CN114231656A - SSR core primer group for identifying purity of cauliflower hybrid and screening method and application thereof - Google Patents
SSR core primer group for identifying purity of cauliflower hybrid and screening method and application thereof Download PDFInfo
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Abstract
The invention discloses an SSR core primer group for identifying the purity of cauliflower hybrids and a screening method and application thereof, belonging to the technical field of molecular markers. The SSR core primer group comprises: BoGMS0624, OI11G11, BoGMS 0941; the alternative core primer group is as follows: BoE607, BoE761, BoGMS2431, BoGMS0808 and BoGMS 1530. According to the method, through DNA fingerprint analysis of 70 hybrid species of cauliflower and broccoli, the evaluation index of the core primer suitable for purity identification of the hybrid species is determined, a set of primers for purity identification of the cauliflower hybrid species is determined, the purity identification of the cauliflower hybrid species is realized by the least primers with wide genome coverage, the workload is greatly reduced, the detection efficiency is improved, and the detection cost is reduced.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to an SSR core primer group for identifying the purity of cauliflower and broccoli hybrids and a screening method thereof.
Background
Cauliflower belongs to Brassica plants in cruciferae, and comprises cauliflower (Brassica oleracea var. borrytis) and broccoli (Brassica oleracea var. italica) and the like, wherein the cauliflower and the broccoli take unique reproductive organ bulbs thereof as commodities and edible organs, contain various vitamins, minerals, crude fibers and the like, particularly contain sulforaphane, and have cancer prevention and anticancer effects. China is a big country for producing cauliflowers and also a big country for consuming the cauliflowers. However, in the history of cauliflower cultivation in China, the production seeds mainly depend on foreign import. More than 90% of broccoli seeds are still imported in the current production, the seed price is extremely high, and the seed quality problem is more prominent in the production practice. The seed purity is the key for evaluating the seed quality, and the seed purity is not high, the activity is reduced, the yield is reduced, the quality is deteriorated, and the economic benefit is seriously influenced. Therefore, it is especially important to vigorously carry out the work of detecting the purity of the seeds.
With the development of molecular marker technology, the molecular marker technology is already applied to the identification of pepper variety purity, and SSR marker is a marking technology with more applications. However, in the conventional studies, in order to solve the purity determination of several hybrid species, a large amount of primer screening work is often required to find a suitable identifying primer. Whether a set of core primers suitable for seed purity identification can be determined, and a known variety purity identification standard DNA fingerprint is constructed, which is important for purity identification of known varieties. With the core primer, complicated primer screening is not needed any more, and even for unknown varieties, only a small amount of screening is needed, and a proper identification primer can be quickly found. The SSR marker has the advantages of simple and convenient operation, stability, reliability and the like, has a large amount of allelic differences, has abundant polymorphism, and has wide application prospect in hybrid seed purity detection.
Two purposes of hybrid purity identification: the most important purpose is to test self-bred seedlings, and only 1 pair of amphiphilic complementary primers is needed; secondly, special strains are tested, and specific primers or primer combinations are needed, so that the probability of the same banding pattern varieties is low enough, and the special strains can be effectively distinguished. Based on these two points, the core primer suitable for purity identification must have the following two characteristics: (1) the heterozygosis rate is high, so that the method has strong capacity of distinguishing selfed seedlings, and the parents complementary primers can be easily screened; (2) the polymorphism is high, so that the capacity of distinguishing the heterotypic strains is high. Only in this way, it can be ensured that only a few primers can be used to find a primer meeting the requirements in the prescreening process. Accordingly, determination of SSR core primers suitable for purity identification of maize hybrids [ J ] agrotechnology, 2007,15 (6): 964. 969 "the primers are classified into 3 types: (1) the polymorphism is high, and the heterozygosity rate is high; (2) the polymorphism is high or medium, and the heterozygosity is high or medium; (3) low or moderate polymorphism, or low heterozygosity. Among them, the type 1 primer is most suitable for purity determination, followed by the type 2 primer, and for the type 3 primer, a primer preferable for purity determination is not generally recommended.
Disclosure of Invention
The invention aims to provide an SSR core primer group for identifying the purity of cauliflower and broccoli hybrid seeds, a screening method and application thereof, so as to solve the technical problem that a large amount of tedious primer screening is required for identifying the purity of the hybrid seeds in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides an SSR core primer group for identifying the purity of cauliflower hybrid seeds, wherein primers in the SSR core primer group are as follows: BoGMS0624, OI11G11, BoGMS 0941;
the BoGMS0624 consists of a BoGMS0624 forward primer and a BoGMS0624 reverse primer, and the base sequence of the BoGMS0624 forward primer is shown in SEQ ID NO: 1, the base sequence of the BoGMS0624 reverse primer is shown as SEQ ID NO: 2 is shown in the specification;
the OI11G11 consists of an OI11G11 forward primer and an OI11G11 reverse primer, and the base sequence of the OI11G11 forward primer is shown in SEQ ID NO: 3, the base sequence of the OI11G11 reverse primer is shown in SEQ ID NO: 4 is shown in the specification;
the BoGMS0941 consists of a BoGMS0941 forward primer and a BoGMS0941 reverse primer, and the base sequence of the BoGMS0941 forward primer is shown as SEQ ID NO: 5, the base sequence of the BoGMS0941 reverse primer is shown as SEQ ID NO: 6 is shown in the specification;
the cauliflower is cauliflower or broccoli.
