CN114540536A - QTL related to low temperature resistance of soybean in bud stage, molecular marker, amplification primer and application - Google Patents
QTL related to low temperature resistance of soybean in bud stage, molecular marker, amplification primer and application Download PDFInfo
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Abstract
The invention relates to the field of soybean molecular breeding, in particular to a QTL (quantitative trait locus) related to low temperature resistance of soybean in a sprouting period, a molecular marker, an amplification primer and application. The QTL is positioned in 38426868bp-38983456bp interval of soybean chromosome 9, the molecular marker is SSR-09-1126, the molecular marker is positioned in 38701572bp-38701609bp interval of soybean chromosome 9, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1. The molecular marker SSR-09-1126 can be used for molecular marker-assisted selective breeding and related functional gene mining, and can utilize wild resources to widen the genetic basis of soybean cultivation.
Description
Technical Field
The invention relates to the field of soybean molecular breeding, in particular to a QTL (quantitative trait locus) related to low temperature resistance of soybean in a sprouting period, a molecular marker, an amplification primer and application.
Background
Seed germination is one of the most important stages in the life cycle of a plant. Typically, the appearance of radicles indicates the completion of seed germination. Low temperature stress is one of the main abiotic stress factors in the growth, development and yield formation process of plants, and the germination of soybean seeds is often stressed at low temperature, so that the emergence rate and emergence uniformity of soybeans are influenced, and further, the yield of the soybeans is influenced. At present, the influence of low temperature on the molecular mechanism of soybean seed germination is largely unknown. The variety improvement is carried out aiming at the low temperature resistance of the bud stage, and the method has important significance for improving the yield and the quality of the soybean. But the low temperature resistance in the bud stage belongs to complex quantitative characters, is controlled by multiple genes, has long traditional breeding period and great difficulty, and needs to consume a large amount of manpower and material resources. With the development of molecular genetics, molecular marker-assisted selective breeding provides a new way for people to accelerate the breeding process. Marker-assisted selective breeding refers to the indirect selection of a target trait by a molecular marker linked to a functional gene during breeding selection. The positioning of Quantitative Trait Loci (QTL) by using molecular markers is an important means for marker-assisted selective breeding. At present, researchers at home and abroad have positioned few low-temperature-resistant QTL at the soybean sprout stage, most of the positioning of the low-temperature-resistant QTL at the soybean sprout stage is primary positioning, and the positioning accuracy is poor due to the fact that the distance between a mark and a target character is too large. Random QTL positioning is a diversified recombination process under different genetic backgrounds of different combinations, so that the positioned QTL has the specificity of hybridization combination, has overlarge marker interval, can be lost along with the change of generations and genetic backgrounds, and is not beneficial to application. It is difficult to perform molecular assisted selection.
Disclosure of Invention
The invention provides a QTL related to soybean sprout stage low temperature resistance, a molecular marker, an amplification primer and application thereof, wherein the QTL locus has a marker interval of 0.56Mb, an LOD value of 4.18 and a contribution rate of 10.59 percent, and the molecular marker SSR-09-1126 related to soybean sprout stage low temperature resistance is identified.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first purpose of the invention is to provide a QTL related to soybean bud stage low temperature resistance, wherein the QTL is located in the 38426868bp-38983456bp interval of soybean chromosome 9.
The invention also provides a SSR molecular marker of the QTL related to soybean bud-stage low temperature resistance, wherein the molecular marker is SSR-09-1126, and the nucleotide sequence of the soybean chromosome 9 is shown in SEQ ID NO.1, and the interval of the molecular marker is 38701572bp-38701609 bp.
The third purpose of the invention is to provide an amplification primer of the SSR molecular marker, wherein the amplification primer is as follows:
SSR-09-1126F：5′-CTCAATCGCGAACCCTAAAC-3′；
SSR-09-1126R：5′-GTGCTCCGAAGGCTGTCTAC-3′。
the fourth purpose of the invention is to provide the application of the amplification primer in the identification of the low-temperature resistance of soybean in the bud stage.
