A method for molecular identification of banana germplasm resources

Abstract

The application belongs to the technical field of molecular biology, and discloses a banana germplasm resource molecular identification method, and specifically discloses a database for banana germplasm resource molecular identification, wherein the database comprises characteristic sequences, and the characteristic sequences comprise at least one of GGKRKDRDNRN, GGKGGDRDNVN, GGGGGGKDK, GGGGKKKKK and the characteristic sequences shown in SEQ ID NO: 5-23. The database for banana germplasm resource molecular identification is disclosed for the first time, and is obtained by analyzing one-generation sequencing peak maps of banana samples, and is more reliable, stable, rich and accurate, and can achieve simple, fast and more accurate determination of the category of bananas.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a method for molecular identification of banana germplasm resources. BACKGROUND

[0002] Banana is a major distribution in the tropics and subtropics of evergreen perennial monocotyledonous large herb of Musa of Scitamineae of Musaceae, and the pulp is edible, and has had three or four thousand years of cultivation history, and is one of the main fruit crops and important food crops in the world. Most of the cultivated species of banana is evolved from two wild species of Musa acuminata Colla. and Musa balbisiana Colla. and interspecific hybridization. According to the method of Simmonds, the gene of the character of Musa acuminata Colla. is called A gene, and the gene of the character of Musa balbisiana Colla. is called B gene. According to the trait classification value and the chromosome ploidy, the banana cultivars can be divided into AA, AAA, AB, AAB, ABB, AAAA, AAAB, AABB, ABBB, BB and BBB genotypes. The genotype is often divided into subgroups, such as the AAA genotype banana is divided into Cavendish, red banana, large honey house and other subgroups according to the character difference, and the subgroup is further divided into multiple cultivars such as 'Baxi', 'Williams', 'North Banana' and the like. Therefore, the general writing method of banana scientific name is "genus name Musa + genotype + subgroup name + variety name", such as 'M.AAA Cavendish cv.Baxi' (AAA genotype, Cavendish subgroup, 'Baxi'), 'M.ABB Pisang Awak cv.Guangfen No1.' (ABB genotype, pink banana subgroup, 'Guangfen No1'). However, this method has a long observation period and is easy to judge incorrectly.

[0003] At present, the common method is to use flow cytometry to determine the chromosome ploidy combined with the method of PCR and electrophoresis running of specific molecular markers to identify the genome typing of banana (a banana A, B genome typing method, CN201910399127.8). However, this method has the following shortcomings: 1. flow cytometry and specific multiple restriction endonucleases are needed, and it is difficult to determine the ploidy of banana in a laboratory without the corresponding instruments and enzymes; 2. the identification result mainly depends on the weak change of the running band of the PCR product and the enzyme digestion product of multiple primers, and the result is easily disturbed by various factors, and the result is unstable; 3. the information obtained by this method is limited, and only the genetic typing of banana germplasm can be carried out, and the specific banana category cannot be further determined.

[0004] Microsatellite (SSR) markers are also molecular genetic markers based on PCR, which have been widely used in the construction of fingerprints of various crop varieties and the identification of hybrid seed purity. Some people screened 5 pairs of SSR primers with higher polymorphism and stability from 199 pairs of primers, and found that the 56 banana germplasm could be completely distinguished by amplification with the 5 pairs of primers (Wang JY, Chen YY, Huang BZ, Yu F, Wu YT. Construction of SSR fingerprint of some banana varieties. Journal of Fruit Tree Research, 2009, 26: 733-738.). However, due to objective and subjective reasons, this method is prone to errors in the process of silver staining band statistics after running the gel, and the repeatability cannot be guaranteed. SUMMARY

[0005] The first aspect of the present application aims to provide a database for molecular identification of banana germplasm resources.

[0006] The second aspect of the present application aims to provide a construction method of the database of the first aspect of the present application.

[0007] The third aspect of the present application aims to provide a kit.

[0008] The fourth aspect of the present application aims to provide the application of the database of the first aspect of the present application or the kit of the third aspect of the present application in the identification or auxiliary identification of banana germplasm resources.

