Application of LFY gene second intron polymorphism in identification of platanus acerifolia variety

Abstract

The invention relates to the technical field of identification of platanus acerifolia varieties, and particularly provides application of polymorphism of a second intron of an LFY gene in identification of platanus acerifolia varieties. A large number of experimental exploration finds that amplification of a specific primer pair developed by aiming at the polymorphism of the second intron of the LFY gene can rapidly distinguish the platanus variety and the genetic relationship of the platanus variety.

Description

Technical Field

[0001] The invention relates to the technical field of identification of plane tree varieties, and in particular to application of the second intron polymorphism of LFY gene in identification of plane tree varieties. Background Art

[0002] The sycamore is a deciduous tree of the genus Platanus in the family Platanaceae of the order Proteaceae. It has a relatively primitive evolutionary status. This family is a small family with only one genus and about 11 species. The sycamore introduced for cultivation in my country is mainly the Platanus diglossa variety, which is a hybrid of the Platanus triglossa and the Platanus uniglossa. The Platanus diglossa is widely used as a street tree because of its advantages such as large crown and dense shade, strong adaptability, rapid growth, and excellent resistance, and enjoys the reputation of "the king of street trees". However, its "fruit drop" problem has always been a defect that affects people's health, and cultivating sycamores with few or no fruits can solve this problem well.

[0003] At present, Wuhan Academy of Landscape Science and other institutions have cultivated a variety of less-fruiting sycamore varieties. The selection and promotion of excellent plant varieties is the premise and foundation for achieving high-yield and high-quality agriculture, and variety identification is an important part of it. As a woody plant, sycamore has a large genome and a long traditional breeding time. How to effectively identify less-fruiting sycamore varieties and their genetic relationships is a technical problem that needs to be solved urgently. Summary of the invention

[0004] Based on this, it is necessary to provide the application of the second intron polymorphism of the LFY gene in identifying sycamore varieties, which can quickly and effectively identify sycamore varieties and their genetic relationships.

[0005] The present invention adopts the following technical solution:

[0006] The invention provides application of the second intron polymorphism of LFY gene in identifying plane tree varieties and their genetic relationships.

[0007] The present invention provides a primer pair for identifying a sycamore variety, wherein the sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; or the sequences of the primer pair are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The present invention can also provide a kit for identifying a sycamore variety, comprising the primer pair for identifying a sycamore variety.

[0008] The present invention can also provide a method for identifying sycamore varieties, comprising the following steps: obtaining a sycamore sample and extracting DNA; amplifying using the primer pair for identifying sycamore varieties, performing gel electrophoresis on the amplified product, and determining the sycamore variety and its genetic relationship through the gel electrophoresis bands:

[0009] When the gel electrophoresis shows a single band smaller than 1000 bp, the LFY gene sequence of the sycamore variety is determined to be from the sycamore gene PaC3G140170, which is 'Yuanke 115', 'Yuanke 23' or 'Sanqiu Platanus';

[0010] When the gel electrophoresis showed a single band larger than 1000 bp, it was determined that the LFY gene sequences in the sycamore varieties were all from the Platanus triloba gene Platanus32797-RA, which was 'Yuan Ke 12' or 'Yiqiu Platanus';

[0011] When the gel electrophoresis shows double bands, and one is greater than 1000bp and the other is less than 1000bp, it is determined that the two LFY gene sequences in the sycamore variety come from the sycamore gene PaC3G140170 and the sycamore gene Platanus32797-RA respectively; if the gel electrophoresis shows double bands (981bp and 1053bp), the sycamore variety is determined to be 'Yuanke 10'; if the gel electrophoresis shows double bands (981bp and 1083bp), the sycamore variety is determined to be 'Yuanke 22' or 'Susheng Fatong'.

[0012] The present invention can also provide a primer pair for identifying a sycamore variety, the sequences of which are respectively shown as SEQ ID NO: 5 and SEQ ID NO: 6. Furthermore, the present invention can also provide a kit for identifying a sycamore variety, comprising the primer pair for identifying a sycamore variety.