The invention provides an alternative SSR core primer group for identifying cauliflower hybrid purity, wherein primers in the alternative SSR core primer group are as follows: BoE607, BoE761, BoGMS2431, BoGMS0808, BoGMS 1530;
the BoE607 consists of a BoE607 forward primer and a BoE607 reverse primer, and the base sequence of the BoE607 forward primer is shown in SEQ ID NO: 7, the base sequence of the BoE607 reverse primer is shown as SEQ ID NO: 8 is shown in the specification;
the BoE761 consists of a BoE761 forward primer and a BoE761 reverse primer, and the base sequence of the BoE761 forward primer is shown in SEQ ID NO: 9, the base sequence of the BoE761 reverse primer is shown as SEQ ID NO: 10 is shown in the figure;
the BoGMS2431 consists of a BoGMS2431 forward primer and a BoGMS2431 reverse primer, and the base sequence of the BoGMS2431 forward primer is shown as SEQ ID NO: 11, the base sequence of the BoGMS2431 reverse primer is shown as SEQ ID NO: 12 is shown in the specification;
the BoGMS0808 consists of a BoGMS0808 forward primer and a BoGMS0808 reverse primer, and the base sequence of the BoGMS0808 forward primer is shown as SEQ ID NO: 13, the base sequence of the BoGMS0808 reverse primer is shown as SEQ ID NO: 14 is shown in the figure;
the BoGMS1530 consists of a BoGMS1530 forward primer and a BoGMS1530 reverse primer, and the base sequence of the BoGMS1530 forward primer is shown as SEQ ID NO: 15, the base sequence of the BoGMS1530 reverse primer is shown as SEQ ID NO: 16 is shown in the figure;
the cauliflower is cauliflower or broccoli.
The invention provides a screening method of an SSR core primer group or an alternative core primer group for identifying the purity of cauliflower hybrids, which comprises the following steps:
(1) respectively extracting DNA of all cauliflower varieties to be detected;
(2) respectively taking the DNA of each cauliflower variety obtained in the step (1) as a template, and respectively carrying out PCR amplification on the DNA of each cauliflower variety by using 22 pairs of SSR primers shown in the table 1;
TABLE 122 pairs of SSR primer sequences
| Serial number | Primer pair | Base sequence of forward primer | Base sequence of reverse primer |
| 1 | BoGMS0624 | SEQ ID NO: 1 is shown in | SEQ ID NO: 2 is shown in |
| 2 | OI11G11 | SEQ ID NO: 3 is shown in | SEQ ID NO: 4 is shown in |
| 3 | BoGMS0941 | SEQ ID NO: 5 is shown in | SEQ ID NO: 6 is shown in |
| 4 | BoE607 | SEQ ID NO: 7 is shown in | SEQ ID NO: 8 is shown in |
| 5 | BoE761 | SEQ ID NO: 9 to | SEQ ID NO: 10 to |
| 6 | BoGMS2431 | SEQ ID NO: 11 to (1) | SEQ ID NO: 12 to |
| 7 | BoGMS0808 | SEQ ID NO: 13 to (9) | SEQ ID NO: 14 to |
| 8 | BoGMS1530 | SEQ ID NO: 15 to | SEQ ID NO: 16 is shown in |
| 9 | HCSSR1 | SEQ ID NO: 17 to | SEQ ID NO: 18 to |
| 10 | BoSF2564 | SEQ ID NO: 19 to | SEQ ID NO: 20 to |
| 11 | BoE718 | SEQ ID NO: 21 is shown in | SEQ ID NO: 22 is shown |
| 12 | BoE450 | SEQ ID NO: 23 to | SEQ ID NO: shown in 24 |
| 13 | BoE699 | SEQ ID NO: 25 is shown | SEQ ID NO: 26 to |
| 14 | BoE723 | SEQ ID NO: 27 to | SEQ ID NO: 28 is shown |
| 15 | BoE134 | SEQ ID NO: 29 to | SEQ ID NO: 30 to |
| 16 | BoE051 | SEQ ID NO: 31 to | SEQ ID NO: 32 is shown in |
| 17 | BoGMS1322 | SEQ ID NO: 33 is shown | SEQ ID NO: 34 shown in |
| 18 | BoGMS1119 | SEQ ID NO: 35 is shown | SEQ ID NO: 36 shown in |
| 19 | BoGMS0501 | SEQ ID NO: 37 shown in | SEQ ID NO: 38 to |
| 20 | BoGMS1004 | SEQ ID NO: 39 to | SEQ ID NO: 40 to |
| 21 | BoGMS1009 | SEQ ID NO: 41 to | SEQ ID NO: 42 is shown in |
| 22 | Na10D03 | SEQ ID NO: 43 is shown in | SEQ ID NO: 44 is shown in |
(3) Performing polymorphism and heterozygosis performance analysis on a PCR amplification product by adopting capillary electrophoresis, selecting a core primer group or an alternative core primer group according to the polymorphism information content PIC and the heterozygosity rate of the primer, wherein the primer is a high polymorphism information primer when the PIC is more than or equal to 0.5, a medium polymorphism information primer when the PIC is more than or equal to 0.25 and less than 0.5, the primer is a high heterozygosity rate primer when the heterozygosity rate is more than or equal to 0.6, and a medium heterozygosity rate primer when the heterozygosity rate is more than or equal to 0.3 and less than 0.6; selecting a primer with PIC more than or equal to 0.5 and clear bands with heterozygosis rate more than or equal to 0.6 as an SSR core primer group for identifying the purity of cauliflower hybrids; selecting a primer with a clear band and 0.25-0.5 PIC and 0.3-0.6 heterozygosity as an alternative SSR core primer group for identifying the purity of cauliflower hybrids.