[0009] The fifth aspect of the present application aims to provide a method for identifying or assisting in identifying banana varieties.

[0010] The sixth aspect of the present application aims to provide the application of the method of the fifth aspect of the present application in the classification of banana germplasm resources.

[0011] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0012] The first aspect of the present application aims to provide a database for molecular identification of banana germplasm resources, wherein the database comprises a characteristic sequence, and the characteristic sequence comprises at least one of GGKRKDRDNRN, GGKGGDRDNVN, GGGGGGKDK, GGGGKKKKK and the characteristic sequence as shown in SEQ ID NO: 5-23.

[0013] Preferably, the banana includes at least one of Yunnan wild banana, Gong banana, Rose banana, Haigong banana, Jiali banana, Lady finger banana, Guangfen No. 1, Red head powder, Immortal powder, Jinfen No. 1, Yinfen No. 1, Big banana, Beihua, Nantianhuang, Nantianhong, Guihua No. 6, Dafeng No. 1, Zhonghua No. 11, Nongke No. 1, Huannongzhongba, Fuxuan No. 1, Zhangzao A, Red banana, Guihong No. 1, Xitou big banana, Jinshan big banana, Zijinghua, Guiji No. 1, Niujiao big banana, Meishihua No. 1, Bengali banana, Jinshouzhi, and Zhonghua No. 9.

[0014] The second aspect of the present application is to provide a method for constructing the database of the first aspect of the present application, comprising the following steps:

[0015] (1) Using the genomic DNA of the known variety information banana as a template, the ITS sequence is amplified by PCR technology to obtain a PCR product;

[0016] (2) After sequencing the PCR product, opening the ".ab1" format file, viewing the sequencing peak chart, selecting the nucleotide sequence of about 420 bp from the 5' end of the PCR product, with a length of 9-11 bp, as a characteristic sequence, and listing the characteristic sequence;

[0017] When the peak type in the sequencing peak chart is a single peak, that is, the sequence in this region is GGGGXXXXXXX, X represents any single peak or nested peak, and the characteristic sequence is directly outputted;

[0018] When the peak type of most peaks in the sequencing peak chart is a nested peak, the nested peak needs to be re-encoded according to the following rules, and the characteristic sequence is outputted:

[0019] Single peak: A, T, C, G;

[0020] Double peak: M = A & C, R = A & G, W = A & T, S = G & C, Y = C & T, K = G & T;

[0021] Triple peak: V = A & G & C, H = A & C & T, D = A & G & T, B = G & C & T;

[0022] Four peaks: N = A & G & C & T;

[0023] (3) According to the known classification of banana, the characteristic sequences of the same classification are grouped, and the corresponding characteristic sequences are marked, to obtain a database for molecular identification of banana germplasm resources.

[0024] Preferably, the primer pair used for amplifying the ITS sequence is ITSL / ITS4.

[0025] Preferably, the nucleotide sequence of the primer pair is shown in SEQ ID NO: 1-2.

[0026] Preferably, the PCR amplification reaction conditions described in (1) are: pre-denaturation at 94-96℃ for 5-10 min; denaturation at 94-96℃ for 20-40 s, annealing at 50-60℃ for 20-30 s, extension at 70-72℃ for 30-40 s, for a total of 32-35 cycles; and extension at 70-72℃ for 10-15 min.

[0027] Preferably, the genomic DNA of the banana described in (1) can be obtained by commonly used DNA extraction methods, including but not limited to CTAB, SDS, phenol-chloroform extraction and extraction using commercially available kits.

[0028] A third aspect of the present invention is to provide a kit comprising primer pairs and a comparison card containing information from a database of the first aspect of the present invention.

[0029] Preferably, the nucleotide sequences of the primer pair are as shown in SEQ ID NO:1-2.

[0030] Preferably, the kit also includes various PCR reagents for amplifying the banana genome sequence.

[0031] A fourth aspect of the present invention is to provide the application of the database of the first aspect of the present invention or the kit of the third aspect of the present invention in the identification or auxiliary identification of banana germplasm resources.