[0013] The present invention can also provide a method for identifying sycamore varieties, comprising the following steps:

[0014] Obtain plane tree samples and extract DNA;

[0015] A primer pair for identifying sycamore varieties (sequences are SEQ ID NO: 5 and SEQ ID NO: 6, respectively) was used for amplification, and the amplified product was detected by agarose electrophoresis. For samples with correct and clear bands, capillary electrophoresis was performed to determine the sycamore varieties and their genetic relationships:

[0016] When the test results show only a single peak of 217 bp, it indicates that the LFY gene sequences in the sycamore varieties are all from the sycamore gene PaC3G140170, which is the 'Yuanke 115' sample, the 'Yuanke 23' sample or the 'Sanqiu Platanus' sample;

[0017] When the test results show two peaks and contain a single peak of 217 bp, it indicates that the two LFY gene sequences in the sycamore variety are from the sycamore gene PaC3G140170 and the sycamore gene Platanus32797-RA, respectively, and are samples of 'Yuanke 10', 'Yuanke 22' or 'Susheng Fatong';

[0018] When the test results show two peaks and do not contain a single peak of 217 bp, it indicates that the LFY gene sequences in the sycamore varieties are all from the Platanus triglossum gene Platanus32797-RA, which is the 'Yuan Ke 12' sample or the 'Platanus uniglossum' sample.

[0019] Compared with the prior art, the core technical contributions of the present invention are:

[0020] The present invention has found through a large number of experimental studies that the second intron of the LFY gene has polymorphism, and the specific primer pair amplification developed for it can quickly distinguish the varieties of plane trees and their genetic relationships. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram of the LFY gene structure.

[0022] Figure 2 Gel electrophoresis diagram of the second intron amplification product of LFY gene in different varieties of Platanus ovata; M is a marker molecular weight marker; samples 1 to 27 from left to right refer to 'Yuanke 115', 'Yuanke 10', 'Yuanke 12', 'Yuanke 22', 'Yuanke 23', 'Plantano occidentalis', 'Plantano occidentalis' and 'Fasusheng Fatong'.

[0023] Figure 3 This is a comparison of the sequencing results of the second intron of the LFY gene of eight Platanus samples amplified using primer pair S257 / S258.

[0024] Figure 4 This is the capillary electrophoresis of the products of 'Yuanke 115' and 'Yuanke 10' amplified using primer pair S424 / S325. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly. The following embodiments are only used to illustrate the present invention, but are not only used to limit the scope of the present invention. Based on the specific embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0026] Sources of some sycamore leaf samples:

[0027] Plane tree species Get the source Test No. Garden Science 115 Wuhan Academy of Landscape Architecture 1 Garden Section 10 Wuhan Academy of Landscape Architecture 10 Garden Science 12 Wuhan Academy of Landscape Architecture 12 Garden Section 22 Wuhan Academy of Landscape Architecture 22 Garden Section 23 Wuhan Academy of Landscape Architecture 23 Plane tree (hybrid) Shanghai Chenshan Botanical Garden 24 Plane tree (hybrid) Hubei Wuhan Tanhualin 25 Fast-growing sycamore Huangpu Road, Wuhan, Hubei 27

[0028] Example 1

[0029] The improved CTAB method (see Li et al 2008: Li Z, Liu G, Zhang J, Zhang J, Bao M. Extraction of high-quality tissue-specific RNA from London plane trees (Platanus acerifolia), permitting the construction of a female inflorescence cDNA library. Funct Plant Biol, 2008, 35: 159-165) was used to extract genomic DNA from 8 plane trees (Yuanke 115, Yuanke 10, Yuanke 12, Yuanke 22, Yuanke 23, Platanus trifoliata, Platanus acerifolia, and Platanus acerifolia) for testing. The extraction quality was detected by 1% agarose gel electrophoresis and the concentration was detected by Biospec-nano microspectrophotometer. After the detection, the concentration was uniformly diluted to 50 ng / μL and stored in a -20°C refrigerator for use.

[0030] Example 2

[0031] The LFY gene sequence was downloaded from the published Platanus genome CoGe (genome number: 67387). There are two LFY gene sequences in the genome, one from Platanus uniflorus and the other from Platanus trilobus. Both LFY gene sequences consist of three exons and two introns. The exon sequences are identical, and the only difference is in the intron sequence. The first intron of the LFY gene sequence from Platanus uniflorus (PaC3G140170) is 221 bp long, and the second intron (intron2) is 7896 bp long; the first intron (intron2) of the LFY gene sequence from Platanus trilobus (Platanus32797-RA) is 213 bp long, and the second intron (intron2) is 8147 bp long, see Figure 1 .