Further, the PCR amplification reaction system in step (2) of the screening method of the SSR core primer group or the alternative core primer group for identifying the cauliflower hybrid purity is 10 muL in total, wherein 5 muL of 2 x mix solution, 0.3 muL of 10 mumol/L forward primer, 0.3 muL of 10 mumol/L reverse primer, 50ng of DNA template and the balance of ultrapure water are complemented; the reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 45s in 35 cycles; extending at 72 ℃ for 10min, storing at 4 ℃, and labeling TAMRA, HEX, ROX or 6-FAM fluorescent group at the 5' end of the forward primer in each pair of primers for PCR amplification.
The invention also provides application of the SSR core primer group for identifying the purity of the cauliflower hybrid in identifying the purity of the cauliflower hybrid.
Further, the SSR core primer group for identifying the purity of the cauliflower hybrid is applied to identifying the purity of the cauliflower hybrid, and SSR primers applied to the identified variety of the cauliflower and each variety of the cauliflower hybrid purity identification pair are shown in Table 2:
TABLE 2 SSR primers for purity identification of cauliflower variety hybrids
The invention also provides application of the alternative SSR core primer group for identifying the cauliflower hybrid seed purity in identifying the cauliflower hybrid seed purity.
Further, the optional SSR core primer group for identifying the purity of the cauliflower hybrid is applied to identifying the purity of the cauliflower hybrid, and the primer pair for identifying the purity of the cauliflower subsous 20003 hybrid is BoE 607; or the primer pair for identifying the purity of the cauliflower W131 hybrid is a primer BoE 607.
Further, the SSR core primer group for cauliflower hybrid purity identification or the alternative SSR core primer group for cauliflower hybrid purity identification is applied to the cauliflower hybrid purity identification, and the application further comprises a reaction system for PCR amplification of each pair of primers in the cauliflower hybrid purity identification, wherein the reaction system accounts for 10 mu L, 5 mu L of 2 x mix liquid, 0.3 mu L of 10 mu mol/L forward primer, 0.3 mu L of 10 mu mol/L reverse primer, 50ng of DNA template and the balance of ultrapure water are supplemented; the reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 45s in 35 cycles; extending for 10min at 72 ℃, storing at 4 ℃, and marking TAMRA, HEX, ROX or 6-FAM fluorescent group at the 5' end of each pair of primers for PCR amplification; analyzing the amplified products of each pair of primers by capillary electrophoresis, wherein the true hybrid with a heterozygous gene band and the false hybrid with a homozygous gene band are respectively obtained, and the purity of the hybrid is calculated according to the following formula: the purity of hybrid is equal to the number of true hybrid/total number of seeds tested x 100%.
The invention also provides application of the SSR core primer group for identifying the purity of the cauliflower hybrid in genetic diversity analysis of cauliflower germplasm resources or identification of the genetic relationship of cauliflower varieties. Or the alternative SSR core primer group for identifying the purity of the cauliflower hybrid is applied to the analysis of genetic diversity of cauliflower germplasm resources or the identification of the genetic relationship of cauliflower varieties.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, through DNA fingerprint analysis of 70 hybrid species of cauliflower and broccoli which are mainly planted at home and abroad, evaluation indexes of screening a core primer group and an alternative core primer group suitable for purity identification of the cauliflower hybrid species are determined, and then a set of cauliflower or broccoli hybrid species purity identification core primer group and an alternative core primer group are determined, so that a foundation is laid for constructing a standard DNA fingerprint spectrum for purity identification of the cauliflower and broccoli hybrid species.
According to the invention, according to two indexes of polymorphism and heterozygosity, the primer BoGMS0624, the primer OI11G11 and the primer BoGMS0941 are determined to be a first-choice core primer group for identifying the purity of cauliflower or broccoli hybrids, the 3 pairs of core primers are utilized to screen, 68 cauliflower varieties accounting for 97 percent of the current major cauliflower varieties can find identification primers with heterozygosis banding patterns, and the 5 primers BoE607, BoE761, BoGMS2431, BoGMS0808 and BoGMS1530 are determined to be alternative core primer groups for identifying the purity of cauliflower or broccoli hybrids, so that the purity of cauliflower hybrids can be identified by using the minimum primers with wide genome coverage, the workload is greatly reduced, the detection efficiency is improved, and the detection cost is reduced.
SEQ ID NO: 1 shows the base sequence of the BoGMS0624 forward primer.
SEQ ID NO: 2 shows the base sequence of the BoGMS0624 reverse primer.
SEQ ID NO: 3 shows the nucleotide sequence of the forward primer OI11G 11.
SEQ ID NO: 4 shows the nucleotide sequence of the reverse primer OI11G 11.
SEQ ID NO: 5 shows the base sequence of the forward primer of BoGMS 0941.
SEQ ID NO: 6 shows the base sequence of the BoGMS0941 reverse primer.
SEQ ID NO: 7 shows the base sequence of the BoE607 forward primer.
SEQ ID NO: shown in FIG. 8 is the base sequence of the BoE607 reverse primer.
SEQ ID NO: 9 shows the base sequence of the BoE761 forward primer.
