InDel-90 Marker Related to the Browning Trait of Pear Pulp and Its Application

Abstract

The present invention belongs to the technical field of pear molecular breeding, and discloses an InDel-90 marker related to the trait of pear pulp browning. The InDel-90 marker is located at the position of 17,958,988-17,959,190 bp on chromosome 2 in the genetic linkage map of 'Mantianhong' × 'Hongxiangsu'. The nucleotide sequence of the InDel-90 marker is as shown in SEQ ID NO.90, with a full length of 203 bp. The forward primer of the InDel-90 marker is 5'-GAAACATCACGTATCGGATT-3', and the reverse primer is 5'-TCAACAATTACAAAAACCCC-3'. Single plants with easily browning pear pulp show the insertion of the InDel-90 marker sequence, while single plants with not easily browning pear pulp show the deletion of the InDel-90 marker sequence. The designed InDel-90 marker of the present invention can be used to identify whether pear pulp is prone to browning, has good application value, and can be used for pear molecular breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of pear molecular breeding, and relates to an InDel-90 marker related to the trait of pear pulp browning and its application. Background Art

[0002] In recent years, fresh-cut fruits have been particularly favored by consumers for their freshness and convenience. However, when the pulp of many pear varieties is exposed to air, it will quickly turn brown, with poor appearance and damaged nutrition, greatly reducing consumers' desire to eat. Exploring genes related to controlling the trait of pear pulp browning and developing molecular markers related to pear pulp browning can accelerate the breeding of pear varieties with pulp anti-browning ability, reduce breeding costs, and have important significance for extending the shelf life of fresh-cut pears, solving the limiting factors in pear fruit processing, and promoting the diversified development of the pear industry. In terms of analyzing the physiological and molecular mechanisms of fruit browning, researchers have found that pulp browning is a physiological and biochemical process in which phenolic substances in the pulp tissue are catalytically oxidized by polyphenol oxidase to quinone under the action of oxygen, and then enzymatically browned into melanin, making the fruit tissue turn brown. Chlorogenic acid is the main browning substrate of PPO. Boss et al. cloned the cDNA of polyphenoloxidase (PPO) from apple peel, and Haruta et al. cloned two DNA fragments encoding apple PPO through PCR reaction and confirmed that PPO in Rosaceae fruits is very similar. Guardo et al. located the genes related to the trait of apple pulp browning on the genome, and among these genes, PPO and PAL are most closely related to pulp browning. Gan Huifang found that when the apple PPO gene was introduced into pears using RNAi technology, the interference effect was not as good as in apples, and the effect of inhibiting pulp browning was poor.

[0003] Up to now, the molecular mechanism of controlling pear pulp browning still needs to be further studied, and there is no practical molecular marker required to assist in screening non-browning individual plants in the hybrid population, making it difficult to provide effective support for the breeding of new anti-browning pear varieties. Summary of the Invention

[0004] The purpose of the present invention is to provide an InDel-90 marker related to the trait of pear pulp browning, which can be used to identify whether pear pulp is prone to browning, has good application value, and can be used in pear molecular breeding.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides an InDel-90 marker related to the trait of pear pulp browning. The InDel-90 marker is located at the position of 17,958,988 - 17,959,190 bp on chromosome 2 in the combined genetic linkage map constructed by 'Mantianhong' × 'Hongxiangsu'. The nucleotide sequence of the InDel-90 marker is as shown in SEQ ID NO.90, with a full length of 203 bp. The forward primer of the InDel-90 marker is 5'-GAAACATCACGTATCGGATT-3', and the reverse primer is 5'-TCAACAATTACAAAAACCCC-3'. Single plants with easily browning pear pulp show the insertion of the InDel-90 marker sequence, while single plants with not easily browning pear pulp show the deletion of the InDel-90 marker sequence.

[0007] The present invention also provides a kit containing a primer pair of the InDel-90 marker related to pear pulp browning.

[0008] The present invention also provides the application of the above kit in the identification of pear pulp browning varieties.