[0032] Primer 6 software was used to design and screen two primer pairs (S257 / S258, S262 / S263) for amplifying the intron2 partial sequence of the LFY gene. The primer sequences were synthesized by Sangon Biotech Co., Ltd.

[0033] Wherein, the sequences of the first primer pair and the second primer pair are shown in the following table:

[0034]

[0035] The eight test sample DNAs obtained in Example 1 were used as templates, and the first primer pair and the second primer pair were used to amplify the partial sequence of intron2 of the LFY gene.

[0036] The PCR reaction system is shown in the following table:

[0037]

[0038]

[0039] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 1 min, for a total of 30 cycles, followed by complete extension at 72°C for 5 min and storage at 16°C.

[0040] 25 μL of amplified products were separated by 1% agarose gel electrophoresis, and the products were recovered and ligated using the FastPure Gel DNA Extraction Mini Kit (Nanjing Novogene, product number: DC301), pMD19-T (TaKaRa, product number: 3271) and DH5α chemical competent cells (Nanjing Novogene, product number: C502), and finally sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0041] From the gel electrophoresis imaging results, the amplification results of primer pair S262 / S263 were not very different, while the amplification results of primer pair S257 / S258 could divide the eight sycamore varieties into three groups. Figure 2 :

[0042] The first group: when gel electrophoresis imaging showed a single band less than 1000 bp, it included 'Yuanke 115' (No. 1), 'Yuanke 23' (No. 23) and Platanus tricolor (No. 24).

[0043] The second group: when the gel electrophoresis imaging showed a single band larger than 1000 bp, it included 'Yuanke 12' (No. 12) and Platanus edulis (No. 25).

[0044] The third group: Gel electrophoresis imaging showed two bands, one larger than 1000bp and the other smaller than 1000bp, including 'Yuanke 10' (No. 10, showing bands of 981bp and 1053bp), 'Yuanke 22' (No. 22, showing bands of 981bp and 1083bp) and 'Susheng Fatong' (No. 27, showing bands of 981bp and 1083bp). The band length of 'Yuanke 10' was shorter than that of 'Yuanke 22' and 'Susheng Fatong'.

[0045] Based on the gel electrophoresis imaging, different varieties of sycamore can be easily distinguished by the number and length of bands.

[0046] The amplified products of the two primer pairs were further connected to the vector and sequenced. The results showed that the amplification of the first primer pair and the second primer pair could pass through the SNP site in the second intron of the LFY gene, thereby distinguishing the eight sycamore samples.

[0047] The sequence alignment results of the amplified fragments of primer pair S262 / S263 can be found in Figure 3 and the table below:

[0048] Nucleotide sites of mutations in the partial sequence of the second intron of LFY gene in Platanus sylvestris

[0049] (S262 / S263 Primers)

[0050]

[0051] InDel site in the partial sequence of the second intron of LFY gene in Platanus sylvestris

[0052] (S262 / S263 Primers)

[0053]

[0054]

[0055] Among them, the sequence results of the amplified fragment of primer pair S257 / S258 showed:

[0056] Yuanke 115 sample (No. 1): Although the gel electrophoresis results showed that the amplified product was a single band less than 1000bp, the sequencing results showed two sequences, 1#1 was 960bp and 1#2 was 981bp, and the bases were different at 32bp, 42bp and 223bp. At 359bp, 1#2 was 21bp more than 1#1.

[0057] Sample Yuanke 23 (No. 23): The sequencing result showed only one sequence, which was the same as the 1#1 sequence of 'Yuanke 115'.

[0058] Sample of Platanus truncatula (No. 24): There are two sequences in the sequencing results, corresponding to the two sequences of 'Yuanke 115', but 24#2 is 21bp more than 24#1 only at 359bp, and 32bp, 42bp and 223bp are the same as 1#1.

[0059] Sample Yuanke 12 (No. 12): The sequencing results showed two sequences. 12#1 had 20 GA more than the genome LFYintron2-1 (from the second intron in the Platanus oleifera gene PaC3G140170) at 814 bp, and 12#2 had 30 GA more than intron2-1.

[0060] Platanus truncatula sample (No. 25): The sequencing results showed two sequences, 25#1 and 25#2, which had 27 and 31 more GAs than intron2-1 at 814 bp, respectively.

[0061] Yuanke 10 sample (No. 10): The sequencing results showed two sequences, 10#1 was consistent with 1#2, and 10#2 was different from 10#1 at 100bp, 163bp, 757bp, and 771bp, especially at 771bp, 10#2 had 15 more GA than 10#1.

