CN109161610B - SSR molecular marker BvRE016 for detecting granularity of beet seeds and application thereof - Google Patents
SSR molecular marker BvRE016 for detecting granularity of beet seeds and application thereof Download PDFInfo
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Abstract
An SSR molecular marker BvRE016 for detecting granularity of beet seeds and application thereof, belonging to the technical field of molecular markers and relating to an SSR molecular marker for detecting granularity of beet seeds and application thereof. The method aims to solve the problem that the seed granularity is not easy to identify due to the lack of the related molecular marker of the beet seed granularity in the prior art. The primer pair for PCR amplification of the molecular marker is BvRE016A and BvRE 016S. The molecular marker BvRE016 of the invention has the consistency of the marker detection result and the seed character performance of 76.76% in all individuals, and respectively reaches 93.33% and 60% in single-particle individuals and multi-particle individuals. The method is used for detecting the granularity of the beet seeds.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and relates to an SSR molecular marker for detecting granularity of beet seeds and application thereof.
Background
The fruits of sugar beet (Beta Vulgaris) are coated with a host lignified calyx to form a more specific "seedball" structure: when the single flower grows, a single lean fruit is in the seed ball, and when a plurality of petiolless florets are gathered to generate the umbrella-shaped inflorescence, a plurality of fruits in the seed ball are closely connected to form a gathered flower and fruit containing a plurality of lean fruits. When describing the beet germplasm resources, the characteristics of seed granularity are described, and the characteristics are divided into single-seed (also called single-embryo) and multi-seed (also called multi-embryo).
The emergence rate of multiple seeds is high, the seedling protection is facilitated, the mechanized cultivation is facilitated for single seeds, and the thinning workload is saved. Meanwhile, the single seed is easy to control the plant spacing, and is beneficial to improving the high yield and the uniformity. With increasing mechanization of seeding, management, and advances in seed coating and pelleting technology, the demand for single seed sugar beet in production is increasing.
At present, most of single-seed beet germplasm in China is obtained by introduction and introduction, and a few single seeds are also independently cultivated, but the research on related molecular markers, genes and molecular mechanisms of beet seed granularity is not reported.
The method develops the seed granularity marker, is applied to seed granularity prediction, is beneficial to the identification, development and utilization of the single seed germplasm of beet in China, is also beneficial to improving the efficiency of single seed cultivation work, and is also beneficial to searching granularity related genes for breeding application and molecular mechanism research.
Disclosure of Invention
The invention provides an SSR molecular marker BvRE016 for detecting beet seed granularity and application thereof, aiming at solving the problems that the existing molecular marker related to beet seed granularity is lacked and the seed granularity is not easy to identify.
The invention develops Simple Sequence Repeat (SSR) candidate markers by using a high-throughput sequencing technology and uses F2The SSR molecular marker BvRE016 related to the granularity of the beet seeds is screened out from the segregating population, the sequence of the marker amplification region is obtained, and the genome positioning is carried out. The molecular marker can be used for detecting single/multiple individuals of beet seeds.
The SSR molecular marker BvRE016 for detecting the granularity of the beet seeds is characterized in that a primer pair for PCR amplification of the molecular marker BvRE016A and BvRE016S has the specific sequences as follows:
BvRE016A：5'-TCCTTCCAACAATCCTCCTG-3'
BvRE016S：5'-CAAACTGCTCGAGTTTGGGT-3'。
the SSR molecular marker BvRE016 for detecting the granularity of the beet seeds is applied to detecting the granularity of the seeds.
The specific detection method comprises the following steps:
firstly, separating and extracting total DNA from young and tender tissues of beet; the young tissue is young leaves, young inflorescences, young leaf stalks, young flower shoots, seeds in a germination stage, roots in a seedling stage or whole seedlings;
secondly, PCR amplification is carried out by taking the DNA obtained in the step one as a template and adopting primers BvRE016A and BvRE016S,
thirdly, separating the PCR amplification product obtained in the second step by polyacrylamide gel electrophoresis (PAGE), judging the result according to the size of the amplification product, and judging the single seed of the beet if only a band of 227bp is detected; when a 383bp band and a 467bp band were detected in addition to the 227bp band, it was judged as a sugar beet seed polyploidy.
In the second step, the primer BvRE016A is 5'-TCCTTCCAACAATCCTCCTG-3', and the primer BvRE016S is 5'-CAAACTGCTCGAGTTTGGGT-3'.
Further, the conditions of the PCR amplification reaction in step two are: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and final extension at 72 deg.C for 5 min.