[0009] Preferably, for the application of the above kit in the identification of pear pulp browning varieties, the specific identification method includes the following steps:

[0010] a. Extract the genomic DNA of the pear material to be detected;

[0011] b. Perform PCR amplification on the genomic DNA of the pear material to be detected using the primer pair of the InDel-90 marker;

[0012] c. Detect the PCR product by electrophoresis. If there are two bands, the pear pulp is easily browning; if there is one band, the pear pulp is not easily browning.

[0013] The present invention also provides the application of the InDel-90 marker related to the trait of pear pulp browning in molecular marker-assisted breeding of pears.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The present invention first performs QTL mapping on the trait of pear pulp browning, locates the major QTL locus PFB, and the SNP molecular marker closest to PFB is Marker02874. The mapping results in two years show good repeatability, verifying the accuracy of the results. Then, 22 pairs of InDel markers are used to verify in the offspring single plants with extreme traits (easily browning and not easily browning). Finally, according to the phenotype and the electrophoresis results of the amplification of 22 pairs of primers, it is determined from the 22 pairs of InDel markers that the InDel-90 marker can be used to identify whether the pear pulp is easily browning, has good application value, and can be used in pear molecular breeding. Description of the Drawings

[0016] Figure 1 This is the phenotypic identification result of the experimental materials of the present invention.

[0017] Figure 2 This is the main-effect QTL mapping diagram related to the pear pulp browning trait.

[0018] Figure 3 This is the mapping result of 22 pairs of InDel markers re-located on Chr2.

[0019] Figure 4 This is the electrophoresis result of the primer amplification product of the InDel-90 marker. Detailed implementation manners

[0020] The following examples are used to illustrate the present invention, but are not used to limit the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified.

[0021] Example 1

[0022] 1. Phenotypic identification

[0023] Using the browning pear variety 'Mantianhong' as the male parent and the non-browning pear variety 'Hongxiangsu' with excellent quality as the female parent for hybridization. In 2020, 109 F1 single-plant fruits were used as materials, and in 2021, 21 more were added, making a total of 130 F1 single-plant fruits as materials to identify the fruit browning degree. The experimental materials were all planted in the Xinxiang Base of Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. At the fruit maturity stage, 3 healthy and uniformly sized pear fruits were randomly selected for the phenotypic identification of the pulp browning degree. The picked fruits were stored at 4°C and the phenotypic identification of the browning degree was carried out within 48 hours. The phenotypic identification of the browning degree refers to the browning degree grade division in the invention patent "Method for Establishing the Standard of Pear Pulp Browning Degree" with the publication number CN 113777250 A (as shown in Figure 1 -a), and the browning grade identification results of the experimental single plants are shown in Table 1. Using Microsoft Excel 2019 to conduct a normal test on the distribution of the browning degree (as shown in Figure 1 -b), the results show that its coefficient of variation is 58.12%, kurtosis is -0.94, and skewness is 0.12, showing a good normal distribution, indicating that the pear pulp browning is a quantitative trait controlled by multiple genes.

[0024] Table 1 Browning grade identification results of the F1 offspring population

[0025]

[0026]

[0027] 2. Construction of Genetic Map and QTL Analysis

[0028] Using the high-density genetic map constructed with 'Mantianhong' × 'Hongxiangsu' and 357 hybrid offspring (Huabai Xue, SuKe Wang et al., Chromosome level high-density integrated genetic maps improve the Pyrus bretschneideri 'DangshanSuli' v1.0 genome[J]. BMC Genomics, (2018) 19:833), the genotype data of the segregation population of the experimental materials of the present invention were screened out.

[0029] Combined with the phenotypic identification results, QTL analysis of pear pulp browning was carried out using MapQTL5 software. The LOD threshold of each linkage group was estimated according to the permutation test (number of times > 1000), and then the interval mapping method was used to detect the position of the markers related to the pear pulp browning trait in the linkage group, and to judge the presence or absence of QTL controlling the pear fruit browning trait and its location area. The results are as Figure 2 shown.

