CN117230234A - InDel molecular marker for identifying homozygous purple broccoli and application thereof - Google Patents
InDel molecular marker for identifying homozygous purple broccoli and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular marker assisted breeding, and particularly provides an InDel molecular marker for identifying homozygous purple broccoli and application thereof. The InDel molecular marker related to the purple property of the broccoli is obtained by amplifying a specific primer with a sequence shown as SEQ ID NO. 2-3. The InDel molecular marker provided by the invention can be used for distinguishing and identifying homozygous and heterozygous purple broccoli and green broccoli, and has the advantages of accuracy and high efficiency. The InDel molecular marker provided by the invention is used for molecular marker assisted selection of homozygous purple broccoli breeding, and has important significance in actual production.
Description
Technical Field
The invention relates to the technical field of molecular marker assisted breeding, in particular to an InDel molecular marker for identifying homozygous purple broccoli and application thereof.
Background
The green pedunculate (Brassica campestris L. Ssp. Chinese Makino var. Com Tsen et Lee) belongs to a variety of brassica rapa cabbage subspecies of Brassicaceae, and can be divided into green peduncles and white peduncles according to the leaf stalk color, and the leaf color of the traditional green peduncles cultivar is green with different degrees. The green pedunculate vegetable has a long cultivation history in China and is rich in genetic resources. Especially in the Yangtze river basin of China, the broccoli occupies an important position in the main cultivated varieties of leaf vegetables, and has important economic value in annual production and supply.
Purple green peduncles are high-end special dishes introduced from abroad in the vegetable market of China in recent years, are purple due to the fact that the leaves of the edible part accumulate rich anthocyanin, and have health-care functions of resisting oxidation, preventing cancers and the like, so that the purple green peduncles are increasingly favored by people. The purple green pedunculate vegetable has high economic added value, and the cultivation of the purple green pedunculate vegetable with rich types becomes a recent attention goal of breeding workers.
How to obtain homozygous purple green peduncles is a primary premise for obtaining breeding target parents, and practical work discovers that it is difficult to distinguish the purple green peduncles menu strains with homozygous and heterozygous genes through field trait observation. The main reason is that the leaf of the homozygous and heterozygous purple broccoli has anthocyanin accumulation, the upper surface of the leaf is purple, and the green broccoli is green (c of figure 1).
Anthocyanin is a flavonoid secondary metabolite, and can be accumulated in plant tissues to enable plants to be purple, red, blue and other different colors. Anthocyanin can protect plant DNA from being damaged in plants, and assist the defense function of plants in adverse conditions such as low temperature, drought and the like. Anthocyanin is used as one of the most important secondary metabolites in plants, has physiological functions in the plants and is beneficial to human health. Anthocyanin is an antioxidant, helps human to delay aging and prevents senile dementia. Meanwhile, the most effective free radical scavenger is used for protecting the human body from being damaged by free radical harmful substances and helping the human body to prevent various diseases related to the free radicals.
At present, the positioning and functional analysis of related genes of anthocyanin accumulation in brassica plants become a research hot spot, and the related research of purple genes of Chinese cabbage vegetables has been advanced to a certain extent. It has been reported that related genes for regulating purple traits of cabbage vegetables are located on chromosomes A02, A03, A07, A09 and the like. However, different research results prove that the purple character regulation and control of the cabbage vegetables have different quality characters and quantitative characters, and the related path for synthesizing the anthocyanin of the cabbage vegetables is complex.
Plant anthocyanin biosynthesis is mainly related to structural genes and regulatory genes. Structural genes include Early Biosynthesis Genes (EBGs) and Late Biosynthesis Genes (LBGs). The role of regulatory genes in the regulation of the transcriptional level of structural genes has been identified in many plant species, and the transcription factors encoded by regulatory genes can regulate the temporal and spatial expression of structural genes. The key genes for regulating and controlling the leaf color purple and green of the broccoli and synthesizing anthocyanin are not reported in the prior study.
In recent years, molecular marker assisted screening has become one of effective auxiliary technologies for crop molecular breeding, and is increasingly used in breeding practice. The molecular marker is a genetic marker based on polymorphism of biological macromolecular nucleic acid, comprises a traditional RFLP, RAPD, SSR, AFLP marker, more efficient SNP, inDel and the like, and has the advantages of large quantity, good polymorphism, difficult interference, no influence on target property expression and the like. The molecular markers closely linked with the plant target genes are developed and utilized, and in a large-population material, the target character single plants can be accurately selected, so that the efficiency of breeding work can be effectively improved.