SEQ ID NO: 10 shows the base sequence of the BoE761 reverse primer.
SEQ ID NO: shown in FIG. 11 is the base sequence of the forward primer of BoGMS 2431.
SEQ ID NO: shown in FIG. 12 is the base sequence of the BoGMS2431 reverse primer.
SEQ ID NO: 13 shows the base sequence of the BoGMS0808 forward primer.
SEQ ID NO: 14 shows the base sequence of the BoGMS0808 reverse primer.
SEQ ID NO: 15 shows the base sequence of the BoGMS1530 forward primer.
SEQ ID NO: 16 shows the base sequence of the BoGMS1530 reverse primer.
SEQ ID NO: 17 shows the base sequence of the HCSSR1 forward primer.
SEQ ID NO: 18 shows the base sequence of the reverse primer of HCSSR 1.
SEQ ID NO: 19 shows the base sequence of the BoSF2564 forward primer.
SEQ ID NO: 20 shows the base sequence of the BoSF2564 reverse primer.
SEQ ID NO: shown in FIG. 21 is the base sequence of the BoE718 forward primer.
SEQ ID NO: 22 shows the base sequence of the BoE718 reverse primer.
SEQ ID NO: 23 shows the base sequence of the BoE450 forward primer.
SEQ ID NO: shown in FIG. 24 is the base sequence of the BoE450 reverse primer.
SEQ ID NO: 25 shows the base sequence of the BoE699 forward primer.
SEQ ID NO: 26 shows the base sequence of the BoE699 reverse primer.
SEQ ID NO: 27 shows the base sequence of the BoE723 forward primer.
SEQ ID NO: 28 shows the base sequence of the BoE723 reverse primer.
SEQ ID NO: 29 shows the base sequence of the BoE134 forward primer.
SEQ ID NO: 30 shows the base sequence of the BoE134 reverse primer.
SEQ ID NO: 31, the base sequence of the BoE051 forward primer is shown.
SEQ ID NO: 32 shows the base sequence of the BoE051 reverse primer.
SEQ ID NO: shown in FIG. 33 is the base sequence of the BoGMS1322 forward primer.
SEQ ID NO: 34 shows the base sequence of the BoGMS1322 reverse primer.
SEQ ID NO: 35, the base sequence of the BoGMS1119 forward primer is shown.
SEQ ID NO: 36 shows the base sequence of the BoGMS1119 reverse primer.
SEQ ID NO: 37 shows the base sequence of the forward primer of BoGMS 0501.
SEQ ID NO: 38 shows the base sequence of the BoGMS0501 reverse primer.
SEQ ID NO: 39 shows the base sequence of the BoGMS1004 forward primer.
SEQ ID NO: 40 shows the base sequence of the BoGMS1004 reverse primer.
SEQ ID NO: shown at 41 is the base sequence of the BoGMS1009 forward primer.
SEQ ID NO: 42 shows the base sequence of the BoGMS1009 reverse primer.
SEQ ID NO: 43 shows the nucleotide sequence of the Na10D03 forward primer.
SEQ ID NO: 44, the nucleotide sequence of the reverse primer Na10D03 is shown.
Drawings
FIG. 1: and (3) performing capillary fluorescence examination electrophoresis images of validity verification of a core primer BoGMS0941 for identifying the purity of cauliflower hybridization combination MS048-1-2 XC 18232. FIG. 1 shows the hybridization combinations MS048-1-2 XC 18232 and F in FIG. 11(Pinus 1821) the amplified band using the primer BoGMS0941, in FIG. 1, a1 is female parent MS048-1-2(234bp), a2 is male parent C18232(224bp), and a3 is F1, wherein 2 complementary bands (224bp/234bp) of the male and female parents appear.
FIG. 2: the cauliflower hybridization combination MS048-1-2 XC 18199 purity-identified core primer OI11G11 validity-verified capillary fluorescence examination electrophoretogram. FIG. 2 shows the amplified bands of the hybridization combination MS048-1-2 XC 18199 and F1 (pine 1963) using primer OI11G11, in FIG. 2, b1 is the parent MS048-1-2(129bp), b2 is the parent C18199(154bp), and b3 is F1 with 2 complementary bands of the parent (129bp/154 bp).
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the following examples are intended to describe the preferred embodiments of the present invention only and are not intended to limit the scope of the present invention, and various changes and modifications of the technical solutions of the present invention, which may be made by those skilled in the art without departing from the spirit of the present invention, are intended to fall within the scope of the present invention defined by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention and are within the scope of the present invention.
The broccoli variety materials used in the following examples are all commercially available.
Example 1
1. Materials and methods
1.1 materials
1.1.1 materials
The test material was 70 parts of hybrid of cauliflower and broccoli (see table 1). Including broccoli and broccoli varieties of main breeding units in the world, such as backbone varieties cultivated by Wuhan Asia non-species limited company, Dalianmi Kyokoku national seedling limited company, Yunnan province agricultural academy, Japanese Bantian seedling Kaisha, and Olympic species limited company in Zhejiang America. The varieties are main varieties for domestic production at present.