[0030] In 2020, the interval length of the QTL locus PFB on Chr2 was 48.301 cM, from 43.476 cM (LOD = 4.0) to 91.768 cM (LOD = 10.58). A total of 72 SNP molecular markers were included in the interval. The highest point of LOD in the interval was located at 76.009 cM (LOD = 15.06). The SNP molecular marker closest to PBF linkage was Marker02874, and the phenotypic variation rate that could be explained was 49.98%, and the contribution rate was 65.6%.

[0031] In 2021, the interval length of the QTL locus PFB on Chr2 was 49.292 cM, from 43.476 cm (LOD = 4.68) to 92.768 cM (LOD = 6.32). A total of 76 SNP molecular markers were included in the interval. The highest point of LOD in the interval was located at 76.009 cM (LOD = 13.77). The SNP molecular marker closest to PBF linkage was Marker02874, and the phenotypic variation rate that could be explained was 86.84%, and the contribution rate was 38.6%.

[0032] The molecular marker Marker02874 is located at the 176,410th bp of the Scaffold of the 'Dangshansuli' genome with the accession number NW_008988236.1, and its base is G. From the mapping results of 2020 and 2021, it can be seen that the QTL mapping shows good repeatability, verifying the accuracy of the results. The QTL locus PFB is the major QTL.

[0033] 3. Fine Mapping

[0034] The mapping results of 2020 and 2021 have good repeatability, but the interval of the major QTL is relatively large. In order to further conduct more precise mapping, 93 pairs of InDel markers were redesigned within the mapped segment and screened and verified in 8 easily browning (the offspring individual plant numbers are 14, 112, 157, 183, 248, 265, 310, and 313) and 8 not easily browning (the offspring individual plant numbers are 19, 50, 77, 97, 113, 263, 337, and 357) offspring individual plants. The 93 pairs of InDel marker sequences were designed according to the 'Dangshansuli' reference genome, as shown in Table 2. PCR amplification conditions: pre-denaturation: 94°C for 2 min; denaturation: 94°C for 30 s; annealing: 55°C for 30 s; extension: 72°C for 1 min; 30 cycles; 72°C for 10 min. The results showed that 22 pairs of InDel markers showed trait segregation in the two groups of extreme trait individual plants. The coincidence degrees of the phenotypes and genotypes of the easily browning individual plant group and the not easily browning individual plant group were 75% and 87.5% respectively. These 22 pairs of InDel markers were re-mapped on Chr2, and the results are shown in Figure 3. These 22 pairs of InDel markers are InDel-18, InDel-19, InDel-25, InDel-37, InDel-38, InDel-40, InDel-41, InDel-42, InDel-43, InDel-45, InDel-54, InDel-62, InDel-63, InDel-67, InDel-69, InDel-75, InDel-85, InDel-86, InDel-90, InDel-91, InDel-92, InDel-93. According to the phenotypes and the electrophoresis results of the amplification of 22 pairs of primers, the InDel-90 marker was finally determined from the 22 pairs of InDel markers to identify whether the pear pulp is prone to browning. Figure 4 The electrophoresis results of the primer amplification products of the two InDel-90 markers are shown.

[0035] Table 2 The primer sequences of 93 pairs of InDel markers used for fine mapping

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] From Figure 4 It can be seen that the electrophoresis results of the first 8 single plants with easy browning in the amplification products of the InDel-90 marker (SEQ ID NO.90) primer all have two bands, indicating that the single plants with easy browning show the insertion of the InDel-90 marker sequence, and their pear flesh turns brown. While the electrophoresis results of the last 8 single plants with difficult browning all have one band, indicating that the single plants with difficult browning show the deletion of the InDel-90 marker sequence, and their pear flesh is not easy to turn brown.

