CN112626256B - Sesame seedling stage drought tolerance molecular marker and application thereof - Google Patents
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Abstract
The invention relates to the technical field of sesame molecular breeding, in particular to a sesame seedling stage drought tolerance molecular marker and application thereof. The invention provides a primer pair A and/or a primer pair B; the primer pair A consists of single-stranded DNA molecules shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer pair B consists of single-stranded DNA molecules shown as SEQ ID NO.3 and SEQ ID NO. 4. A PCR reagent comprising primer pair a and/or primer pair b. Drought tolerance is a very complex comprehensive property, is greatly influenced by environmental factors, and has low selection efficiency in the traditional breeding method. The primer pair A and/or the primer pair B can improve the selection efficiency of the drought tolerance of the sesame in the seedling stage and accelerate the breeding of the sesame drought tolerance variety.
Description
Technical Field
The invention relates to the technical field of sesame molecular breeding, in particular to a sesame seedling stage drought tolerance molecular marker and application thereof.
Background
Sesame is one of the most important oil crops in the world, and sesame seeds are rich in proteins, vitamins, sesamin, sesamolin and other special antioxidants, so that the sesame is a health food which is deeply favored by consumers. While sesame is one of the more drought-resistant than other oil crops, excessive drought severely affects sesame growth and yield formation. In recent years, seasonal or regional drought damage frequently occurs in northern arid and semiarid regions and southern hilly red soil regions of China, and the method has become one of main factors limiting sesame production. The root system of the sesame is not very developed, the seedling stage and the flowering stage are very sensitive to drought stress, the sesame occurs in summer and drought in the sowing stage, the emergence of the sesame and the growth vigor of the plant after the emergence of the sesame are affected, and the improvement of the drought tolerance in the seedling stage has important significance for stable production of the sesame. Cultivation of drought-tolerant sesame varieties by genetic improvement is the most effective measure for alleviating drought damage.
Existing researches show that the characters related to the enhancement of plant drought tolerance are mostly complex quantitative characters controlled by multiple genes, such as root length, root biomass, coleoptile length and stem length in seedling stage, plant height, root length and yield related characters in adult stage and the like. Plant roots play a vital role in the process of absorbing and transporting moisture and nutrient elements, developed root systems can remarkably improve drought tolerance of plants, and relative root length traits (root length under stress treatment/root length under control treatment) are often used for measuring drought tolerance of plants. At present, although researchers have identified a plurality of sites related to drought tolerance in the seedling stage or the flowering stage of sesame by using a whole genome association analysis method, genetic research on the characteristics of sesame roots is not reported, and related molecular markers are not reported. Therefore, the development of the molecular marker closely linked with the major gene locus has great potential application value for sesame drought-tolerant breeding by digging the related major QTL of the root trait of sesame under drought stress, and improves the selection efficiency of drought tolerance.
Disclosure of Invention
Therefore, the invention aims to overcome the defect of low drought-tolerant sesame selection efficiency of the traditional breeding method in the prior art, thereby providing a primer pair A and/or a primer pair B, a PCR reagent and a kit, improving the drought-tolerant sesame selection efficiency in the seedling stage and accelerating the breeding of sesame drought-tolerant varieties.
Primer pair a and/or primer pair b; the primer pair A consists of single-stranded DNA molecules shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer pair B consists of single-stranded DNA molecules shown as SEQ ID NO.3 and SEQ ID NO. 4.
A PCR reagent comprising the primer pair A and/or the primer pair B.
A kit comprising the primer pair a and/or the primer pair b described above, or the PCR reagent described above; the single-stranded DNA molecules shown in SEQ ID No.1-SEQ ID No.4 are packaged independently.
The invention also provides a method for predicting drought tolerance of the sesame to be detected in the seedling stage and/or the relative root length of the sesame, which comprises the following steps: taking genome DNA of sesame varieties A and B to be detected as templates, and respectively carrying out PCR amplification by using a primer pair A and a primer pair B; if the amplified product of the sesame variety A to be detected subjected to PCR amplification by the primer pair A has a DNA fragment of 205bp (optionally 205 bp), and the amplified product of the sesame variety A to be detected subjected to PCR amplification by the primer pair B has a DNA fragment of 171 bp; the amplified product of the sesame variety B to be detected, which is amplified by the primer pair A through PCR, has a DNA fragment of 184bp (optionally, 184 bp), and the amplified product of the sesame variety B to be detected, which is amplified by the primer pair B through PCR, has a DNA fragment of 164 bp; the drought tolerance of the sesame variety A to be detected is higher than that of the sesame variety B to be detected, and/or the relative root length of the sesame variety A to be detected is longer than that of the sesame variety B to be detected; relative root length = root length under stress treatment/root length of control treatment.