[0032] Preferably, the bananas include at least one of the following: A-Kuan banana, Yunnan wild banana, Gong banana, Rose banana, Hai-Gong banana, Jia-Li banana, Fu-Fu-Zhi banana, Guang-Fen No. 1, Hong-Tou-Fen, Bu-Si-Fen, Jin-Fen No. 1, Yin-Fen No. 1, Fen-Da-Jia, Bei-Jia, Nan-Tian-Huang, Nan-Tian-Hong, Gui-Jia No. 6, Da-Feng No. 1, Qi-Wei, Zhong-Jia No. 11, Nong-Ke No. 1, Huanong Zhong-Ba, Fu-Xuan No. 1, Zhang-Zao A, Hong-Xiang banana, Gui-Hong banana No. 1, Qi-Tou banana, Jin-Shan banana, Zi-Jing banana, Gui-Ji banana No. 1, Niu-Jiao banana, Mei-Mei banana No. 1, Bengal vegetable banana, Jin-Shu-Zhi, and Zhong-Jia No. 9.

[0033] A fifth aspect of the present invention is to provide a method for identifying or assisting in the identification of banana varieties, comprising the steps of using the database of the first aspect of the present invention or the kit of the third aspect of the present invention.

[0034] Preferably, the method includes the following steps:

[0035] (a1) Using the genomic DNA of the banana to be tested as a template, the ITS sequence was amplified by PCR technology to obtain the PCR product;

[0036] (a2) after sequencing the PCR product, opening the ".ab1" format file, viewing the sequencing peak chart, selecting the nucleotide sequence of about 420bp from the 5' end of the PCR product with a length of 9-11bp as the comparison characteristic sequence, and listing the comparison characteristic sequence;

[0037] When the peak type in the sequencing peak chart is a single peak, i.e. the sequence in this region is GGGGXXXXXXX, X represents any single peak or nested peak, the characteristic sequence is directly outputted;

[0038] When the peak type of most peaks in the sequencing peak chart is a nested peak, the nested peak needs to be re-encoded according to the following rules, and the characteristic sequence is outputted:

[0039] Single peak: A, T, C, G;

[0040] Double peak: M = A & C, R = A & G, W = A & T, S = G & C, Y = C & T, K = G & T;

[0041] Triple peak: V = A & G & C, H = A & C & T, D = A & G & T, B = G & C & T;

[0042] Four peak: N = A & G & C & T;

[0043] (a3) comparing the comparison characteristic sequence with the comparison card in the database of the first aspect of the application or the kit of the third aspect of the application, to obtain the information of the banana variety.

[0044] Preferably, the reaction conditions for the PCR amplification in (a1) are 94-96℃ pre-denaturation for 5-10min; 94-96℃ denaturation for 20-40s, 50-60℃ annealing for 20-30s, 70-72℃ extension for 30-40s, a total of 32-35 cycles; 70-72℃ extension for 10-15min.

[0045] Preferably, the primer pair used for amplifying the ITS sequence is ITSL / ITS4.

[0046] Preferably, the nucleotide sequence of the primer pair is shown in SEQ ID NO: 1-2.

[0047] Preferably, the genomic DNA of the banana in (a1) can be extracted by the currently commonly used DNA extraction methods, including but not limited to CTAB, SDS, phenol chloroform extraction method and using commercially available kits for extraction.

[0048] Preferably, the genomic DNA of the banana can be extracted from the tissues of the banana (such as peel, pulp, flower, root and pseudostem, etc.) and banana leaves.

[0049] In addition to the difference in the 420bp position region of the ITS sequence, the application purposes can also be achieved by using other differences on the sequence.

[0050] The sixth aspect of the application provides application of the method of the fifth aspect of the application in classification of banana germplasm resources.

[0051] Preferably, the banana comprises at least one of Awi, Yunnan wild banana, Geng banana, Rose banana, Haigong banana, Jiali banana, Lady finger banana, Guangfen No.1, Hongtoufen, Bufen, Jinfen No.1, Yinfen No.1, Plantain, Beixia, Nantianhuang, Nantianhong, Guijia No.6, Dafeng No.1, Zhongjia No.11, Nongke No.1, Huannongzhongba, Fuxuan No.1, Zhangzao A, Hongbanana, Guihongjia No.1, Qitouplantain, Jinshanplantain, Zijiangjia, Guijijia No.1, Niujiangplantain, Meishijia No.1, Bengali cooking banana, Jishouzhi and Zhongjia No.9.