The invention has the beneficial effects that:
the SSR molecular marker BvRE016 related to the beet seed granularity is obtained, can be used independently or in combination with other primers, and can be used for pre-judging the beet seed granularity through PCR detection; in any growth period of the beet, the young leaves or other young tissues of the beet are taken to extract genome DNA as a material, so that the workload is reduced, and the period of seed graininess judgment is shortened; can detect multiple beet individuals and judge the purity of the germplasm on the grain character.
The invention carries out genotyping on multiple species and single species of individuals of a separation generation respectively, and verifies the consistency of the marker and the performance of the seed granularity. The molecular marker BvRE016 of the invention is found that the consistency of the marker detection result and the representation of the seed granularity character in all individuals reaches 76.76%, and respectively reaches 93.33% and 60% in single-particle individuals and multiple-particle individuals, which indicates that BvRE016 has a linkage relation with the seed granularity character and can be used for detecting the seed granularity character.
And (3) comparing the sequence of the marker amplification region with the beet genome, and carrying out genome positioning on the marker. The results indicate that the BvRE016 molecular marker is located in the 30888225-30887980 region of chromosome 9 in the sugar beet genome.
The BvRE016 molecular marker is used for auxiliary selection of single-seed/multi-seed germplasm of the beet, prediction of individual and later-generation granularity of the beet, and auxiliary theoretical research of flowering and seedball formation of the beet and excavation of related genes of the granularity of the beet seeds.
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FIG. 1 shows the results of genotyping single individuals of sugar beet seeds;
FIG. 2 shows the results of genotyping of sugar beet seed from a number of individuals.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the SSR molecular marker BvRE016 for detecting the granularity of the beet seeds in the embodiment has the primer pairs BvRE016A and BvRE016S for PCR amplification of the molecular marker, and has the specific sequences as follows:
BvRE016 A：5'-TCCTTCCAACAATCCTCCTG-3'
BvRE016 S：5'-CAAACTGCTCGAGTTTGGGT-3'。
the molecular marker of the present embodiment can be used to determine the seed granularity of sugar beet germplasm or an individual. When in use, the young leaves or other young tissues of the annual plants are directly taken to carry out DNA extraction and marking detection, and seeding, mother root cultivation, mother root planting and biennial plant character investigation work which takes two years are not needed; or extracting DNA from young leaves, inflorescences or other young tissues of the biennial plants and detecting the markers. When the method is used for judging the granularity of the whole germplasm, the judgment can be directly carried out according to the PCR result by combining with statistical analysis, or the trait investigation is carried out on key individuals in the germplasm for supplement, and the detection on all individuals is not needed. When the method is used for judging the fertility of the beet individual, young leaves, young inflorescences or other young tissues can be taken at the early stage of bolting or budding or other early stages of budding according to the PCR result to be judged in advance, so that the scope of the individual character investigation is reduced, and unnecessary single/multiple individuals are eliminated in advance.
The second embodiment is as follows: the application of the SSR molecular marker BvRE016 for detecting the granularity of the beet seeds in the embodiment is used for detecting the granularity of the seeds.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the specific detection method comprises the following steps:
firstly, separating and extracting total DNA from young and tender tissues of beet;
secondly, using the DNA obtained in the first step as a template, adopting primers BvRE016A and BvRE016S to perform PCR amplification,
thirdly, separating the PCR amplification product obtained in the second step by polyacrylamide gel electrophoresis, and judging that the single beet seed is single if only a band of 227bp is detected according to the size judgment result of the amplification product; when a 383bp band and a 467bp band were detected in addition to the 227bp band, it was judged as a sugar beet seed polyploidy. The rest is the same as the second embodiment.
The fourth concrete implementation mode: the third difference between the present embodiment and the specific embodiment is that: in the step one, the tender tissue is tender leaves, tender inflorescences, tender petioles, tender flower shoots, seeds in a germination stage, roots in a seedling stage or whole seedlings. The rest is the same as the third embodiment.
The fifth concrete implementation mode: this embodiment is different from one of the second to fourth embodiments in that: in the second step, the primer BvRE016A is 5'-TCCTTCCAACAATCCTCCTG-3', and the primer BvRE016S is 5'-CAAACTGCTCGAGTTTGGGT-3'. The other is the same as one of the second to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the second to fifth embodiments in that: the conditions of the PCR amplification reaction in the second step are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and final extension at 72 deg.C for 5 min. The other is the same as one of the second to fifth embodiments.