1.1.2 SSR primers
According to 'Wangfeng et al' determination of SSR core primer suitable for purity identification of corn hybrid [ J ]. Proc of agricultural biotechnology, 2007,15 (6): 964. 969. the screening principle of the core primers, by online primary screening and laboratory re-screening, 22 pairs of SSR primers with good comprehensive properties of polymorphism, stability, repeatability and the like are determined in 98 pairs of SSR primers, and the 22 pairs of primers are as follows: BoGMS0624, Ol11G11BoGMS0941, BoE607, BoE761, BoGMS2431, BoGMS0808, BoGMS1530, HCSSR1, BoSF2564, BoE718, BoE450, BoE699, BoE723, BoE134, BoE051, BoGMS1322, BoGMS1119, BoGMS0501, BoGMS1004, BoGMS1009, Na10D 03.
The BoGMS0624 consists of a BoGMS0624 forward primer and a BoGMS0624 reverse primer.
The Ol11G11 consists of an Ol11G11 forward primer and an Ol11G11 reverse primer.
The BoGMS0941 consists of a BoGMS0941 forward primer and a BoGMS0941 reverse primer.
The BoE607 consists of a BoE607 forward primer and a BoE607 reverse primer.
The BoE761 consists of a BoE761 forward primer and a BoE761 reverse primer.
The BoGMS2431 consists of a BoGMS2431 forward primer and a BoGMS2431 reverse primer.
The BoGMS0808 consists of a BoGMS0808 forward primer and a BoGMS0808 reverse primer.
The BoGMS1530 consists of a BoGMS1530 forward primer and a BoGMS1530 reverse primer.
The HCSSR1 consists of an HCSSR1 forward primer and an HCSSR1 reverse primer.
The BoSF2564 consists of a BoSF2564 forward primer and a BoSF2564 reverse primer.
The BoE718 consists of a BoE718 forward primer and a BoE718 reverse primer.
The BoE450 consists of a BoE450 forward primer and a BoE450 reverse primer.
The BoE699 consists of a BoE699 forward primer and a BoE699 reverse primer.
The BoE723 consists of a BoE723 forward primer and a BoE723 reverse primer.
The BoE134 consists of a BoE134 forward primer and a BoE134 reverse primer.
The BoE051 consists of a BoE051 forward primer and a BoE051 reverse primer.
The BoGMS1322 was composed of a BoGMS1322 forward primer and a BoGMS1322 reverse primer.
The BoGMS1119 consists of a BoGMS1119 forward primer and a BoGMS1119 reverse primer.
The BoGMS0501 consists of a BoGMS0501 forward primer and a BoGMS0501 reverse primer.
The BoGMS1004 consists of a BoGMS1004 forward primer and a BoGMS1004 reverse primer.
The BoGMS1009 consists of a BoGMS1009 forward primer and a BoGMS1009 reverse primer.
The Na10D03 consisted of a Na10D03 forward primer and a Na10D03 reverse primer.
The SSR primers are further evaluated and screened by the 22 pairs of primers to determine a core primer group or an alternative core primer group suitable for identifying the purity of the cauliflower or broccoli hybrids, and further screened to determine SSR primers corresponding to the purity identification of the hybrids of 70 cauliflower varieties (see table 3). And detecting the sizes of the amplified fragments with different allelic variation by using a DNA analyzer.
1.2 methods
1.2.1 DNA extraction
Randomly taking 5 single young tissues of each variety, and adopting a CTAB method to extract DNA from each single plant.
1.2.2 PCR amplification
Carrying out PCR amplification on each cauliflower or broccoli variety in 70 cauliflower varieties in the table 1 by using the 22 pairs of primers, wherein the 5' tail end of the forward primer in each pair of primers is marked with TAMRA, HEX, ROX or 6-FAM fluorescent group, and the reaction system and the reaction program for carrying out PCR amplification on each cauliflower or broccoli variety by each pair of primers are as follows:
PCR amplification reaction System: a total of 10. mu.L reaction volume was prepared with 5. mu.L of 2 Xmix, 0.3. mu.L of 10. mu. mol/L forward primer, 0.3. mu.L of 10. mu. mol/L reverse primer, 50ng of DNA template, and the balance ultrapure water.
PCR amplification reaction procedure: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45s in a cycle, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 45s for 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃. PCR amplification was performed on a PCR machine AB 9700.
1.2.3 capillary fluorescent electrophoresis examination analysis
The PCR products were denatured and analyzed by capillary fluorescence electrophoresis on an ABI 3130 sequencer, with 2 replicates per reaction.
1.2.4 statistical analysis
The data of the size of allelic variation of each sample at each site was read using the fragment analysis software of the DNA analyzer. As cauliflower is a codominant characteristic of a diploid and an SSR marker, the genotype of each test variety at each primer site is recorded as X/X by using the allelic variation of a homozygous site, and the allelic variation of a heterozygous site is recorded as X/Y, wherein X, Y is the sizes of two different allelic variation fragments at the site, a small fragment is in front of a large fragment, and a large fragment is behind the large fragment. The size of the null allelic variation was recorded as 0/0.
Polymorphism information amount PIC (polymorphic information content, PIC) for evaluation of primer Polymorphism level: 1-sigma P as PICi2 ijWhere Pi and Pj represent the frequency of the ith and jth alleles, respectively, and i ≠ j.
When PIC is more than or equal to 0.5, the primer is high polymorphism information primer, when PIC is more than or equal to 0.25 and less than 0.5, the primer is medium polymorphism information primer, and when PIC is less than 0.25, the primer is low polymorphism information primer.
Evaluation of degree of heterozygosity at the primer site indicators for heterozygosity:
the heterozygosity rate is the number of varieties with heterozygosity bands/total number of varieties on the primer site.