The fifth purpose of the invention is to provide a method for identifying the low temperature resistance of soybean sprouts by using the amplification primer, which comprises the following steps:
s1, extracting the genomic DNA of the soybean material to be identified;
s2, carrying out PCR amplification on the soybean material to be identified by using an SSR-09-1126 amplification primer;
s3, carrying out electrophoretic analysis on the amplification product, identifying as a soybean sprout stage low temperature resistant material if the electrophoretic band type of the soybean material amplification product to be identified belongs to the No.1 band type in the SSR-09-1126, and identifying as a soybean sprout stage low temperature sensitive material if the electrophoretic band type of the soybean material amplification product to be identified belongs to the No. 2 band type in the SSR-09-1126.
The sixth purpose of the invention is to provide the application of the SSR molecular marker in the low-temperature-resistant soybean bud stage breeding.
Compared with the prior art, the invention has the following beneficial effects:
1. the method takes cultivated soybeans and wild beans as parents to construct a genetic population, positions 1 QTL site related to low temperature resistance of soybean sprout, has a marker interval of 0.56Mb, an LOD value of 4.18 and a contribution rate of 10.59 percent, identifies a molecular marker SSR-09-1126 related to low temperature resistance of soybean sprout, and can be used for molecular marker-assisted selective breeding and excavation of related functional genes. Meanwhile, wild resources can be utilized, and the genetic basis of soybean cultivation is widened.
2. The invention utilizes the incubator to carry out low-temperature stress treatment on the soybean in the sprouting period, and takes the germination rate of soybean seeds subjected to low-temperature treatment as an index for evaluating the low-temperature resistance of the soybean sprouts in the sprouting period. And constructing a genome wide introductions line (CSSLs) population by crossing and backcrossing for multiple generations with the Sulnong 14 as a recurrent parent and the wild bean ZYD00006 as a donor parent. Each plant row only contains a few wild bean introduced fragments, so that the QTL interval can be shortened, the positioning precision and accuracy are improved, and the molecular markers related to low temperature resistance of the soybeans are identified in the interval, which is necessary for the molecular marker-assisted selective breeding and functional gene research of the low temperature resistance of the soybeans and has important significance for enriching the genetic diversity of soybean cultivars.
Drawings
FIG. 1 is a drawing of the construction process of mapping population.
Fig. 2 is a flowchart of the population re-sequencing.
Fig. 3 is a maternal chromosome coverage depth profile.
FIG. 4 is a distribution density chart of SNP markers and Bin markers of each chromosome.
FIG. 5 is a polyacrylamide gel electrophoresis image of a material used for developing molecular markers;
wherein, the picture A is an electrophoresis picture of the SSR-09-1126 primer resistance materials, and each lane from left to right respectively shows Gongye 03-7239, Geng' an Xiao jin Huang, Hei nong 24, brown bean, Ji Yun 39, Gongye 03-5570, Chang Ji soya bean, Tie Gao Sihuang, Huang Bao Zhu, Jinmanya soya bean, Ji Yun 93, Gong Ye 04L-141, Jilin 20, Yuanbao jin, Tie Gao black, Dabaimei eyebrow, Tong nong 13, Qing Gong Siding, Ji Yun 71, Gong Ye 04-L15, Huang jin Yuan, Bai Dou, tender Feng 11, Ji 97, Qianjin, Xiao Dou 406, Ji Yun 66, Wu Dou and cat eye bean;
FIG. B is an electrophoretogram of SSR-09-1126 primer-sensitive materials, wherein each lane from left to right shows black river 25, Gongnong P06-12, Tongnong 15, Hengnong 69, J2512, Jiyun 69, Gongnong P06-6, Jiyun 441, Jiyun 303, Mongolian bean 31, Hengnong 31, Henghe 29, Changnong 14, black river 35, Changnong 25, black river 11, black river 31, Jiyun 299, Hefeng 37, black river 14, Jinong 36, black river 13, Changbai bean 30, Suiyong 15, Jinong 32, Anserin egg, Changnong 29, black river 43, Suiyong 28, and Suiyouwu bean No. 2.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Example 1
QTL related to low temperature resistance of soybean in bud stage, molecular marker, amplification primer and application
Acquisition of QTL (quantitative trait locus), molecular marker and amplification primer related to low temperature resistance of soybean in sprouting period
1. Construction of mapping populations
In 2006, a whole genome introgression line population is constructed by taking the seinhong 14(SN14) as a recurrent parent and taking the wild soybean ZYD00006 as a donor parent through continuous backcrossing and selfing. See FIG. 1 for a specific generation.