The invention also provides a sesame seedling stage drought tolerance related molecular marker, which is any one of the following:
(I) Consists of a DNA molecule 1 and a DNA molecule 2;
(II) DNA molecule 1 or DNA molecule 2;
the DNA molecule 1 is obtained by using the genomic DNA of sesame as a template and amplifying the primer pair A; the DNA molecule 2 is obtained by using the genome DNA of sesame as a template and amplifying the primer pair B;
the length of the DNA molecule 1 is 205bp or 184bp;
the length of the DNA molecule 2 is 164bp or 171bp.
The application of the sesame seedling stage drought-resistant molecular marker in any one of the following 1) to 10):
1) Identifying or assisting in identifying drought tolerance of sesame;
2) Preparing a product for identifying or assisting in identifying the drought tolerance of sesame;
3) Detecting or assisting in detecting the relative root length of sesame;
4) Preparing a product for detecting or assisting in detecting the relative root length of sesame;
5) Detecting or assisting in detecting whether the sesame material to be detected contains drought-enduring major gene locus qRRL12;
6) Preparing a product for detecting or assisting in detecting whether the sesame material to be detected contains drought-enduring major gene locus qRRL12;
7) Screening or auxiliary screening of drought-enduring sesame seeds in a seedling stage;
8) Preparing a product for screening or assisting in screening drought-resistant sesame at a seedling stage;
9) Predicting the relative root length phenotype of sesame;
10 Preparing a product for predicting the relative root length phenotype of sesame or sesame plants;
relative root length = root length under stress treatment/root length of control treatment.
Use of primer pair a and/or primer pair b, PCR reagents or kits in any of the following 1) -7):
1) Identifying or assisting in identifying drought tolerance of sesame;
2) Detecting or assisting in detecting the relative root length of sesame;
3) Detecting or assisting in detecting whether the sesame contains the drought tolerance molecular marker;
4) Preparing a product for detecting or assisting in detecting whether sesame contains the drought tolerance molecular marker;
5) Detecting or assisting in detecting whether the sesame material to be detected contains drought-enduring major gene locus qRRL12;
6) Screening or auxiliary screening of drought-enduring sesame seeds in a seedling stage;
7) Predicting a relative root growth phenotype;
relative root length = root length under stress treatment/root length of control treatment.
Optionally, the primer pair A and the primer pair B are used for carrying out PCR amplification on the genome DNA of the sesame material to be detected, if the primer pair A is used for amplifying to obtain a 205bp fragment and the primer pair B is used for amplifying to obtain a 171bp fragment, the sesame material to be detected contains a drought-enduring major gene locus qRRL12.
Optionally, the stress is drought stress; the drought stress is PEG stress.
Optionally, the sesame is golden jute, hubei bamboo white or existing unnamed sesame materials.
Optionally, the genome template is a sesame genome template in a seedling stage.
The technical scheme of the invention has the following advantages:
1. the primer pair A and/or the primer pair B provided by the invention; the primer pair A consists of single-stranded DNA molecules shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer pair B consists of single-stranded DNA molecules shown as SEQ ID NO.3 and SEQ ID NO. 4. Drought tolerance is a very complex comprehensive property, is greatly influenced by environmental factors, and has low selection efficiency in the traditional breeding method. The primer pair A and/or the primer pair B can improve the selection efficiency of the drought tolerance of the sesame in the seedling stage and accelerate the breeding of the sesame drought tolerance variety.