[0052] The application has the following beneficial effects:

[0053] The application discloses a database for molecular identification of banana germplasm resources, which is obtained by analyzing a first sequencing peak map of a banana sample, and is more reliable, stable, rich and accurate, and achieves simple, fast and more accurate identification of the category of banana.

[0054] The method for identifying or assisting in identifying the banana variety provided by the application only uses a pair of primers (ITSL and ITS4) for PCR and direct first sequencing, and the method is simple and fast in identifying the corresponding variety information of the to-be-tested banana, and can replace the cumbersome processes of flow cytometer identification ploidy, multiple primer PCR and restriction enzyme digestion and gel running to a certain extent.

[0055] The method provided by the application uses polymorphism on the ITS sequence sequencing peak map as a molecular marker of banana, can achieve relatively accurate germplasm identification, can assist the traditional morphological classification method, and greatly improves the identification efficiency of banana germplasm. As shown in Table 1, different genotypes of bananas (such as AA, AAA, ABB, AAB and AAAB), different subgroups within the same genotype of bananas (such as the red banana subgroup and the Cavendish banana subgroup of the AAA genotype), and different cultivated varieties of bananas in the same genotype or the same subgroup within the same genotype (such as Yunnan wild banana, Jiali banana and Lady finger banana in AA, different cultivated varieties in ABB plantain, and two different varieties in the red banana subgroup) can be effectively distinguished by the method. Several AAA Cavendish bananas with the same or similar sources can be identified by the method, as shown in Table 1, and several AAA Cavendish bananas can be classified into one group; Guangfen No.1 and Hongtoufen are classified into one group, and Jinfen No.1 and Yinfen No.1 are classified into one group, which indicates that the method has high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 Flow chart of the method for molecular identification of banana germplasm resources.

[0057] Figure 2 Examples of re-coding of single peaks and nested peaks and characteristic sequences.

[0058] Figure 3 Identification of banana samples using prior art, wherein A is identification of A and B genome content using ITS fragment RsaI enzyme digestion, and B is identification of the number of B genome using copia-IRAP fragment AluI enzyme digestion.

[0059] Figure 4 Characteristic sequences for identification of banana samples using the method of Example 1. DETAILED DESCRIPTION

[0060] The present application will be described in detail with specific examples, but the scope of the present application is not limited.

[0061] The materials, reagents, etc. used in the present example are commercially available unless otherwise specified.

[0062] Example 1

[0063] A method for molecular identification of banana germplasm resources, comprising the following steps:

[0064] (1) Database establishment

[0065] 1) Leaf samples were taken from 38 banana germplasm resources, including various genome types, different subgroups of homogenomic types, and different cultivars within the same subgroup (Table 1).

[0066] 2) Banana DNA was extracted, ITS was amplified and sequenced

[0067] A small piece of banana leaf (about 1 cm 2), put in 1.5 mL centrifuge tube, grind with electric grinding rod for about 30 s, add 600 μL DNA extraction solution (100 mM Tris-HCL, 50 mM EDTA of pH 8.0, 500 mM NaCl, 2% SDS, 1% PVP40), 65 °C water bath for 15 min, 12000 rpm centrifuge for 5 min, take the supernatant to a new 1.5 mL centrifuge tube, add equal volume of isopropanol, shake up and down, 12000 rpm centrifuge for 5 min, discard the supernatant, add 500 μL 70% ethanol, 12000 rpm centrifuge for 5 min, discard the ethanol, put the centrifuge tube in 65 °C oven and dry for 10 min, add 100 μL sterile water, and put in -20 °C refrigerator for standby. Amplify the ITS sequence with primers ITSL (5'-TCGTAACAAGGTTTCCGTAGGTG-3' (SEQ ID NO: 1)) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2)). Perform PCR amplification with 2X SanTaq PCR Master Mix (with Blue Dye) kit (purchased from Sheng Wu Bioengineering, item number B532061-0040). The PCR reaction system is as follows: 2X SanTaq PCR Mix 25 μL, primer ITSL 2 μL, primer ITS4 2 μL, 1-2 μL DNA solution, sterile ultrapure water to 50 μL. The PCR reaction conditions are as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s, a total of 35 cycles; 72 °C extension for 10 min. After the PCR reaction is completed, the product (about 700 bp in size) is sent to Beijing Qikong Biotechnology Co., Ltd. for sequencing with primer ITSL (5'-TCGTAACAAGGTTTCCGTAGGTG-3' (SEQ ID NO: 1)).