2. Group genotype detection
Performing re-sequencing on the population to detect the genotype (the flow is shown in figure 2), wherein the steps comprise:
(1) and (3) extracting DNA of parent and progeny strains by using a CTAB method. And after the sample is detected to be qualified, randomly breaking the DNA by using an ultrasonic crushing method, repairing the tail end of a DNA fragment, adding A to the 3' end, adding a sequencing joint, purifying and performing PCR amplification to complete the construction of a sequencing library. The library is qualified by quality inspection and then sequenced by an Illumina HiSeqTM sequencing platform.
(2) And (4) repositioning sequencing reads obtained by re-sequencing on a reference genome for subsequent variation analysis. And (3) comparing the short sequence obtained by the second generation high-throughput sequencing with a reference genome by using BWA software. The position of the Clean Reads on the reference genome was located by alignment, and information such as sequencing depth (fig. 3) and genome coverage of each sample was counted to detect the mutation.
(3) For the results obtained from BWA alignment, Duplicate was removed using Picard's Mark Duplicate tool, masking the effect of PCR-duplication. And (3) performing InDel reaction by using the GATK, namely performing local re-alignment on sites near the alignment result with the insertion deletion, and correcting the error of the alignment result caused by the insertion deletion. Base quality values were recalibrated using GATK (Base Recalibration), and Base quality values were corrected. Mutation detection (variant calling) was performed using GATK, mainly including SNP and InDel. Stringent filtration of SNPs: SNP cluster filtration (filtration if 2 SNPs exist within 5 bp), and SNP filtration near InDel (filtration of SNPs within 5bp near InDel); and adjacent INDEL filtering (two INDEL filters with a distance of less than 10 bp). The number of SNP tags available for the final screening was 580524.
(4) And carrying out sliding scanning on the chromosome by using the obtained 580524 SNPs, wherein 17 SNPs are taken as one window, 1 SNP is taken as a step length, the SNP typing of aa in the sliding window is aa when the number of SNPs is more than 12, the SNP typing of bb in the sliding window is bb when the number of SNPs is more than 14, and the other cases are ab for genotype filling and correction.
(5) And after the mark filling and correction are completed, carrying out Bin division according to the recombination condition of the filial generation. The samples were arranged in order according to the physical location of the chromosome, and when any sample had a typing transition, it was considered that a recombination breakpoint had occurred, then the SNPs between the recombination breakpoints were classified into bins, and were screened for bins, and finally 3196 bins were used as markers for mapping for localization (fig. 4).
3. Phenotypic data acquisition
In this example, the germination percentage of soybeans after low-temperature treatment was used as an evaluation index of low-temperature resistance in the soybean sprout stage.
Germination phenotype acquisition tests are as follows: setting each material for 3 times, selecting round, full and full seeds without diseases and insect pests with consistent sizes, sterilizing with 1% sodium hypochlorite solution, rinsing with distilled water for three times, placing 40 seeds in a sterilized 9cm culture dish, laying a layer of sterilized filter paper on each of the upper and lower layers, placing in a 20 ℃ incubator for imbibition for 12h, taking out, changing water, and transferring to a 6 ℃ incubator. The number of sprouts was investigated on day 6 after the completion of imbibition.