2. The invention provides a method for predicting drought tolerance of sesame to be detected in a seedling stage and/or relative root length of sesame, which comprises the following steps: taking genome DNA of sesame varieties A and B to be detected as templates, and respectively carrying out PCR amplification by using a primer pair A and a primer pair B; if the amplified product of the sesame variety A to be detected subjected to PCR amplification by using the primer pair A has a DNA fragment of 205bp, and the amplified product of the sesame variety A to be detected subjected to PCR amplification by using the primer pair B has a DNA fragment of 171 bp; the amplified product of the sesame variety B to be detected, which is amplified by the primer pair A through PCR, has a 184bp DNA fragment, and the amplified product of the sesame variety B to be detected, which is amplified by the primer pair B through PCR, has a 164bp DNA fragment; the drought tolerance of the sesame variety A to be detected is higher than that of the sesame variety B to be detected, and/or the relative root length of the sesame variety A to be detected is longer than that of the sesame variety B to be detected; relative root length = root length under stress treatment/root length of control treatment. The method can rapidly and simply predict drought tolerance of sesame in seedling stage and/or relative root length of sesame.
3. The invention provides a sesame seedling stage drought tolerance molecular marker, which is any one of the following: (I) consisting of DNA molecule 1 and DNA molecule 2; (II) DNA molecule 1 or DNA molecule 2; the DNA molecule 1 is obtained by using the genomic DNA of sesame as a template and amplifying the primer pair A; the DNA molecule 2 is obtained by using the genome DNA of sesame as a template and amplifying the primer pair B; the length of the DNA molecule 1 is 205bp or 184bp; the length of the DNA molecule 2 is 164bp or 171bp. The invention provides a codominant molecular marker linked with drought tolerance major gene loci related to relative root length of sesame for the first time, the marker amplification is simple and stable, materials with qRRL12 loci can be rapidly detected through the amplification of the linked molecular marker DNA molecules 1 and 2, the relative root length phenotype is predicted, and materials with relatively drought tolerance in the emergence period are screened for variety improvement.
4. The invention provides application of a primer pair A and/or a primer pair B, a PCR reagent or a kit in detecting or assisting in detecting whether sesame materials to be detected contain drought-enduring major gene loci qRRL12; the molecular marker linked with qRRL12 obtained by the invention can rapidly determine whether qRRL12 sites exist in materials through the primer pair A and/or the primer pair B, the PCR reagent or the kit, and can be used for molecular marker assisted selection of the sites.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is an electrophoretogram of the amplification products of ZMG009 in the parent and sesame drought tolerance identification material; m is a molecular weight marker, 1-16 are sesame materials to be detected, 17 and 18 are parent golden jute and Hubei bamboo mountain white respectively, and the arrow indicates a polymorphic band;
FIG. 2 is an electrophoretogram of the amplification products of ZMG010 in parent and sesame drought tolerance identification materials; m is a molecular weight marker, 1-16 are sesame materials to be detected, 17 and 18 are parent golden jute and Hubei bamboo mountain white respectively, and the arrow indicates a polymorphic band;
FIG. 3 is a comparison of the relative root lengths of different genetic profiles at qRRL12 locus; z and J respectively represent materials with qRRL12 locus of Hubei Zhushan white and golden jute genotypes; * Represent significant differences at the level of 0.001; the ordinate is the relative root length.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Jin Huangma in Sun Jian, lemeiwang, rao Yueliang, yan Tingxian, yan Xiaowen, zhou Gongying. Effects of bud period Al (3+) stress on sesame seedling growth, evaluation of aluminium toxicity resistance of germplasm resources [ J ]. Chinese oil crop theory, 2014,36 (05): 602-609;
hubei Zhushanbai is disclosed in Liu Aili, wang Linhai, li Donghua, zhou, jiang Shiyang, zhang Xiurong. Studies of the change of physiological indexes of sesame of different moisture resistance genotypes in response to moisture damage stress [ J ]. Nuclear agronomy, 2019,33 (02): 372-378.
15% PEG:75g PEG-6000+100mL distilled water was dissolved and the volume was set to 500mL.