[0068] 3) List characteristic sequences according to polymorphic regions of peak map

[0069] After obtaining the sequencing results, open the ".ab1" format file, view the sequencing peak map, and check whether the sequencing results are normal. If there is obvious sample contamination, abnormal peak type, or insufficient sequencing length, retest is required. When the peak type of the region near 420 bp in the sequencing peak map is "single peak", the sequence of this region ("GGGGXXXXXXX", "X" represents any "single peak" or "nested peak") is directly recorded in the "characteristic sequence list".

[0070] When the peak type of most peaks in the sequencing peak map is "nested peak" (i.e., two, three, or four sequencing peaks appear at the same position), these "nested peaks" need to be re-encoded according to the following rules to list the characteristic sequences:

[0071] Single peak: A, T, C, G;

[0072] Double peak: M=A&C, R=A&G, W=A&T, S=G&C, Y=C&T, K=G&T;

[0073] Three peaks: V=A&G&C, H=A&C&T, D=A&G&T, B=G&C&T;

[0074] Four peaks: N = A&G&C&T.

[0075] like Figure 2 As shown, record the sequence near the 420bp position in the sequencing peak diagram (usually starting with "GGGG"). Single peaks can be recorded directly, while clustered peaks are recoded into characteristic sequences according to the rules and then recorded.

[0076] 4) Establish a list of characteristic sequences for banana germplasm, grouping those with identical characteristic sequences together.

[0077] A list was created based on the known classifications of bananas. Bananas within the same classification were grouped together with identical characteristic sequences, and their corresponding characteristic sequences were labeled. The constructed characteristic sequence database for molecular identification of banana germplasm resources is shown in Table 1.

[0078] As shown in Table 1, the characteristic sequences of bananas with different genotypes, such as AA, AAA, ABB, AAB, and AAAB, are all different. Within the same genotype, different subgroups of bananas, such as the Yunnan wild banana, Jiali banana, and Furenzhi banana within the AA genotype, all have different characteristic sequences. The ABB pink banana can be divided into three groups (Guangfen No. 1 and Hongtou pink are the same, Jinfen No. 1 and Yinfen No. 1 are the same, and the pink plantain is different from all of these). Several AAA Cavendish bananas have the same characteristic sequence. The characteristic sequences of the red banana subgroup, which is also of the AAA genotype, are different from those of Cavendish bananas (and also different from Guihong banana No. 1). Other bananas, such as Qitou plantain, Sabah plantain, Niujiao plantain, and Jinshouzhi plantain, all have unique characteristic sequences. This result is consistent with known banana classifications, indicating that the library construction was successful and that this method can be used effectively to identify banana categories.

[0079] (2) Identification

[0080] Extract DNA from the banana germplasm to be tested, amplify its ITS sequence and sequence it. List the characteristic sequences of the bananas to be identified based on the polymorphic regions of the peak diagram. The experimental procedure is the same as steps 2) to 3) in (1). After obtaining the characteristic sequences of the bananas to be identified, search for the sequence in the constructed characteristic sequence database for molecular identification of banana germplasm resources. If there is germplasm with the same sequence, it means that the banana to be identified is the same as that germplasm or belongs to the same category; if there is no germplasm with the same sequence, it means that the banana to be identified is different from all the known bananas in the list.