4. Low temperature resistant QTL analysis in soybean sprout period
And (3) carrying out QTL positioning on the low temperature resistance of the population at the bud stage by using an ICIM (complete interval mapping method) method in ICIMapping4.1 software, selecting an ICIM-ADD module for analysis, and adopting a CSL template for the introduction line population. Setting LOD value to be more than or equal to 2.5, and carrying out QTL analysis on the low temperature resistance of the soybean in the bud stage. A low-temperature-resistant QTL interval in the soybean sprout stage is found on chromosome 9, and the physical position is 38426868bp-38983456bp, as shown in Table 1.
TABLE 1 Soybean sprout stage Low temperature resistant QTL interval
5. Molecular marker identification
60 parts of soybean existing varieties are utilized to identify molecular markers related to soybean bud-stage low temperature resistance in an interval, a molecular marker related to the bud-stage low temperature resistance is identified, the molecular marker is located in the 38701572bp-38701609bp interval of soybean chromosome 9 and named as SSR-09-1126, and the nucleotide sequence of the molecular marker is shown as SEQ ID No. 1.
SEQ ID NO.1：
GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA phenotypic data for the above 60 existing varieties of soybean are shown in Table 2;
TABLE 2 materials phenotype for development of molecular markers
The amplification primers of the molecular marker SSR-09-1126 are as follows:
SSR-09-1126F：5′-CTCAATCGCGAACCCTAAAC-3′(SEQ ID NO.2)；
SSR-09-1126R：5′-GTGCTCCGAAGGCTGTCTAC-3′(SEQ ID NO.3)。
II, molecular marker SSR-09-1126 and application of amplification primer thereof
1. Identification of low-temperature resistant material in soybean sprout stage
S1, extracting the genomic DNA of the soybean material to be identified;
the CTAB method for extracting the soybean leaf genome DNA comprises the following steps:
(1) putting 2-4g of soybean leaves into a 1.5ml centrifuge tube, adding 4 steel balls with the diameter of 2 mm, freezing in liquid nitrogen, and then grinding for 30 times/s for one minute;
(2) adding 700 mu L of CTAB extracting solution preheated at 65 ℃ in advance after grinding is finished, and fully and uniformly mixing;
(3) putting the centrifugal tube added with CTAB into a 65 ℃ water bath kettle, carrying out water bath for 60 minutes, and reversing and uniformly mixing the centrifugal tube and the water bath at intervals of 15 minutes;
(4) adding 700 mu L of chloroform solution into the centrifuge tube after the water bath is finished, reversing and uniformly mixing, and centrifuging at 12000rpm/min for 15 min;
(5) sucking the supernatant, putting the supernatant into another prepared 1.5mL centrifuge tube, adding 700 mu L chloroform again, mixing the supernatant and the chloroform slightly, then centrifuging the mixture at 12000rpm/min for 15 min;
(6) taking another new 1.5ml centrifuge tube, adding 700 μ L of pre-cooled-20 deg.C isopropanol, collecting supernatant from the centrifuged solution, slowly dropping into the centrifuge tube containing isopropanol solution, and centrifuging at 8000rpm/min for 2 min;
(7) the supernatant was decanted and the pellet was retained. Adding 700 μ L of anhydrous ethanol, blowing for several times, sucking out the anhydrous ethanol, adding 700 μ L of 75% ethanol, blowing for several times, sucking out 75% ethanol, and air drying the DNA in a ventilated place;
(8) adding 100 μ L of sterilized water into the air-dried DNA, placing in a refrigerator at 4 deg.C until the DNA is completely dissolved, and storing in a refrigerator at-20 deg.C.
S2, carrying out PCR amplification on the soybean material to be identified by using an SSR-09-1126 amplification primer;
the PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4;
TABLE 3 PCR reaction System
TABLE 4 PCR reaction procedure
S3, carrying out electrophoretic analysis on the amplification product, identifying as a soybean sprout stage low temperature resistant material if the electrophoretic band type of the soybean material amplification product to be identified belongs to the No.1 band type in the SSR-09-1126, and identifying as a soybean sprout stage low temperature sensitive material if the electrophoretic band type of the soybean material amplification product to be identified belongs to the No. 2 band type in the SSR-09-1126.