The leaf color of the purple green peduncles is affected by various conditions, such as temperature, illumination and the like, and by utilizing a field character observation and identification method, the purple green peduncles menu strains with heterozygous or homozygous genes are difficult to distinguish by naked eyes. By utilizing molecular Marker Assisted Selection (MAS), a marker closely linked with purple characters of the green-stem vegetable leaves is developed, homozygous and heterozygous green-stem vegetables can be screened in a plant seedling stage, the stability of homozygous parents of the purple green-stem vegetables can be effectively accelerated, and the method has important significance in shortening the breeding process, saving the breeding cost and accelerating the promotion of the breeding work of the purple green-stem vegetables in China.
Disclosure of Invention
The invention obtains the hybrid F by taking leaf purple green peduncle vegetable and common green peduncle vegetable as parents according to the re-sequencing data of the leaf purple green peduncle vegetable and the common green peduncle vegetable 1 F (F) 1 F obtained by selfing 2 Sequencing data from the pool of mixed populations identified a deletion or insertion mutation that was a polymorphic marker associated with the purple broccoli leaf color gene. Based on the above, the invention develops an InDel molecular marker, and aims to identify homozygous or heterozygous purple green stem menu strains by PCR amplification of the band difference.
In a first aspect, the invention provides an InDel molecular marker gene fragment related to purple traits of broccoli leaves, wherein polymorphism of the InDel molecular marker gene fragment is a deletion/insertion sequence, and the InDel molecular marker gene fragment is amplified by a specific primer with a sequence shown as SEQ ID NO. 2-3.
Forward primer: TTCTTGTGCCGAGCATGGAT (SEQ ID NO. 2);
reverse primer: GTGTGTTTTCAGGACGGTGC (SEQ ID NO. 3).
The nucleotide sequence of the insertion sequence of the InDel molecular marker gene fragment provided by the invention is shown as SEQ ID NO. 1.
The forward primer and the reverse primer cover the region to be amplified, which is the whole sequence of the special sequence of the homozygous purple green pedunculate She Sexiang gene disclosed by the invention, and is tightly linked with the purple gene of the green pedunculate.
In a second aspect, the invention provides a specific primer pair for detecting the InDel molecular marker gene fragment, wherein the sequence of the specific primer pair is shown as SEQ ID NO. 2-3.
In a third aspect, the present invention provides a kit comprising a specific primer pair as described above.
The kit also includes other reagents for PCR amplification including, but not limited to, PCR reaction buffers, DNA polymerase, dNTPs, ddH 2 O, etc.
In a fourth aspect, the invention provides the application of the InDel molecular marker gene fragment or the specific primer pair or the kit in the identification or auxiliary identification of homozygous purple and heterozygous purple properties of broccoli leaves.
The invention provides application of the InDel molecular marker gene fragment or the specific primer pair or the kit in early screening of green-stem vegetable seedling stage and predicting leaf color in the adult plant stage.
And the application of the InDel molecular marker gene fragment or the specific primer pair or the kit in homozygous purple broccoli genetic breeding.
The application of the identification gene homozygous leaf purple broccoli genetic breeding provided by the invention is based on the amplification result of the primer pair shown in SEQ ID NO.2-3, and the homozygous leaf purple broccoli material can be obtained by screening in the early seedling stage of planting for genetic breeding.
Under the influence of cultivation environments such as temperature and illumination, if the green-stem vegetables which are all purple in the leaves are observed only through the field characters, the green-stem vegetables are homozygous single plants (a of fig. 1) or heterozygous single plants (b of fig. 1) which are difficult to distinguish accurately. The invention provides an accurate and efficient method for screening homozygous purple broccoli by using molecular markers.
In a fifth aspect, the invention provides a method for identifying the leaf color purple character as homozygous green-stem dish, which comprises the steps of carrying out PCR (polymerase chain reaction) amplification on green-stem dish DNA by utilizing a specific primer with a sequence shown as SEQ ID NO.2-3, and identifying the green-stem dish to be detected as homozygous green-stem dish if an amplification product of 1063bp exists through agarose gel electrophoresis analysis; if the amplification product of 250bp exists, identifying the green broccoli to be detected as green broccoli; if two amplification products of 250bp and 1063bp exist in the amplification, the purple broccoli to be detected is identified as heterozygous purple broccoli.