[0062] Sample Yuanke 22 (No. 22): There are two sequences in the sequencing results. 22#1 is highly consistent with the genome LFY intron2-2 (the second intron of the LFY gene Platanus32797-RA from Platanus triglobosa), with only 3 fewer GA repeats at 873bp than intron2-2. 22#2 is the same as 1#2.

[0063] Sequence alignment of the fragment amplified by primers S262 / S263 showed:

[0064] The sequencing results showed that the sequence of the 'Yuanke 115' sample was consistent with that of the 'Yuanke 23' sample; the 'Plantanum multiflorum' sample was different from the former two at 152bp, 588bp, 1157bp and 1206bp.

[0065] The sequencing results showed that the 'Plantalum oxyphylla' sample had only one sequence; the 'Yuanke 12' sample had two sequences, one consistent with intron2-2 and the other consistent with the 'Plantalum oxyphylla' sample sequence.

[0066] The sequencing results showed that the 'Yuanke 10' sample was a single sequence, which was consistent with the intron2-2 sequence; the 'Yuanke 22' sample was the same as the 'Sushengfatong' sample, both of which had two sequences, one consistent with the intron2-2 sequence and the other consistent with the 'Yuanke 115' sample sequence.

[0067] Example 3

[0068] By analyzing the amplification results of primer pair S257 / S258, it was found that in the 3' region near the second intron of the LFY gene, there was one SSR site with GA repeats in the Platanus tricolor samples, while there were 6 GA repeats in the Platanus unicolor samples.

[0069] A pair of SSR primers (S324 / S325) were designed based on the GA repeat site in the intron2 sequence of the LFY gene of Platanus truncatula, and the 5' end of the upstream primer was modified with a 6-FAM (blue) fluorescent group. The specific primer sequences are shown in the table below.

[0070] name Primer sequences S324 5'-CTTATGAGAAGGCATGCACCAAAG-3'(SEQ ID NO:5) S325 5'-GCATATCTGAATACCTGGTTCGTCAC-3'(SEQ ID NO:6)

[0071] The 8 test sample DNAs obtained in Example 1 were used as templates, and the third primer pair was used to amplify the partial sequence of intron2 of the LFY gene. The PCR reaction system was the same as the amplification system of the first primer pair mentioned above, and the PCR reaction program was adjusted to: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, renaturation at 60°C for 45s, extension at 72°C for 50s, for a total of 35 cycles; final extension at 72°C for 5min, and storage at 4°C. The PCR product was first detected by agarose electrophoresis, and subsequent capillary electrophoresis was performed for samples with correct and clear bands. Before loading, 9μL of a mixture of formamide and molecular weight internal standard (mixed at a volume ratio of 100:1) and 1μL of 10-fold diluted PCR product were added to each well, and then capillary electrophoresis was performed using ABI3730XL DNA genetic analyzer, and the raw electrophoresis data was collected using DataCollection software.

[0072] The vector NTI software was used for multiple sequence alignment, and the differences in nucleotide sequences between the eight test samples and the control PaC3G140170 and Platanus32797-RA were sorted out, and single nucleotide polymorphisms (SNPs) and insertion and deletion (Indel) sites were analyzed. The raw data obtained by the genetic analyzer were analyzed using the Fragment (Plant) fragment in the Genemarker software, and the position of the molecular weight internal standard in each lane was compared with the position of the peak of each sample to directly obtain the precise length (unit bp) of the SSR amplified fragment.

[0073] The experimental results show that:

[0074] The samples of 'Yuanke 115', 'Yuanke 23' and 'Plantago serrata' (No. 24) have only one peak at 217 bp, so their LFY genes all come from the Platanus serrata gene PaC3G140170;

[0075] The samples of 'Yuanke 10', 'Yuanke 22' and 'Susheng Fatong' all have two peaks, and one of the peaks is at 217 bp, so their LFY genes come from the gene PaC3G140170 of Platanus uniglobosa and the gene Platanus tricholoma.

[0076] The samples 'Yuan Ke 12' and 'Plantanum sibiricum' (No. 25) also have two peaks, but both of their peaks are not at 217 bp. The 'Yuan Ke 12' sample has 20 and 30 more GA repeats, respectively, and the 'Plantanum sibiricum' sample has 27 and 31 more GA repeats, respectively, so their LFY genes all come from the Platanus sibiricum gene Platanus32797-RA.