Example 1, obtaining of drought-enduring major Gene locus qRRL 12-linked molecular markers in sesame seedling stage
1. Positioning of sesame drought-enduring major gene locus qRRL12
1. Construction of high-density genetic map
F constructed by taking golden jute and Hubei bamboo mountain white as parents 9 Recombinant inbred line population, combined with genome resequencing technology, is subjected to high-density genetic map construction. The kit for extracting plant genome DNA of Shanghai chemical company ((B518261, sang) was usedon Biotech co., ltd., shanghai) extracts 180F 9 Family and parent DNA were subjected to library construction and high throughput sequencing according to standard methods provided by Illumina corporation, the sequencing platform was Illumina Hiseq 2000, the parent sequencing depth was 10×, and the average sequencing depth of the population family was 4×. After data filtration, reference genome comparison, mutation analysis and colony genotyping, the minimum allele frequency (minor allele frequency, MAF) is less than or equal to 0.2, the relative heterozygosity is less than or equal to 0.2, and the proportion of the deletion genotypes is less than or equal to 0.2<And 0.5 is used for screening polymorphic markers for genetic map construction. A total of 466,911 SNPs and 72,981 InDels were obtained and distributed on 13 chromosomes. Sesame reference genome was downloaded from sesame genome database (http:// www.sesame-bioinfo/Sinbase2.0 /).
The 539,802 (466,911 SNPs+72,981 InDels) polymorphic markers obtained by screening are distinguished into linkage groups according to the chromosome positions of the polymorphic markers, the genotype of a parent is deduced according to the allele linkage relation between adjacent markers, and the genotype is compared with the genotype of the parent obtained by actual sequencing to find that the consistency accords with expectations. Based on the inferred parent genotypes, the source of each allele is determined, then the missing genotypes are filled in using hidden markov model (Hidden Markov Model, HMM) based algorithms (Xie et al 2010), genetic distances are calculated, and then genetic map construction is performed on linkage groups. Finally 1354 marker loci were successfully mapped to 13 chromosomes covering 538,090 polymorphic markers.
2. Investigation of relative root length characters in seedling stage
Drought tolerance stress, using 15% PEG6000 solution (configuration method see above) for parent and Hubei Zhushan Baijinyuang F containing 180 families 9 The recombinant inbred line population is subjected to germination stress treatment, water is used as a control, root length phenotype data are measured after five days of germination, 20 plants are measured for each family, the stress treatment test is repeated twice, and three times of treatments are arranged for each family. The relative root length of each material was calculated as follows = root length under stress treatment/root length under control treatment. Stress treatment experiments were performed in plant incubators. The data comparison analysis shows that the parent Hubei bamboo mountain white is PEGThe relative root length under stress treatment is significantly higher than that of golden jute. The average variation distribution of the population family relative to root length shows super-parent separation phenomenon, which indicates that the character is a compound polygene genetic mode, and the relative root length shows higher genetic transmission which reaches 75 percent.
3. Positioning of drought tolerance QTL
QTL localization was performed on the relative root length trait using the complex interval mapping (composite interval mapping, CIM) function of Window QTL Cartographer Version 2.5 software. LOD value greater than or equal to 2.5 is used as a standard for QTL detection. As a result, a major gene locus qRRL12 was detected on chromosome 12 of sesame, which was detected in two independent replicates, and which was located between InDel markers c12b031-8 and c12b036-9, and the average LOD value reached 7.81, explaining 13.37% of phenotypic variation. qRRL12 alleles from Hubei Zhushan white have the effect of increasing relative root length.
2. Development of drought-enduring major gene locus qRRL12 linked molecular marker
A pair of specific primers are respectively designed by utilizing 500bp sequences beside a genome where InDel markers c12b031-8 and c12b036-9 are positioned, wherein the specific primers are ZMG009F (5'-caaaccctcgttggaagtgg-3', SEQ ID NO. 1) and ZMG009R (5'-gcttgaacctagatatgatgtctg-3', SEQ ID NO. 2) and ZMG010F (5'-gcatctcttcattcaatatagatg-3', SEQ ID NO. 3) and ZMG010R (5'-cacgaccaagaccttgattc-3', SEQ ID NO. 4), and corresponding InDel marker fragments can be amplified by utilizing the two pairs of primers, wherein the amplified fragment length of ZMG009 in Hubei Zhushan white is 205bp, and the amplified fragment length in golden jute is 184bp; the length of the amplified fragment of ZMG010 in Hubei Zhushan Bai is 171bp, and the length of the amplified fragment in Jinhuang is 164bp.