In the method for identifying the leaf color purple character as homozygous broccoli, the PCR amplification reaction procedure is 95 ℃ for 3min;95 ℃, 15s,56-60 ℃, 15s,72 ℃, 20-40s,30-35 cycles; 72 ℃ for 5min; in the reaction system of PCR amplification, the concentration of the forward primer and the reverse primer in the specific primer pair is 10 pmol/. Mu.L.
Preferably, the reaction system of the PCR amplification is as follows: 2xRapid Taq Master Mix5. Mu.L, forward primer (10. Mu.M) 0.2. Mu.L, reverse primer(10μM)0.2μL，gDNA 1μL，ddH 2 O 3.6μL。
As a specific embodiment of the invention, as a preferable scheme of the invention, the method for identifying the purple property of the broccoli comprises the following steps:
(1) Genomic DNA is extracted from a plant sample to be tested.
(2) Carrying out PCR amplification by adopting a primer shown in SEQ ID No.2-3, detecting whether 1063bp products exist in the PCR amplification products by agarose gel electrophoresis, and if 1063bp amplification products exist, identifying the to-be-detected broccoli as homozygous purple broccoli; if the amplification product of 250bp exists, identifying the green broccoli to be detected as green broccoli; if amplification products of 250bp and 1063bp exist, identifying the broccoli to be detected as heterozygous purple broccoli.
In the step (1), the plant sample to be tested may be the whole plant or a part of the plant, such as leaf, stem, etc. Plant tender leaves are preferably used in the invention.
The green peduncles in the invention refer to green peduncles of brassica vegetables in brassicaceae. The purple broccoli refers to a broccoli with leaf purple character.
Detection of PCR amplification products may be accomplished by techniques conventional in the art, such as electrophoresis detection, DNA sequencing, and the like. The invention preferably uses agarose gel electrophoresis method to detect.
The invention has the beneficial effects that:
the InDel molecular marker related to the purple property of the green peduncle vegetable leaves can be used for distinguishing and identifying homozygous and heterozygous purple peduncle vegetable and green peduncle vegetable, has high accuracy, can be used as a molecular marker auxiliary selection tool for breeding of homozygous purple peduncle vegetable parents, and has important significance in breeding practice.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photograph illustration of the leaf color traits of green pedunculate vegetable seedling stage to be identified in the present invention. Wherein a is homozygous purple broccoli, b is heterozygous purple broccoli, c is green broccoli, d is F 2 And (5) separating group field photographs.
FIG. 2 shows the result of electrophoresis of PCR amplification products of purple homozygous, heterozygous broccoli and green broccoli in example 2 of the present invention, wherein the bands of DNAmaroker are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.
FIG. 3 is F in example 3 of the present invention 2 And screening and identifying agarose gel electrophoresis detection results of the generation group materials are shown in a schematic diagram. The DNAmarker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom.
FIG. 4 shows the result of agarose gel electrophoresis of random purple broccoli samples in example 4 of the present invention. The DNA marker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 development of InDel molecular markers related to homozygous purple trait of broccoli
In order to better and faster screen and obtain homozygous purple green peduncle resources, promote the molecular marker to assist in the implementation of the selective breeding practice, the resequencing data of leaf purple green peduncle and common green peduncle and offspring F are obtained 2 Sequencing data analysis of the population pool found an insertion sequence leading to a change in leaf color of broccoliAnd the insertion sequence is tightly linked with the purple gene of the broccoli.
According to the found difference gene fragments of the purple green cabbages and the green cabbages, the forward InDel BrBur-F is designed, the sequence is shown as SEQ ID NO.2, and the reverse primer InDel BrBur-R is shown as SEQ ID NO. 3. The forward and reverse primers are used for amplifying the green-stem gDNA, and the length of a fragment obtained by amplification in the homozygous purple green-stem gDNA is 1063bp (the sequence is shown as SEQ ID NO. 1).
Example 2 method for identifying leaf color of broccoli Using InDel molecular marker
The embodiment provides a method for identifying the leaf color of broccoli by using the InDel molecular marker of the embodiment 1, which is specifically as follows:
1. extracting genomic DNA (gDNA) from a plant sample to be tested;
(1) Taking a leaf sample of the broccoli with the size of a nail cover, placing the leaf sample into a 2ml centrifuge tube, adding 2 steel balls with the size of 4mm, and adding 200 mu LTPS solution;
(2) Crushing the mixture in the step (1) for 40s in a 40Hz crusher;
(3) 600 μLTPS was added to a 2ml centrifuge tube and water-bath at 75deg.C for 30min;
(4) Centrifuging at 12000rpm for 10min;
(5) Adding isopropyl alcohol with equal volume into the supernatant, and standing at-20deg.C for more than 0.5 hr;
(6) Centrifuging at 12000rpm for 5min, and removing supernatant;
(7) Adding 1ml of 75% alcohol, standing for 10min, centrifuging at 12000rpm for 2min to remove alcohol, and drying;
(8) 100ul of ddH was added 2 O, uniformly mixing, and preserving at-20 ℃. The concentration of the DNA to be detected is 100-200 ng/. Mu.L.