[0081] The flow chart of the banana germplasm resource molecular identification method is shown in Figure 1

[0082] Table 1: 420bp characteristic sequences and grouping of 38 known banana germplasm resources

[0083]

[0084]

[0085]

[0086] Effect embodiment

[0087] In order to further verify the accuracy of the method, the banana germplasm resource molecular identification method provided in Example 1 is used to identify two banana trees randomly selected from the national banana planting resource garden without a plaque, which are named "banana 1" and "banana 2". At the same time, morphological, genomic typing (reference patent document CN110157830A) are used to identify and analyze the two banana varieties.

[0088] The characteristic sequences of "banana 1" and "banana 2" are detected by the identification method of Example 1, and the results are shown in Figure 4 The characteristic sequences of "banana 1" and "banana 2" are "GGGGKKKKRGV" and "GGGGGKKKKGG", respectively, which are the same as the existing database of Mysore 1 group and Cavendish banana (Table 1), indicating that "banana 1" and "banana 2" are Mysore (ABB) and Cavendish banana (AAA), respectively, and it can be judged that "banana 1" is highly similar to Mysore 1, red head powder and immortal powder, and is obviously different from gold powder 1, powder banana and other Mysore.

[0089] According to the traditional Simonds method, the scores of 15 characteristics of "banana 1" and "banana 2" are scored, and the morphological method identification is carried out, and the results are shown in Table 2. The comprehensive scores of "banana 1" and "banana 2" are 60 and 18, respectively, belonging to ABB and AAA genome type. And according to the characteristics of the 15 characteristics, it is speculated that "banana 1" may be Mysore (ABB), and "banana variety 2" may be Cavendish banana (AAA).

[0090] By using ITS primer (ITSL and ITS4) to amplify ITS (about 690bp in size), RsaI enzyme digestion and gel electrophoresis, etc. The results of the genomic typing identification of banana are shown in Figure 3 ​As shown in Fig. 1A, the inventors found that "banana 1" had four bands, about 560 bp, 370 bp, 190 bp and 130 bp, respectively, indicating that it contained both A and B genomes; "banana 2" had one band about 560 bp and one band about 130 bp, indicating that it only contained A genome. Since "banana 1" contained both A and B genomes, the number of A and B genomes was unknown, the inventors further used the copia-IRAP molecular marker to detect the number of B genomes (Liangyu, Jiani, Yingkang, Jianjun, Chong, Gou. Establishment of a molecular marker identification system for genome types of banana cultivars [J]. Molecular Plant Breeding, 2022, 20(09):3011-3018.). Amplification (about 420 bp) was performed using copia-IRAP primers (Bfor: 5'-AGGGTTCGAAGTATAGGTTCGG-3'(SEQ ID NO: 3), Brev: 5'-AATGTTTAAGTAGAGGGCAAGACG-3'(SEQ ID NO: 4)), and Alu I enzyme was used for enzyme digestion, as shown in Fig. 1B. Figure 3 As shown in Fig. 1B, two bands (200 bp up and down) appeared closely together after enzyme digestion, indicating that the number of B genomes was greater than or equal to 2. According to the above experiments, it was speculated that "banana 1" contained both A and B genomes, and the number of B genomes was greater than 1, so it might be ABB or ABBB; while "banana 2" only contained A genome, and might be AA, AAA or AAAA. This result intersected with the morphological identification result, and it was determined that "banana 1" and "banana 2" were pink banana (ABB) and Cavendish banana (AAA).

[0091] The above results show that the identification results using the method of Example 1 are consistent with the identification results using morphology and the commonly used genetic identification method, indicating that the identification method of Example 1 has good accuracy.

[0092] Table 2 Morphological identification results

[0093]

[0094] The embodiments of the application are described in detail above with reference to the drawings, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A database for molecular identification of banana germplasm resources, the database comprising characteristic sequences including GGKRKDRDNRN, GGKGGDRDNVN, GGGGGGKDK, GGGGKKKKK and characteristic sequences as shown in SEQ ID NO:5~23.