The result of the detection of the amplification product by polyacrylamide gel electrophoresis is shown in FIG. 5, wherein the graph A is an electrophoresis graph of SSR-09-1126 primer resistance materials (wild 03-7239, Gentianxiaojinhuang, Heiyong 24, brown bean, Jiyu 39, wild 03-5570, Changji soybean, four yellow pod, Huangbaozhu, Jinmanhua soybean, Jiyu 93, wild 04L-141, Jilin 20, Yuanbao gold, black pod, white eyebrow, Tonggong 13, Qinggang four vertex, Jiyu 71, wild 04-L15, gold dollar, white bean, tender and abundant 11, Jiyu 97, Fleming, small white bean, Jiyu 406, Jiyu 66, Wuchang bean, cat eye bean);
FIG. B is an electrophoretogram of SSR-09-1126 primer-sensitive materials (Black river 25, Gongnong P06-12, Tongnong 15, Henong 69, J2512, Jiyu 69, Gongnong P06-6, Jiyu 441, Jiyu 303, Mongolian bean 31, Henong 31, Henghe 29, Channong 14, Henong 35, Channong 25, Black river 11, Black river 31, Jiyu 299, Hefeng 37, Black river 14, Jinong 36, Black river 13, Changdense bean 30, Suiyong 15, Jinong 32, Anseri, Channong 29, Black river 43, Suiyong 28, Suiyou No. 2).
The phenotypic genotyping results for the materials used to develop the molecular markers are shown in table 5;
TABLE 5 phenotypic genotyping results for materials used to develop molecular markers
The SSR-09-1126 molecular markers have obvious difference in single-factor variance analysis results of germination rates of the No.1 banding pattern and the No. 2 banding pattern in the bud stage, as shown in Table 6;
TABLE 6SSR-09-1126 molecular marker No.1 band type and No. 2 band type single factor analysis results
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
1. The QTL related to soybean cold tolerance in the bud stage is characterized in that the QTL is positioned in the 38426868bp-38983456bp interval of the 9 th chromosome of soybean.
2. An SSR molecular marker of a QTL related to soybean sprout stage low temperature resistance according to claim 1, wherein the molecular marker is SSR-09-1126, the molecular marker is positioned in the 38701572bp-38701609bp interval of the soybean chromosome 9, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
3. A set of amplification primers for the SSR molecular marker according to claim 2, wherein said amplification primers are as follows:
SSR-09-1126F：5′-CTCAATCGCGAACCCTAAAC-3′；
SSR-09-1126R：5′-GTGCTCCGAAGGCTGTCTAC-3′。
4. use of the amplification primer of claim 3 for identification of low temperature resistance at soybean sprout stage.
5. The method for identifying the low temperature resistance of soybean sprouts by using the amplification primer of claim 3, which is characterized by comprising the following steps:
s1, extracting the genomic DNA of the soybean material to be identified;
s2, carrying out PCR amplification on the soybean material to be identified by using an SSR-09-1126 amplification primer;
s3, carrying out electrophoretic analysis on the amplification product, identifying as a soybean sprout stage low temperature resistant material if the electrophoretic band type of the soybean material amplification product to be identified belongs to the No.1 band type in the SSR-09-1126, and identifying as a soybean sprout stage low temperature sensitive material if the electrophoretic band type of the soybean material amplification product to be identified belongs to the No. 2 band type in the SSR-09-1126.
6. The use of the SSR molecular marker of claim 2 in soybean sprout stage cold tolerance trait breeding.
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|---|---|---|---|---|
| CN116751882A (en) * | 2023-06-01 | 2023-09-15 | 江苏省农业科学院 | KASP (KASP sequence identity) marker closely linked with drought tolerance of soybean in germination period and application thereof |
| CN116751882B (en) * | 2023-06-01 | 2023-12-22 | 江苏省农业科学院 | KASP (KASP sequence identity) marker closely linked with drought tolerance of soybean in germination period and application thereof |
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