When the heterozygosis rate is more than or equal to 0.6, the primer is a high heterozygosis rate primer, and when the heterosis rate is more than or equal to 0.3 and less than 0.6, the primer is a medium heterosis rate primer;
the primer with low heterozygosity is used when the heterozygosity is less than 0.3.
Therefore, the primers with PIC more than or equal to 0.5 and clear bands and heterozygosity more than or equal to 0.6 are selected as SSR core primer groups for identifying the purity of cauliflower hybrids; selecting a primer with a clear band and 0.25-0.5 PIC and 0.3-0.6 heterozygosity as an alternative SSR core primer group for identifying the purity of cauliflower hybrids.
2. Results
2.1 selection of primers suitable for identifying purity of Cauliflower or broccoli hybrid
DNA fingerprint maps (shown in tables 3-4) of 70 varieties of cauliflower and broccoli are established by utilizing 22 pairs of SSR primers, and the gene frequency of the primers, basic information of gene distribution, polymorphism and heterozygosis performance are analyzed according to map data statistics, as shown in table 4.
Based on the detection results and analysis in tables 3 and 4, the primers were classified into 3 types according to the classification criteria:
(1) the primers with high polymorphism and high heterozygosity, namely PIC (positive temperature coefficient) is more than or equal to 0.5, and the heterozygosity is more than or equal to 0.6, and the clear bands are SSR core primer groups for identifying the purity of cauliflower hybrids: BoGMS0624, BoGMS0941, OI11G 11.
(2) In the polymorphism, in the heterozygosity, a primer with PIC of more than or equal to 0.25 and less than 0.5 and the heterozygosity of more than or equal to 0.3 and less than 0.6 is selected as an alternative SSR core primer group for identifying the purity of cauliflower hybrids: BoE607, BoE761, BoGMS2431, BoGMS0808, and BoGMS 1530.
(3) The other primers have low polymorphism or low heterozygosity, and are not selected.
PCR amplification, detection and polymorphism analysis are carried out on 70 cauliflower varieties (cauliflower and broccoli) by adopting three pairs of primers BoGMS0624, BoGMS0941 and OI11G11 according to the method 1.2, wherein 68 cauliflower varieties have heterozygous banding patterns (see Table 3), and 2 varieties (serial number 69: W131 and serial number 70: Pinus massoniana 20003 in Table 1) do not have heterozygous banding patterns, so that the purity of hybrids of 68 cauliflower varieties in the 70 cauliflower main-carried varieties can be identified by the screened three pairs of primers BoGMS0624, BoGMS0941 and OI11G11, which account for 97% of the cauliflower main-carried varieties, therefore, the three pairs of primers can be used as core primer groups for identifying the purity of cauliflower hybrids, and the three pairs of primers realize the identification of the cauliflower hybrids with the minimum genome coverage, thereby greatly reducing the workload, improving the detection efficiency and reducing the detection cost.
The remaining 2 cauliflower cultivars (W131 and Pinus densiflora 20003) were PCR amplified, detected and analyzed for polymorphisms according to the method 1.2 above using the 5 primers described in the second category, wherein the 2 cultivars detected by the primer BoE607 all had heterozygous banding patterns (see Table 3), indicating that: the second primer group BoE607, BoE761, BoGMS2431, BoGMS0808 and BoGMS1530 can be used as an alternative core primer group for identifying the purity of cauliflower hybrids.
3. Core primer validity verification for cauliflower hybrid purity identification
PCR amplification, capillary fluorescence detection and analysis are carried out on the parent and the parent of 2 cauliflower hybridization combinations (MS048-1-2 XC 18232) (No. 61: Pinus 1821 in the variety name table 3) and F1 (No. 63: Pinus 1963 in the variety name table 3) which are randomly selected by adopting 3 pairs of SSR primers (a primer BoGMS0624, a primer BoGMS0941 and a primer OI11G11) of the screened core primer group according to the 1.2 method, and the result shows that complementary bands can be screened from 1 pair of primers BoGMS0941 of the cauliflower hybridization combinations (MS048-1-2 XC 18232); complementary bands can be respectively screened by using 2 pairs of primers BoGMS0624 and OI11G11 in a cauliflower hybridization combination (MS048-1-2 XC 18199), so that 3 core primers BoGMS0624, BoGMS0941 and OI11G11 screened by the method have high effectiveness and can be used as a core primer group for identifying the purity of cauliflower hybrids.
As shown in fig. 1-2:
in FIG. 1, the amplified bands of the hybridization combinations MS048-1-2 XC 18232 and F1 (pine 1821) using the primers BoGMS0941 are shown, wherein a1 is the female parent MS048-1-2(234bp), a2 is the male parent C18232(224bp), and a3 is F1, wherein 2 complementary bands (224bp/234bp) of the male parent appear.
FIG. 2 shows the amplified bands of the hybridization combinations MS048-1-2 XC 18199 and F1 (pine 1963) using primer OI11G11, b1 as the female parent MS048-1-2(129bp), b2 as the male parent C18199(154bp), and b3 as F1 with 2 complementary bands of the male and female parents (129bp/154 bp).