2. The database according to claim 1, characterized in that, The bananas mentioned include at least one of the following: Yunnan wild banana, Gong banana, Rose banana, Haigong banana, Jiali banana, Furenzhi banana, Guangfen No. 1, Hongtoufen, Busifen, Jinfen No. 1, Yinfen No. 1, Fendabanana, Beibanana, Nantianhuang, Nantianhong, Guijiao No. 6, Dafeng No. 1, Qiwei, Zhongjiao No. 11, Nongke No. 1, Huanong Zhongba, Fuxuan No. 1, Zhangzao A, Hongxiangbanana, Guihongbanana No. 1, Qitoubanana, Jinshanbanana, Zijingbanana, Guijibanana No. 1, Niujiaobanana, Meishibanana No. 1, Bengal vegetable banana, Jinshouzhi, and Zhongjiao No.

9.

3. The method for constructing a database for molecular identification of banana germplasm resources as described in claim 1 or 2, comprising the following steps: (1) Using the genomic DNA of bananas with known varietal information as a template, the ITS sequence was amplified by PCR technology to obtain the PCR product; (2) After sequencing the PCR product, open the ".ab1" format file, view the sequencing peak diagram, select the nucleotide sequence of the PCR product from the 420th bp from the 5' end, with a length of 9~11 bp, as the characteristic sequence, and list the characteristic sequence. When the peak shape in the sequencing peak diagram is a single peak, that is, the sequence of the region is GGGGXXXXXXX, where X represents any single peak or nested peaks, the characteristic sequence is directly output. When most peaks in the sequencing peak diagram are overlapping peaks, the overlapping peaks need to be recoded according to the following rules, and the characteristic sequence should be output: Single peak: A, T, C, G; Double peak: M=A&C, R=A&G, W=A&T, S=G&C, Y=C&T, K=G&T; Three peaks: V=A&G&C, H=A&C&T, D=A&G&T, B=G&C&T; Four peaks: N = A&G&C&T; (3) List the bananas according to their known classifications. Group the bananas with the same characteristic sequences in the same classification and label the corresponding characteristic sequences to obtain a database for molecular identification of banana germplasm resources.

4. The construction method according to claim 3, characterized in that, The primer pair used to amplify the ITS sequence is ITSL / ITS4; the nucleotide sequence of the primer pair is shown in SEQ ID NO:1~2.

5. A kit comprising primer pairs and a comparison card containing information from the database of claim 1 or 2; the nucleotide sequences of the primer pairs are shown in SEQ ID NO: 1-2.

6. The application of the database of claim 1 or 2 or the kit of claim 5 in the identification or auxiliary identification of banana germplasm resources.

7. A method for identifying or assisting in the identification of banana varieties, comprising the steps of using the database of claim 1 or 2 or the kit of claim 5.

8. The method according to claim 7, characterized in that, The method includes the following steps: (a1) Using the genomic DNA of the banana to be tested as a template, the ITS sequence was amplified by PCR technology to obtain the PCR product; (a2) After sequencing the PCR product, open the ".ab1" format file, view the sequencing peak diagram, select the nucleotide sequence of the PCR product from the 420th bp from the 5' end, with a length of 9~11 bp, as the characteristic sequence to be compared, and list the characteristic sequences to be compared. When the peak shape in the sequencing peak diagram is a single peak, that is, the sequence of the region is GGGGXXXXXXX, where X represents any single peak or nested peaks, the characteristic sequence is directly output. When most peaks in the sequencing peak diagram are overlapping peaks, the overlapping peaks need to be recoded according to the following rules, and the characteristic sequence should be output: Single peak: A, T, C, G; Double peak: M=A&C, R=A&G, W=A&T, S=G&C, Y=C&T, K=G&T; Three peaks: V=A&G&C, H=A&C&T, D=A&G&T, B=G&C&T; Four peaks: N = A&G&C&T; (a3) The feature sequence to be compared is compared with the database of claim 1 or 2 or the comparison card of claim 5 to obtain information on banana varieties.

9. The method according to claim 8, characterized in that, The PCR amplification reaction conditions described in (a1) are: pre-denaturation at 94-96℃ for 5-10 min; denaturation at 94-96℃ for 20-40 s, annealing at 50-60℃ for 20-30 s, extension at 70-72℃ for 30-40 s, for a total of 32-35 cycles; and extension at 70-72℃ for 10-15 min.

10. The application of the method according to any one of claims 7 to 9 in the classification of banana germplasm resources.

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