2. PCR amplification
The PCR amplification reaction system was 2xRapid Tag Master Mix:5. Mu.L, forward primer F0.2. Mu.L, reverse primer R0.2. Mu.L, gDNA 1. Mu.L, ddH 2 O:3.6μL。
The PCR amplification procedure was as follows: 95 ℃ for 3min;95 ℃, 15s,56 ℃, 15s,72 ℃, 40s,35 cycles; 72 ℃ for 5min.
3. Detection and characterization of PCR amplified products
The PCR amplification product is detected by agarose gel electrophoresis, and the agarose gel preparation and electrophoresis operation steps are as follows:
(1) Configuration of 1.5% agarose gel
(1) 1.5g agarose was weighed and 100ml of 0.5% TAE buffer was added;
(2) heating until agarose is completely dissolved, adding 7 mu L of nucleic acid dye, and uniformly mixing;
(3) immediately pouring glue and inserting a comb;
(4) the gel was allowed to solidify at room temperature.
(2) Agarose gel electrophoresis
Electrophoresis was performed using the gel prepared in (1) under 140V for 20min. Judging whether a 1063bp product exists in the PCR amplification product, and if the 1063bp amplification product exists, identifying the to-be-detected broccoli as homozygous purple broccoli; if the amplification product of 250bp exists, identifying the green broccoli to be detected as green broccoli; if amplification products of 250bp and 1063bp exist, the broccoli to be detected is identified as heterozygous purple broccoli (figure 2).
Example 3 identification of the leaf color of broccoli Material for BSA-seq Using InDel molecular markers
Pair P using the procedure of example 2 1 (homozygous purple broccoli parent), P 2 (homozygous green broccoli parent), F 1 (P 1 And P 2 Obtained by hybridization) and F 2 (F 1 Selfing) randomly selecting 49 parts of materials in a seedling stage, identifying, respectively carrying out PCR amplification by using gDNA (deoxyribonucleic acid) of leaf materials of different green peduncles as templates and using primers shown as SEQ ID NO.2-3, wherein the electrophoresis detection result is shown in a figure 3, and 9 parts of materials (No. 1, 2, 8, 14, 15, 30, 34, 37 and 38) have PCR products with the length of 1063bp, so that the characteristics of the homozygous purple green peduncles are met; wherein 8 parts of materials (No. 3, no. 4, no. 44-49) show PCR products with the length of 250bp, which accord with the characteristics of green broccoli; wherein, 32 parts of materials (No. 5-7, no. 9-13, no. 16-29, no. 31-33, no. 35, no. 36, no. 39-43) show PCR products with the lengths of 250bp and 1063bp, which accord with the characteristics of heterozygous purple broccoli.
Identifying the molecular markerThe fixed result corresponds to the actual plant and the leaf color is identified, and the result shows that the single plants 1 and 2 are purple broccoli DH lines and are homozygous materials; individual plants 8, 14, 15, 30, 34, 37, 38 are F 2 Substituting a single plant, enabling leaves to show purple, enabling agarose gel electrophoresis results to be consistent with homozygous purple DH lines of numbers 1 and 2, and identifying the homozygous purple broccoli; the numbers 3 and 4 are the high-generation inbred lines of the homozygous green-leaf broccoli, and the single plants 44-49 are F 2 The green leaf-replacing green pedunculate vegetable phenotypic material and the agarose gel electrophoresis result are consistent with the green leaf color phenotype. Number 5 and 6 are F 1 The plant is a hybrid purple broccoli, and the agarose gel electrophoresis result is consistent with that of the hybrid purple broccoli; materials 7, 9-13, 16-29, 31-33, 35, 36, 39-43 are F 2 The generation of single plants is She Sebiao, the purple color is achieved, and agarose gel electrophoresis results are consistent with those of single plants 5 and 6, and the heterozygous purple broccoli is identified. Therefore, inDel BrBur-F and InDel BrBur-R can be used as markers for accurately distinguishing whether the leaf color of the broccoli material is homozygous, heterozygous purple or green.