4. Method for identifying purity of cauliflower hybrid
Further screening 3 pairs of core primers (BoGMS0624, BoGMS0941, and OI11G11) of the core primer set screened by the invention, screening hybrid purity identification primers corresponding to 68 cauliflower varieties in 70 main cultivation cauliflower varieties (Table 3) at present, respectively carrying out PCR amplification and capillary electrophoresis according to the method 1.2 in the embodiment 1, respectively analyzing the amplification products of each pair of primers, wherein the hybrid seeds are heterozygous gene bands, the selfed seedlings are homozygous gene bands, and the purity of the hybrid seeds is calculated according to the following formula: the purity of the hybrid is equal to the number of the hybrid/the total number of the detected seeds multiplied by 100%.
5. Conclusion
The SSR marking technology can distinguish the truth of a sample to be identified by detecting whether the sample has a gene specific fragment of a certain variety, so that the purity of the seed is detected, and the SSR marking technology has the advantages of rapidness, accuracy, stability, safety, no influence of environmental factors and the like. At present, SSR markers are widely applied to purity identification of hybrids of crops such as rice, cotton, corn and the like. The actual genotype number of the primers in the research is generally lower than the theoretical genotype number, the polymorphism is low, the average PIC value is 0.43, the heterozygosity rate is not high, and the average heterozygosity rate is 0.36, so that the narrow genetic background of the conventional cauliflowers is disclosed, and the genetic background is mainly concentrated on a plurality of backbone parents. This increases the difficulty of distinguishing hybrid species and is not conducive to detecting the purity of hybrid species.
The purity identification of hybrid seeds includes the first detection of hybrid seed mixing caused by selfing of female parent and mistaken harvesting of male parent, and the second identification of heterozygote caused by heterogenous pollen pollution or mechanical mixing. When the selfed seedlings are detected, 1 pair of biparental complementary primers are needed, and then the special-shaped strains are detected by using the specific primers or the primer combination. The primer heterozygosis rate is high, and the biparental complementary primer can be screened more easily to be used for detecting the selfed seedling; the primer has high polymorphism, can reduce the frequency of appearance of the same banding pattern, and is favorable for distinguishing the heterotypic strains.
In the experiment, 3 pairs of codominant SSR markers BoGMS0624, BoGMS0941 and OI11G11 are screened out and used as core primers for detecting the purity of cauliflower varieties. The 3 pairs of primers have high heterozygosis rate, 97 percent of cauliflower main cultivars can find primers with heterozygosis banding patterns, the requirement of selfing seedling inspection is met, and the 3 pairs of primers have high polymorphism and higher impurity inspection capability. Therefore, the 3 pairs of primers can be recommended as a first-choice core primer group for identifying the purity of cauliflower hybrids. The other 5 pairs of primers BoE607, BoE761, BoGMS2431, BoGMS0808 and BoGMS1530 with medium expression can be used as an alternative core primer group for identifying the purity of cauliflower hybrids and can be used for solving other varieties which cannot be identified by the 3 pairs of primers. At present, most of the existing researches are directed at screening of purity identification primers of specific varieties, however, the proper primers are selected from the core primer group or the alternative core primer set for identifying the purity of hybrid varieties of the specific varieties of cauliflowers, so that the workload can be greatly reduced, the detection efficiency can be improved, and the detection cost can be reduced.
TABLE 370 corresponding primers and genotypes for identifying the basic information of the cauliflower varieties to be tested and their hybrid purities
Note: in Table 3, two or more pairs of primers are shown for a certain variety of cauliflower, which indicates that the purity of hybrid species is identified by using any pair of primers, and that any pair of primers can achieve the effect of identifying the purity of hybrid species.
Table 4 basic evaluation information of SSR primers for 70 broccoli varieties in table 3
Sequence listing
<110> institute of food crops of academy of agricultural sciences of Yunnan province
Institute of tropical ecological agriculture, Yunnan Academy of Agricultural Sciences
Research on quality standard and detection technology of agricultural academy of sciences of Yunnan province
SSR core primer group for identifying purity of cauliflower hybrid and screening method and application thereof
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
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ccgttggaga tgactgactg 20
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<213> Artificial Sequence (Artificial Sequence)
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<400> 32
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Claims (10)
1. An SSR core primer group for identifying the purity of cauliflower hybrid seeds is characterized in that primers in the SSR core primer group are as follows: BoGMS0624, OI11G11, BoGMS 0941;
the BoGMS0624 consists of a BoGMS0624 forward primer and a BoGMS0624 reverse primer, and the base sequence of the BoGMS0624 forward primer is shown in SEQ ID NO: 1, the base sequence of the BoGMS0624 reverse primer is shown as SEQ ID NO: 2 is shown in the specification;
the OI11G11 consists of an OI11G11 forward primer and an OI11G11 reverse primer, and the base sequence of the OI11G11 forward primer is shown in SEQ ID NO: 3, the base sequence of the OI11G11 reverse primer is shown in SEQ ID NO: 4 is shown in the specification;
the BoGMS0941 consists of a BoGMS0941 forward primer and a BoGMS0941 reverse primer, and the base sequence of the BoGMS0941 forward primer is shown as SEQ ID NO: 5, the base sequence of the BoGMS0941 reverse primer is shown as SEQ ID NO: 6 is shown in the specification;
the cauliflower is cauliflower or broccoli.
