip and DPPH removing ability.Chinese agricultural science 43(23) 4814. supplement 4822; reference 1 to alcaligenes odorata: 2010, breeding and cultivation techniques of vegetable type sweet potato Hubei agricultural science 49(8), 1823, 1830; hubei potato No. 2, national identification number: the national reference is made to sweet potato 2015017; reference document No. 1 fun purple sweet potato: zhuyuling, Van ze Min, etc. 2017, Fuyang purple sweet potato No. 1, the No. 1 of the Chinese breed of edible purple sweet potato; reference document No. 1 funai potato: wangxiumei, Van ze Min, et al.2017. Breeding and supporting cultivation technology of leaf vegetable type sweet potato Fucai potato No. 1. China seed industry No. 9; anhui potato 373 reference: exemplary, Wanxiang, Xuxuxu-Xuehao, etc. 2018, variety innovation and sweet potato industry development, Jiangsu agricultural bulletin 34(6), 1401 and 1409; the materials can be obtained by the public from the research institute of grain crops of agricultural academy of sciences of Hubei province.

1.2 isolation of the sequences

Sweet potato and its wild species I.trifida NBS sequences were downloaded from NCBI databases (https:// www.ncbi.nlm.nih.gov/nuccore) and Sweetpatato GARDEN (http:// sweet potatoo-garden.kazusa.or.jp/index. html).

1.3 primer design

SSR sites for NBS sequences were searched using SSRhunter software. The SSR primer design standard is that the number of bases per repeating unit is 3-5, and the repeating times are more than or equal to 5. SSR primers were designed using Primer5 software and synthesized by Tianyihui Biotechnology (Wuhan) Inc. (Table 1).

TABLE 130 pairs NBS-SSR markers

Table1 Thirty-one NBS-SSR markers

1.4 SSR marker detection

Extracting total DNA of genome by using an improved CTAB method. The PCR reaction system is 10 × buffer (Mg)²⁺) 5.0. mu.L, dNTPs (10mmol/L) 4.0. mu.L, forward primer 1.0. mu.L, reverse primer 1.0. mu.L, genomic DNA (50-60 ng/. mu.L) 1.0. mu.L, Easy-Taq DNApolymerase 0.5. mu.L, ddH2O to a total volume of 50. mu.L. The reaction amplification program is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 deg.C for 1min, renaturation at 50 deg.C for 1min, extension at 72 deg.C for 2min, and 36 cycles; extension at 72 ℃ for 5 min. The PCR amplification product was electrophoresed on 0.5% native polyacrylamide gel, and observed after silver staining.

2.2 development and detection of NBS-SSR markers

Finding SSR sites of the sequences of the NBS, developing and designing primers according to the sequences of the SSR sites, and developing NBS-SSR marker 31 pairs (Table 1). 31 pairs of primers are amplified in sweet potato disease-resistant material Eyema 11 and

susceptible material Eyema

13, and 6 pairs of primers have better and stable polymorphism between the two materials (as shown in figure 1). The 6 pairs of polymorphic primers identified and screened are primer pairs NBS2, NBS4, NBS8, NBS11, NBS14 and NBS17 respectively. As can be seen in FIG. 1, the amplification results of the disease-resistant material Ipomoea batatas Hu of sweet potato 11 and the disease-sensitive material Ipomoea batatas Hu 13 of sweet potato by the primer pairs NBS2, NBS4, NBS8, NBS11, NBS14 and NBS17 are obviously different in the bands, which indicates that the disease-resistant material Ipomoea batatas Hu 11 and the disease-sensitive material Ipomoea batatas 13 of sweet potato can be distinguished and identified by the primer pairs NBS2, NBS4, NBS8, NBS11, NBS14 and NBS 17.

Example 2

2.1 plant Material

27 sweet potatoes shown in Table 2 were selected for the test, and the resistance to the vine cutting disease of each sweet potato was shown in Table 2. Provided by the research institute of food crops of agricultural science institute of Hubei province. (whether the standard of the resistance degree of the vine cutting disease is high resistance, namely the disease index is less than or equal to 20; the disease resistance is more than 20 and less than or equal to 40; the disease resistance is more than 40 and less than or equal to 60; the disease index is more than 60 and less than or equal to 80; and the disease resistance is more than 80 and less than or equal to 100)

TABLE 2 materials names corresponding to 27 sweet potatoes and their vine-cutting resistance

2.2 SSR marker detection

The total genomic DNA of the sweet potatoes in the 27 samples was extracted by the modified CTAB method.

The extracted DNAs were used as templates and primer pair NBS4 was used as primers for PCR amplification, and the PCR amplification products were electrophoresed on 0.5% native polyacrylamide gel, and then observed after silver staining, and the results are shown in FIG. 2. As is apparent from FIG. 2, the NBS4 primer was used to amplify the total genomic DNA from the sweet potato of 27 above, and the electrophoretic bands were significantly different at positions between 150 and 300 bp.

The extracted DNAs were used as templates and primer pair NBS14 was used as primers for PCR amplification, and the PCR amplification products were electrophoresed on 0.5% native polyacrylamide gel, followed by silver staining and then observed, with the results shown in FIG. 3. As is apparent from FIG. 3, the NBS14 primer is used to amplify the total genomic DNA of the sweetpotato of 27 above, and the position of the electrophoretic band between 250-400bp is obviously different.