The reaction system of the labeling PCR was 10. Mu.L: 10 Xbuffer (containing Mg 2+ ) 1. Mu.L, 2.5mM dNTP1. Mu.L, 0.2. Mu.M each of the forward and reverse primers, 0.1. Mu.L of 1U/. Mu.L Taq polymerase (R001B, takara Biomedical Technology), 1. Mu.L of 10 ng/. Mu.L of DNA template, and water to 10. Mu.L.
PCR amplification was performed on a Bio-Rad thermal cycler T100 amplification apparatus, with the amplification procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 40s, extension at 72℃for 30s, and cycling for 36 times; finally, the extension is carried out for 5min at 72 ℃. The amplified products were separated by electrophoresis on an 8% denaturing polyacrylamide gel, and after electrophoresis, the gel was silver stained and recorded by photographing.
Example 2qRRL12 use of molecular markers
Taking genome DNA of 16 parts of sesame materials (numbered 1-16) leaves collected from Jiangxi provinces of golden jute and Hubei Zhushan white and the subject group as templates, and carrying out PCR amplification and electrophoresis of amplification products by using the developed marking primers ZMG009F and ZMG009R and ZMG010F and ZMG010R, wherein in golden jute and 9 parts of sesame materials (numbered 4,6,7,8,10,13,14,15,16), the ZMG009F and the ZMG009R can amplify a band with the fragment size of 184bp, and the ZMG010F and the ZMG010R can amplify a band with the fragment size of 164 bp; in Hubei Zhushanbai and another 7 parts of sesame material ( accession number 1,2,3,5,9,11,12), ZMG009F and ZMG009R amplified a 205bp band, and ZMG010F and ZMG010R amplified a 171bp band (FIG. 1; FIG. 2). The relative root length of the materials was investigated using the method described above and found that the relative root length of 7 parts of the material identical to the Hubei bamboo mountain Bai Kuozeng strip was significantly greater than the 9 parts of the material identical to the golden jute amplification strip (FIG. 3).
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Sequence listing
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Claims (6)
1. A primer pair, which is characterized by comprising a primer pair A and a primer pair B; the primer pair A consists of single-stranded DNA molecules shown as SEQ ID NO.1 and SEQ ID NO.2, and the primer pair B consists of single-stranded DNA molecules shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. A PCR reagent comprising the primer set a and the primer set b of claim 1.
3. A kit comprising the primer set a and the primer set b of claim 1, or the PCR reagent of claim 2; wherein, the single-stranded DNA molecules shown in SEQ ID NO.1-SEQ ID NO.4 are packaged independently.
4. A method for predicting drought tolerance of sesame to be detected in seedling stage and/or relative root length of sesame is characterized by comprising the following steps: PCR amplification is carried out by using genome DNA of sesame varieties A and B to be detected as templates and respectively using the primer pair A and the primer pair B in claim 1; if the amplified product of the sesame variety A to be detected subjected to PCR amplification by using the primer pair A has a DNA fragment of 205bp, and the amplified product of the sesame variety A to be detected subjected to PCR amplification by using the primer pair B has a DNA fragment of 171 bp; the amplified product of the sesame variety B to be detected, which is amplified by the primer pair A through PCR, has a 184bp DNA fragment, and the amplified product of the sesame variety B to be detected, which is amplified by the primer pair B through PCR, has a 164bp DNA fragment; the drought tolerance of the sesame variety A to be detected is higher than that of the sesame variety B to be detected, and/or the relative root length of the sesame variety A to be detected is longer than that of the sesame variety B to be detected; relative root length = root length under drought stress treatment/root length of control treatment.
5. Use of the primer pair of claim 1, the PCR reagent of claim 2 or the kit of claim 3 in any one of the following 1) -4):
1) Identifying or assisting in identifying drought tolerance of sesame in seedling stage;
2) Detecting or assisting in detecting the relative root length of sesame in the seedling stage;
3) Screening or auxiliary screening of drought-enduring sesame seeds in a seedling stage;
4) Predicting the relative root length phenotype of the sesame seedling stage;
relative root length = root length under drought stress treatment/root length of control treatment.
6. The method of claim 4, or the use of claim 5, wherein the drought stress is PEG stress.
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| 芝麻耐旱性的鉴定方法及关联分析;黎冬华 等;《作物学报》;1425-1433 * |
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