[0048] The above-described embodiments are only the preferred embodiments of the present invention, which are only used to explain the present invention and do not limit the scope of implementation of the present invention. For those skilled in the art of this technology, of course, other implementation manners can be easily made through substitution or change according to the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention. SEQUENCE LISTING <110> Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences <120> InDel-90 Marker Related to the Trait of Pear Pulp Browning and Its Application <130> 2022 <160> 93 <170> PatentIn version 3.3 <210> 1 <211> 202 <212> DNA <213> Artificial Sequence (InDel-01) <400> 1 aattaataat tactcgatag ataatgcttt tgtttgattt aattgctcat aaaatagaat 60 aatatagtac attaaataat ttacaattta tttttcatta aaaaataacg tagattcatg 120 aattttagaa gtttgactgt atattcacat aattcaagaa aaaaggaaaa attgggggtg 180 ttgggatagc agagccgtta gc 202 <210> 2 <211> 205 <212> DNA <213> Artificial sequence (InDel-02) <400> 2 cgtatcaagg tttgtaatta gatgtttgca aaataaagag ttatatagaa taagaaggtg 60 tattaagagt cgtagtatat gattgagaag tgttaagaag actcttctct aaaaaatgag 120 attctttgtg gaccttctat catatcatat ttttataaca atgttttaat gtttgtacgg 180 tttgaattgt aaagtgacag agagt 205 <210> 3 <211> 203 <212> DNA <213> Artificial sequence (InDel-03) <400> 3 aaacatatca ctcccacaaa ttaggaagca tattgttaac gtagatcctt tttgaccctc 60 aaaaaagtat aggcatggaa aaagaccgag taaagctcaa gaagatcaag gtccaaagaa 120 ggaatggaaa tccaaagccc aatggaagta ggaagagtat ttccgagctt tatagctgat 180 agctcaccag cctagcttga cta 203 <210> 4 <211> 202 <212> DNA <213> Artificial sequence (InDel-04) <400> 4 taaacacgct acgaaattcc ataaaagtaa cgggcctcaa aaccaatttg cttcaggact 60 ttgagtcaac aatttgaatg ggcaggccca aagggaggga gtgtcctccg ggtccaaagt 120 ccaaaaggcg cgtccttcta agagctctgc tgaaagaaaa gaatttagaa ctcgaagact 180 gagagagatg acgatagacg ac 202 <210> 5 <211> 201 <212> DNA <213> Artificial sequence (InDel-05) <400> 5 cggaccaagt tgttcaagca gttgcaagaa tagcaatccc acaaagaaat ttatcacctg 60 aaaaataccc agtcgcatat aaatgaaacc gtaagtaaac taactacatt gattgattca 120 gaatctgcat agacgaagaa gctaaatcac aaaattatta gagtgctgaa gctacacaat 180 aattagaaac gtagcctaaa a 201 <210> 6 <211> 202 <212> DNA <213> Artificial sequence (InDel-06) <400> 6 tgccaaggta tttcccttga aaccaagatc tttagatatg ctttcaagag gttcattaac 60 taccctgcac aaagcaagag tagaaaaaga aaaggaaggt atattactct cttatgcctg 120 aacataaaac aaactatgca aaagagaggg gaagcagact gtaagcacca acccgagatg 180 gtagccaaac aagaatgaag at 202 <210> 7 <211> 201 <212> DNA <213> Artificial sequence (InDel-07) <400> 7 tctgccattg tccttttaaa atttgctact tttgtggtaa attcatcacg aaagtacaaa 60 aatgaacacc agagcatcac aattgatagt acccagcata ctataaacta cagatgctca 120 ataaattgag ttacctgaat tttcttccac gtcaagagga