2. An alternative SSR core primer group for identifying cauliflower hybrid purity is characterized in that primers in the alternative SSR core primer group are as follows: BoE607, BoE761, BoGMS2431, BoGMS0808, BoGMS 1530;
the BoE607 consists of a BoE607 forward primer and a BoE607 reverse primer, and the base sequence of the BoE607 forward primer is shown in SEQ ID NO: 7, the base sequence of the BoE607 reverse primer is shown as SEQ ID NO: 8 is shown in the specification;
the BoE761 consists of a BoE761 forward primer and a BoE761 reverse primer, and the base sequence of the BoE761 forward primer is shown in SEQ ID NO: 9, the base sequence of the BoE761 reverse primer is shown as SEQ ID NO: 10 is shown in the figure;
the BoGMS2431 consists of a BoGMS2431 forward primer and a BoGMS2431 reverse primer, and the base sequence of the BoGMS2431 forward primer is shown as SEQ ID NO: 11, the base sequence of the BoGMS2431 reverse primer is shown as SEQ ID NO: 12 is shown in the specification;
the BoGMS0808 consists of a BoGMS0808 forward primer and a BoGMS0808 reverse primer, and the base sequence of the BoGMS0808 forward primer is shown as SEQ ID NO: 13, the base sequence of the BoGMS0808 reverse primer is shown as SEQ ID NO: 14 is shown in the figure;
the BoGMS1530 consists of a BoGMS1530 forward primer and a BoGMS1530 reverse primer, and the base sequence of the BoGMS1530 forward primer is shown as SEQ ID NO: 15, the base sequence of the BoGMS1530 reverse primer is shown as SEQ ID NO: 16 is shown in the figure;
the cauliflower is cauliflower or broccoli.
3. A screening method of SSR core primer group or alternative core primer group for identifying purity of cauliflower hybrid is characterized by comprising the following steps:
(1) respectively extracting DNA of all cauliflower varieties to be detected;
(2) respectively taking the DNA of each cauliflower variety obtained in the step (1) as a template, and respectively carrying out PCR amplification on the DNA of each cauliflower variety by using 22 pairs of SSR primers shown in the table 1;
TABLE 122 pairs of SSR primer sequences
(3) Performing polymorphism and heterozygosis performance analysis on a PCR amplification product by adopting capillary electrophoresis, selecting a core primer group or an alternative core primer group according to the polymorphism information content PIC and the heterozygosity rate of the primer, wherein the primer is a high polymorphism information primer when the PIC is more than or equal to 0.5, a medium polymorphism information primer when the PIC is more than or equal to 0.25 and less than 0.5, the primer is a high heterozygosity rate primer when the heterozygosity rate is more than or equal to 0.6, and a medium heterozygosity rate primer when the heterozygosity rate is more than or equal to 0.3 and less than 0.6; selecting a primer with PIC more than or equal to 0.5 and clear bands with heterozygosis rate more than or equal to 0.6 as an SSR core primer group for identifying the purity of cauliflower hybrids; selecting a primer with 0.25-0.5 PIC and 0.3-0.6 heterozygosity rate as an alternative SSR core primer group for identifying the purity of cauliflower hybrids.
4. The screening method according to claim 3, wherein the reaction system of the PCR amplification in the step (2) is 10. mu.L in total, wherein 5. mu.L of a2 x mix solution, 0.3. mu.L of a 10. mu. mol/L forward primer, 0.3. mu.L of a 10. mu. mol/L reverse primer, 50ng of a DNA template, and the balance of ultra pure water are made up; the reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 45s in 35 cycles; extending at 72 ℃ for 10min, storing at 4 ℃, and labeling TAMRA, HEX, ROX or 6-FAM fluorescent group at the 5' end of the forward primer in each pair of primers for PCR amplification.
5. The use of the SSR core primer set for cauliflower hybrid purity identification according to claim 1 in cauliflower hybrid purity identification.
6. The use according to claim 5, wherein the SSR primers used for the purity identification of the identified cauliflower cultivars and each cauliflower cultivar hybrid are shown in Table 2:
TABLE 2 SSR primers for purity identification of cauliflower variety hybrids
7. Use of the alternative SSR core primer set for cauliflower hybrid purity identification according to claim 2 in cauliflower hybrid purity identification.
8. The use according to claim 7, wherein the primer pair for identifying the purity of the cauliflower subsous 20003 hybrid is BoE 607; or the primer pair for identifying the purity of the cauliflower W131 hybrid is BoE 607.
9. The use of any one of claims 5 to 8, further comprising a total of 10 μ L of reaction system for PCR amplification of each pair of primers in the cauliflower hybrid purity determination, wherein 5 μ L of 2 x mix solution, 0.3 μ L of 10 μmol/L forward primer, 0.3 μ L of 10 μmol/L reverse primer, 50ng of DNA template, and the balance of ultrapure water; the reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 45s in 35 cycles; extending for 10min at 72 ℃, storing at 4 ℃, and marking TAMRA, HEX, ROX or 6-FAM fluorescent group at the 5' end of each pair of primers for PCR amplification; analyzing the amplified products of each pair of primers by capillary electrophoresis, wherein the true hybrid with a heterozygous gene band and the false hybrid with a homozygous gene band are respectively obtained, and the purity of the hybrid is calculated according to the following formula: the purity of hybrid is equal to the number of true hybrid/total number of seeds tested x 100%.
10. The use of the SSR core primer group for purity identification of cauliflower hybrids as claimed in claim 1 in genetic diversity analysis of cauliflower germplasm resources or identification of cauliflower variety genetic relationship; or the optional SSR core primer group for cauliflower hybrid purity identification according to claim 2, in genetic diversity analysis of cauliflower germplasm resources or identification of cauliflower variety genetic relationship.
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