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1:
obtaining a large amount of beet sequences through high-throughput sequencing, excavating unreported SSR markers from the beet sequences, screening more than one hundred candidate markers with good quality and polymorphism, and simultaneously constructingF establishing the graininess of sugar beet seeds2Isolating the population.
Selecting multiple species and single species from separation generations to construct a mixed pool, and screening candidate markers by using a group mixed separation analysis (BSA) method to obtain SSR markers showing polymorphism among the mixed pools. And an SSR molecular marker related to the granularity of the beet seeds is selected from the SSR molecular markers and named as BvRE 016.
(ii) detection of seed graininess by the marker
Firstly, respectively extracting DNA of 30 young beet leaves;
secondly, using the DNA obtained in the first step as a template, and adopting primers BvRE016A and BvRE016S to perform PCR amplification, wherein a PCR reaction system is shown in Table 1:
primer BvRE 016A: TCCTTCCAACAATCCTCCTG
Primer BvRE 016S: CAAACTGCTCGAGTTTGGGT
TABLE 1
The conditions of the PCR amplification reaction are as follows:
pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and final extension at 72 deg.C for 5 min.
Thirdly, a result detection method:
and (3) separating the PCR amplification product obtained in the step two by using polyacrylamide gel electrophoresis, wherein the gel crosslinking degree is 8%, the voltage during electrophoresis is 100V, and the time is 5 h. And after electrophoresis, staining the membrane for 15-20 min by using 10mg/L Ethidium Bromide (EB) solution.
After examining the seed properties of segregating generations, 30 individuals of single-grain and multi-grain individuals were genotyped with markers. The result of genotyping a single particle is shown in FIG. 1, where M represents DNA 50bp marker, and P represents DNA1To representMaternal (Single seed), P2Represents a male parent (multiparticulate), and the number of the male parent is 1-30, and the male parent is 30 single individuals. The results of the genotyping of the multiparticulates are shown in FIG. 2, where M denotes DNA 50bp marker, P1Denotes maternal (Single grain), P2Represents a male parent (multiparticulate), and the numbers 1 to 30 are more than 30 multiparticulates.
And (4) genotyping a plurality of seeds and a single seed individual of a separation generation respectively, and verifying the consistency of the marker and the seed performance. The molecular marker BvRE016 is found that the consistency of the marker detection result and the representation of the seed character in all individuals reaches 76.76 percent, and the consistency reaches 93.33 percent and 60 percent in single-particle individuals and multi-particle individuals respectively. The BvRE016 shows that the BvRE016 has a linkage relation with the seed granularity and can be used for detecting the seed granularity.
TABLE 2 detection results of the seed graininess related markers on different grainy plants
According to the size judgment result of the amplified product, if only a band of 227bp is detected, judging that the single seed of the beet is single seed; when a 383bp band and a 467bp band were detected in addition to the 227bp band, it was judged as a sugar beet seed polyploidy.
(II) obtaining sequences of amplified regions of the tags
The labeled amplification sequence was obtained by PCR product recovery, T-A cloning and sanger sequencing.
The BvRE016 amplified sequence is sequenced as follows (the cross-hatched part is SSR repeated unit):
single event-related band: 227bp
TTGACTATGATTTAATGGATCAACTGTTAAATGAAGGGTGTTGGTTACAAACAGCTGATGGATCAAACCTTCTGCAGTTGAGTCAGGGTCCTGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTTCGAGTCCACCCACCTCTCGCGGTTTAAGCTACCCATCTTTTCCCTTCCTTGGTTCGGACCCAAACTCGAGCAGTTTG
Multigrain-related band 1: 383bp
TTGACTATGATTTAATGGATCAACTGTTAAATGAAGGGTGTTGGTTACAAACAGCTGATGGATCAAACCTTCTGCAGTTGAGTCAGGGTCCTGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAG GGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCA GGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTC AGGGTTCGAGTCCACCCACCTCTCGCGGTTTAAGCTACCCATCTTTTCCCTTCCTTGGTTCGGACCCAAACTCGAGCAGTTTG
Multigrain-related band 2: 467bp
TTGACTATGATTTAATGGATCAACTGTTAAATGAAGGGTGTTGGTTACAAACAGCTGATGGATCAAACCTTCTGCAGTTGAGTCAGGGTCCTGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAG GGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCA GGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTC AGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGTCAGGGT CAGGGTCAGGGTTCGAGTCCACCCACCTCTCGCGGTTTAAGCTACCCATCTTTTCCCTTCCTTGGTTCGGACCCAAACTCGAGCAGTTTG
(III) analysis and genomic mapping of sequences of tagged amplified regions
The amplified product and a plant non-redundant nucleic acid database are subjected to blast N comparison analysis, and the result shows that the amplified product of BvRE016 is similar to the sequence of a predicted cytotoxic protein of beet, the similar part is the 1 st to 137bp of the amplified fragment, and the sequence similarity of the part is 99%.