tccgtcaact cttttgtatc 180 catgtcaatg gtatttgaat g 201 <210> 8 <211> 203 <212> DNA <213> Artificial sequence (InDel-08) <400> 8 gagatcaatg cttttcaatt tctgaagcga ctgttgcata aaaacaaaga caaattgaaa 60 ttaatatcca aaagaaagat ctcataaata ataataataa taaataaaaa aaaataattg 120 attatacctt ggaaccctcc caaacttgta gcagttggct aaactgcatg tctaaaacaa 180 ctagtcttgg ttgattaaga aag 203 <210> 9 <211> 204 <212> DNA <213> Artificial sequence (InDel-09) <400> 9 ctataaattc atacaaacta aattttacag ttttttttca aaaccttacc caaaagtgat 60 cctaagtcct aacatcctct aattttaaaa aataaaatat atataaaaaa aacttctctc 120 tctaatccac attccttttt cctctctctc tccttcccat tttaaaatta aaataaaaat 180 tattttcaca caccaaagtg tggg 204 <210> 10 <211> 206 <212> DNA <213> Artificial sequence (InDel-10) <400> 10 ataaattcat acaaactaaa ttttacagtt ttttttcaaa accttaccca aaagtgatcc 60 taagtcctaa catcctctaa ttttaaaaaa taaaatataa aaagaaaaaa aacttctctc 120 tctaatccac attccttttt cctctctctc tccttcccat tttaaaatta aaataaaaat 180 tattttcaca caccaaagtg tggggc 206 <210> 11 <211> 204 <212> DNA <213> Artificial sequence (InDel-11) <400> 11 tctctctaat ccacattcct ttttcctctc tctctccttc ccattttaaa attaaaataa 60 aaattatttt cacacaccaa agtgtggggc atatgctagt ctatctaaga aaacaagtag 120 gtttagtctc acaatgagtt aacaataaca tagctcaaac tcgtctttga tcaaaactta 180 tgtttgtttt taaatgggag ggag 204 <210> 12 <211> 202 <212> DNA <213> Artificial sequence (InDel-12) <400> 12 agctcaaact cgtctttgat caaaacttat gtttgttttt aaatgggagg gagggaacgt 60 gagaatgaga gcgggaatag aggtttttgt tttttatttt ttatttttta ttttttaata 120 taaaaaaatc acttgtagtt caagcatcat aaaaaacaac aaaatttaat tttatgaaat 180 tacattatct cacattattt tt 202 <210> 13 <211> 201 <212> DNA <213> Artificial sequence (InDel-13) <400> 13 tgatttagtg atttttcaaa atttatcgga atttaaatat ttttaggtta aaatgttcat 60 aaaattaatt taggataatc tacataaatt ttttttttaa aagttaattt aaaaaaaaaa 120 aagccttaat tcattctttg ataatctgga gctaaaattt taagccagaa gggttggagt 180 agaaaagctg tttttgggct a 201 <210> 14 <211> 203 <212> DNA <213> Artificial Sequence (InDel-14) <400> 14 taatttgggg atacaaaaat aggtgagtga cacaaggcaa agctacaccc gacacgaaga 60 tcttaatagt gcctttgcca gtgaagcctc ttgggaacgt tcttccttct catactccta 120 ctcgaggaac accgaggccc aatgaaggaa gatatggtgc aatttgacag ttacgacact 180 gaatacactg gatatcagct aac 203 <210> 15 <211> 209 <212> DNA <213> Artificial Sequence (InDel-15) <400> 15 ttgtaatatg cccaaacagg cgtatgtatg caacaggaca acacattgaa cgcaccacaa 60 ttatctatga gaattattga tcctcgagtc aaaaggccac gagcataaag attaacggaa 120 aaaattaatg attaaccaaa taatggaatc aatgtcaact catgaaacag tagggatagc 180 aaaaggaaac aattagacaa gacaagaca 209 <210> 16 <211> 214 <212> DNA <213> Artificial sequence (InDel-16) <400> 16 tatgaaagat gccaatctcc taatgaagtg aaagaggctt taatgtttct ttataatgag 60 tacatgtcaa tctagttgga tttcggattc caaatctaaa acaccgtttg gtataccagt 