Example 4 identification of purchased and introduced broccoli resources and other purple broccoli Using InDel molecular markers
8 parts of purple and green broccoli materials were identified by the method of example 2, and the numbers were 1 to 8, wherein the number 7 was the product of Zixiu broccoli commercial variety produced by Junde seed Limited of Zhangzhou, market acquisition and the number 8 was the product of Zibao broccoli commercial variety produced by Junde seed Limited of Zhangzhou, market acquisition.
Respectively carrying out PCR amplification by using leaf material gDNA of different green pedunculate seedling stages as templates and primers shown in SEQ ID NO.2-3, wherein the electrophoresis detection result is shown in figure 4: wherein 6 parts of materials (No. 1 and No. 3-7) have PCR products with the length of 1063bp, and the materials are identified as homozygous purple broccoli; wherein, 1 part of material (number 2) shows a PCR product with the length of 250bp, which is green broccoli; wherein 1 part of the material (No. 8) shows PCR products with the lengths of about 250bp and 1063bp, and the PCR products are identified as heterozygous purple broccoli.
Comparing the molecular marker identification result with She Sebiao type in plant field, the result shows that the numbers 1 and 3-6 are purple broccoli DH line (figure 4). And the number 2 is a high-generation inbred line of the green leaf broccoli. And the numbers 7 and 8 are purple broccoli introduced in the market. The identification shows that the number 7 is the introduction of the homozygous purple broccoli and the number 8 is the introduction of the heterozygous purple broccoli. Therefore, inDel BrBur-F and InDel BrBur-R can be used for identifying purple green vegetables purchased in the market and are applied as screening marks for distinguishing homozygous or heterozygous purple green vegetable resource introduction materials.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The InDel molecular marker gene fragment related to the purple property of the broccoli is characterized in that the polymorphism of the InDel molecular marker gene fragment is a deletion/insertion sequence, and the InDel molecular marker gene fragment is amplified by a specific primer with a sequence shown as SEQ ID NO. 2-3.
2. The InDel molecular marker gene segment according to claim 1, wherein the nucleotide sequence of said insert sequence is shown in SEQ ID No. 1.
3. A specific primer pair for detecting the InDel molecular marker gene fragment according to any one of claims 1-2, wherein the sequence of the specific primer pair is shown as SEQ ID No. 2-3.
4. A kit comprising the specific primer pair of claim 3.
5. Use of the InDel molecular marker gene fragment of any one of claims 1-2 or the specific primer pair of claim 3 or the kit of claim 4 in the identification or auxiliary identification of homozygous purple, heterozygous purple traits of broccoli.
6. Use of the InDel molecular marker gene fragment of any one of claims 1-2 or the specific primer pair of claim 3 or the kit of claim 4 for early screening and predicting leaf color in the adult stage of broccoli seedling stage.
7. Use of the InDel molecular marker gene fragment of any one of claims 1-2 or the specific primer pair of claim 3 or the kit of claim 4 in homozygous green-stem dish genetic breeding.
8. The use according to claim 7, wherein the homozygous purple broccoli can be identified and selected for genetic breeding in the seedling stage based on the amplification result of the primer pair shown in SEQ ID NO. 2-3.
9. A method for identifying the leaf color purple character as homozygous green-stem vegetable is characterized in that a specific primer with a sequence shown as SEQ ID NO.2-3 is utilized to carry out PCR amplification on the DNA of the green-stem vegetable, and if an amplification product of 1063bp exists, the green-stem vegetable to be detected is identified as homozygous green-stem vegetable; if the amplification product of 250bp exists, identifying the green broccoli to be detected as green broccoli; if two amplification products of 250bp and 1063bp exist in the amplification, the purple broccoli to be detected is identified as heterozygous purple broccoli.
10. The method of claim 9, wherein the PCR amplification reaction is performed at 95℃for 3min;95 ℃, 15s,56-60 ℃, 15s,72 ℃, 20-40s,30-35 cycles; 72 ℃ for 5min; in the reaction system of PCR amplification, the concentration of the forward primer and the reverse primer in the specific primer pair is 10 pmol/. Mu.L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311151429.6A CN117230234A (en) | 2023-09-07 | 2023-09-07 | InDel molecular marker for identifying homozygous purple broccoli and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311151429.6A CN117230234A (en) | 2023-09-07 | 2023-09-07 | InDel molecular marker for identifying homozygous purple broccoli and application thereof |
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| CN202311151429.6A Pending CN117230234A (en) | 2023-09-07 | 2023-09-07 | InDel molecular marker for identifying homozygous purple broccoli and application thereof |
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