The PCR reaction system is 10 Xbuffer (Mg2+)5.0 μ L, dNTPs (10mmol/L)4.0 μ L, forward primer 1.0 μ L, reverse primer 1.0 μ L (NBS4 and NBS14), genomic DNA (50-60ng/μ L)1.0 μ L, Easy-Taq DNApolymerase 0.5 μ L, ddH2O to make up the total volume of 50 μ L. The reaction amplification program is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 deg.C for 1min, renaturation at 50 deg.C for 1min, extension at 72 deg.C for 2min, and 36 cycles; extension at 72 ℃ for 5 min.

Genetic clustering analysis is carried out on (0, 1) matrix statistical data of 27 sweet potato germplasm resources according to a UPGMA method, and a genetic relationship dendrogram of 27 germplasm materials is constructed (shown in figure 4). The Fukan purple sweet potato No. 1, the Fucai potato No. 1, the cinnamon powder No. 3, the Wan potato 373 and the Ningcai potato No. 1 are totally 5 varieties which are gathered into one category, including 1 high sense, 1 sense, 3 sense and 1 anti-moderate. The total 5 varieties of the commercial potato No. 9, the sweet potato No. 24, the Ji potato No. 982, the sweet potato No. 16 and the Hubei potato No. 11 are gathered into one category, which comprises 4 high resistance and 1 medium resistance. The clustering result is basically consistent with the resistance of 16 sweet potato varieties.

When the vine cutting disease resistance or the genetic relationship of the sweet potato to be detected needs to be identified, the genomic DNA of the sweet potato to be detected can be extracted according to the method, the primer pair NBS4 and the primer pair NBS14 are used as primers for amplification, the amplification result is compared with the genetic relationship dendrogram in the figure 4 to obtain the species which is closest to or consistent with the amplification result, and the vine cutting disease resistance or the genetic relationship of the sweet potato to be detected can be judged.

The primers NBS4 and NBS14 can provide help for breeding of the anti-vine cutting disease, and a foundation is laid for quickly and accurately acquiring a disease-resistant parent to breed a new resistant variety.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

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Claims

1. A primer composition for identifying sweet potato vine cutting disease resistance is composed of at least one of the following 6 pairs of specific primer pairs:

A1) the primer pair NBS2 consists of a primer NBS2-F and a primer NBS 2-R;

the primer NBS2-F is a single-stranded DNA molecule shown in sequence 1 of the sequence table;

the primer NBS2-R is a single-stranded DNA molecule shown in sequence 2 of the sequence table;

A2) the primer pair NBS4 consists of a primer NBS4-F and a primer NBS 4-R;

the primer NBS4-F is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

the primer NBS4-R is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

A3) the primer pair NBS8 consists of a primer NBS8-F and a primer NBS 8-R;

the primer NBS8-F is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;

the primer NBS8-R is a single-stranded DNA molecule shown in sequence 6 of the sequence table;

A4) the primer pair NBS11 consists of a primer NBS11-F and a primer NBS 11-R;

the primer NBS11-F is a single-stranded DNA molecule shown in a sequence 7 in a sequence table;

the primer NBS11-R is a single-stranded DNA molecule shown in a sequence 8 in a sequence table;

A5) the primer pair NBS14 consists of a primer NBS14-F and a primer NBS 14-R;

the primer NBS14-F is a single-stranded DNA molecule shown in a sequence 9 in a sequence table;

the primer NBS14-R is a single-stranded DNA molecule shown in a sequence 10 in a sequence table;

A6) the primer pair NBS17 consists of a primer NBS17-F and a primer NBS 17-R;

the primer NBS17-F is a single-stranded DNA molecule shown in a sequence 11 in a sequence table;

the primer NBS17-R is a single-stranded DNA molecule shown in sequence 12 of the sequence table.

2. The primer composition for identifying sweet potato vine disease resistance of claim 1, which consists of a primer pair NBS4 and a primer pair NBS 14.

3. Use of the primer composition of claim 1 or 2 for identifying or assisting in identifying sweet potato vine cutting disease resistance.

4. The use of the primer composition of claim 1 or 2 in the breeding of high-vine-disease-resistant sweet potatoes.

5. A product for identifying or assisting in identifying sweet potato vine cutting disease resistance, which contains the primer composition of claim 1 or 2.

6. Use of the product of claim 5 for identifying or aiding in identifying sweet potato vine cutting resistance.

7. The use of the product of claim 5 in the breeding of high-vine-disease-resistant sweet potatoes.

8. A method for identifying or assisting in identifying sweet potato vine cutting disease resistance comprises the following steps: 1) carrying out PCR amplification by using the primer composition of claim 1 or 2 by using the genomic DNA of the sweet potato as a template to obtain a PCR amplification product;

9. Use of the method of claim 8 for identifying or aiding in identifying sweet potato vine top disease resistance.

10. The method of claim 8, wherein the method is applied to breeding of the sweet potatoes with high resistance to the vine cutting disease.