The markers were mapped in the sugar beet genome based on sequence similarity, which indicated that BvRE016 mapped to the 30888225 and 30887980 regions of chromosome 9 in the sugar beet genome.
Sequence listing
<110> university of Heilongjiang
<120> SSR molecular marker BvRE016 for detecting granularity of beet seeds and application thereof
<160> 5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer BvRE016S
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> primer BvRE016A
<400> 2
<210> 3
<211> 227
<212> DNA
<213> Artificial sequence
<220>
<223> Single-particle-associated-band-corresponding amplification sequence
<400> 3
ttgactatga tttaatggat caactgttaa atgaagggtg ttggttacaa acagctgatg 60
gatcaaacct tctgcagttg agtcagggtc ctggtcaggg tcagggtcag ggtcagggtc 120
agggtcaggg tcagggtcag ggtcagggtt cgagtccacc cacctctcgc ggtttaagct 180
acccatcttt tcccttcctt ggttcggacc caaactcgag cagtttg 227
<210> 4
<211> 383
<212> DNA
<213> Artificial sequence
<220>
<223> amplification sequence corresponding to Multigrain-related band 1
<400> 4
ttgactatga tttaatggat caactgttaa atgaagggtg ttggttacaa acagctgatg 60
gatcaaacct tctgcagttg agtcagggtc ctggtcaggg tcagggtcag ggtcagggtc 120
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 180
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 240
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 300
agggttcgag tccacccacc tctcgcggtt taagctaccc atcttttccc ttccttggtt 360
cggacccaaa ctcgagcagt ttg 383
<210> 5
<211> 467
<212> DNA
<213> Artificial sequence
<220>
<223> amplified sequences corresponding to the Multigrain-related band 2
<400> 5
ttgactatga tttaatggat caactgttaa atgaagggtg ttggttacaa acagctgatg 60
gatcaaacct tctgcagttg agtcagggtc ctggtcaggg tcagggtcag ggtcagggtc 120
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 180
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 240
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 300
agggtcaggg tcagggtcag ggtcagggtc agggtcaggg tcagggtcag ggtcagggtc 360
agggtcaggg tcagggtcag ggtcagggtt cgagtccacc cacctctcgc ggtttaagct 420
acccatcttt tcccttcctt ggttcggacc caaactcgag cagtttg 467
Claims (2)
1. An SSR molecular marker BvRE016 related to the granularity detection of beet seeds is characterized in that a primer pair for PCR amplification of the molecular marker is BvRE016A and BvRE016S, and the specific sequence is as follows:
BvRE016 A：5'- TCCTTCCAACAATCCTCCTG -3'
BvRE016 S：5'- CAAACTGCTCGAGTTTGGGT -3'；
wherein the detection sequence of the seed multi-particle related molecular marker is shown as SEQ ID NO: 4 and SEQ ID NO: 5, the detection sequence of the single seed related molecular marker is shown as SEQ ID NO: 3, respectively.
2. Use of an SSR molecule marker BvRE016 agent according to claim 1 for detecting seed granularity.
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| US20030115624A1 (en) * | 2000-03-10 | 2003-06-19 | Terry L. Irene | Quantitative trait loci for insect resistance in soybeans |
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| CN102461460A (en) * | 2010-12-03 | 2012-05-23 | 黑龙江大学 | Creation of beet novel cytoplasmic male sterile (P-CMS) line |
| CN102461461A (en) * | 2010-12-03 | 2012-05-23 | 黑龙江大学 | Creation of beet novel cytoplasmic male sterile (M-CMS) line |
| CN108291234A (en) * | 2015-09-04 | 2018-07-17 | 主基因有限公司 | Multiple sporinite forms gene |
| CN105603096A (en) * | 2016-02-25 | 2016-05-25 | 华中农业大学 | Co-dominance InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof |
| CN107267660A (en) * | 2017-08-21 | 2017-10-20 | 黑龙江大学 | Detect SSR molecular marker and its application of beet stamen fertility |
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| Title |
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| Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet);Katherine C Dent 等;《J Microbiol Methods》;20040131;第56卷(第1期);第17-26页 * |
| 表型与分子标记相结合分析130份糖用甜菜种质的遗传多样性;王希 等;《中国农学通报》;20181030;第34卷(第30期);第26-36页 * |
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