[0077] The detection results of the above SSR molecular markers are consistent with the sequencing results of Example 2. For some experimental results, see Figure 4 .

[0078] Through the above experimental research, the present invention discovered for the first time that the polymorphism of the second intron of the LFY gene can be used for the identification of sycamore varieties and their genetic relationships, especially to facilitate the rapid identification of new varieties of sycamore with few fruits and their genetic relationships, and to provide theoretical support for enriching sycamore germplasm resources and understanding the parental information of sycamore with two balls, laying a good foundation for the promotion of new varieties of sycamore with few fruits.

[0079] Through SNP site analysis, the present invention found that both primer pairs can distinguish between 'Yuanke 10' and 'Yuanke 22', 'Yuanke 12' and 'Plantus unicornus' samples. Among them, primer pair S257 / S258 can distinguish between 'Yuanke 115', 'Yuanke 23' and 'Plantus tricornus' samples, and primer S262 / S263 can distinguish between 'Yuanke 23' and 'Plantus tricornus' samples. According to the analysis of sequencing results, it was speculated that the two LFY intron sequences of ‘Yuanke 10’, ‘Yuanke 22’ and ‘Susheng Fatong’ were derived from the gene PaC3G140170 of Platanus truncatus and the gene Platanus 32797-RA of Platanus truncatus, respectively. The LFY intron sequences of ‘Yuanke 115’ and ‘Yuanke 23’ were derived from the gene PaC3G140170 of Platanus truncatus, and the two LFY intron sequences of ‘Yuanke 12’ were derived from the gene Platanus 32797-RA of Platanus truncatus. The LFY intron sequence of ‘Platanus truncatus’ was derived from the gene Platanus 32797-RA of Platanus truncatus, while the two LFY intron sequences of ‘Platanus truncatus’ were derived from the gene PaC3G140170 of Platanus truncatus.

[0080] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of the second intron polymorphism of LFY gene in identifying sycamore varieties.

2. A primer pair for identifying a sycamore variety, characterized in that: The sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; or The sequences of the primer pair are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

3. A kit for identifying sycamore varieties, characterized in that: The invention comprises the primer pair for identifying sycamore varieties as described in claim 2.

4. A method for identifying a sycamore variety, characterized in that: The steps include: Obtain plane tree samples and extract DNA; Amplification is performed using the primer pair for identifying the sycamore variety shown in claim 2, gel electrophoresis is performed on the amplified product, and the sycamore variety is determined by the gel electrophoresis bands: When the gel electrophoresis showed a single band less than 1000 bp, it was determined that the LFY gene sequences in the sycamore varieties were all from the sycamore gene PaC3G140170; When the gel electrophoresis showed a single band larger than 1000 bp, it was determined that the LFY gene sequences in the sycamore varieties were all from the Platanus triglossum gene Platanus32797-RA; When gel electrophoresis showed double bands, with one band larger than 1000 bp and the other smaller than 1000 bp, it was determined that the LFY gene sequences in the sycamore varieties came from the sycamore gene PaC3G140170 and the sycamore gene Platanus32797-RA, respectively.

5. The method for identifying the species of Plane trees according to claim 4, characterized in that: The method also includes a step of sequencing and identifying the amplified product.

6. A primer pair for identifying a sycamore variety, characterized in that: The sequences of the primer pair are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

7. A kit for identifying sycamore varieties, characterized in that: The invention comprises the primer pair for identifying sycamore varieties as described in claim 6.

8. A method for identifying a sycamore variety, characterized in that: The steps include: Obtain plane tree samples and extract DNA; Amplification is performed using the primer pair for identifying the sycamore variety as shown in claim 6, and the amplified product is detected by agarose gel electrophoresis. For samples with correct and clear bands, capillary electrophoresis is performed to determine the sycamore variety: When only a single peak of 217 bp appeared in the test results, it indicated that the LFY gene sequences in the sycamore varieties all came from the sycamore gene PaC3G140170; When the test results show two peaks and contain a single peak of 217 bp, it indicates that the two LFY gene sequences in the sycamore variety come from the sycamore gene PaC3G140170 and the sycamore gene Platanus32797-RA respectively; When the detection results showed two peaks and did not contain a single peak of 217 bp, it indicated that the LFY gene sequences in the sycamore varieties all came from the Platanus triglossum gene Platanus32797-RA.

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