120 tcatgtgtta tggtatcatg aatatttgat tgatactgta gccattcaac tctaaagagc 180 tgtggttagt ttatattgtg acgcgtagac tgat 214 <210> 17 <211> 208 <212> DNA <213> Artificial sequence (InDel-17) <400> 17 gaaagctacc actccaacct tctcttttga ccctgtctac tggtgtagca gcttgcaata 60 gtaaggtacc aactccatgc agcttctctc tctctctctc tctctccccc cacaaagtcc 120 ctctataaat ttcatcaatc caaccaaagg attcatcaaa ccaaagtctt caaatttcaa 180 gtctttttct ccaagaaacc acattatt 208 <210> 18 <211> 217 <212> DNA <213> Artificial sequence (InDel-18) <400> 18 agaatcccaa aatttattga ggtttcaaaa cttgaattat cctcgggtga ctaatttcac 60 ttcgaaagat gggacccaat tagtttgaaa ataagcctag tctagacact ttccaacgac 120 actgtctaag tactggtttg gtattgatat atgcttttat aaaaaaataa gcataaaaaa 180 aaaaactgag ctaaaaaaaa tgtttggtaa acattta 217 <210> 19 <211> 217 <212> DNA <213> Artificial sequence (InDel-19) <400> 19 ttttcgtcgt attgtaaaac tggttcaaca ttgcaggatt tctcttctct acgaaaaaat 60 aacctttaaa tcgcgcggct aaaagaacaa tttagatggt gcgaggcaat ggctttcaca 120 ctaaaggaga ttatcaagtt gtgcatgtag ttcttgtatg gaaggcatgt gtatgatgcc 180 ttgtacctct tccgctgaat actcgtcctt cagcagg 217 <210> 20 <211> 203 <212> DNA <213> Artificial sequence (InDel-20) <400> 20 gaatagtata gttcgtgttg acgggttctg caaataaagc ttcattttcg tcattaccat 60 tggcaactct tgctgaattg cagcagctac ctgttccaga gtaacattat gagacttagc 120 tgttgttatc aaatatgtag accgcaaaac aagaacccta gcagtcaaat ttatgatggc 180 cctccctaac atggctctcc act 203 <210> 21 <211> 219 <212> DNA <213> Artificial sequence (InDel - 21) <400> 21 ttgaaacgtc ctctgcagaa gatggaaaaa aatgaacaaa aagttggtgc tctagctggc 60 agacctgcaa taaatagtgg tttcaattac aatggaaact tttctgtcct acaacataat 120 gttcacaacg tatgcggctc aaattcatac atgagaataa gttgcatatc acattacatc 180 aaatatcaaa gatactgccc gctatcatta aagaggaca 219 <210> 22 <211> 203 <212> DNA <213> Artificial sequence (InDel - 22) <400> 22 ccgcttgctg ctgctcagat aacggagttt tctgcagagg aaagaaagca ctacatcaaa 60 attcatatcg agaggatccg atccgatccg atctgaagca agcctaattg caataaagcg 120 gagaaaattg cacaaaactc agatgtagac gaaaaaggcg ttgtttcaat gaagaatttt 180 gaatctatgg caacgaggag gag 203 <210> 23 <211> 213 <212> DNA <213> Artificial sequence (InDel-23) <400> 23 atggctccag atttcggaac gccgggtttg cttgccgcca tttctcaatc ctcttcaact 60 cacaacaaag tcggttcata caacggttgc agtcgccgtc gccgtcgccg tcacgtcatc 120 cacaattcgc ctcctctcct ccgccctctt ttctcccagt cgagtttttt ctcagtcctt 180 cgctccctct tccgacgata aagatttaaa cgc 213 <210> 24 <211> 201 <212> DNA <213> Artificial sequence (InDel-24) <400> 24 ttttttttaa ttattatacc tttattttaa aaacattatt tgaatttatt taaattttat 60 aatttgtggg atattgatca atgcataatg tgcagagttt tttttatttt attttttttt 120 aacaaataat attatctaaa ttgaggggga gggtgggctt agcctcgtaa tagactagca 180 ataatgtggt ttaaattcac t 201 <210> 25 <211> 201 <212> DNA <213> Artificial sequence (InDel-25) <400> 25 cttgtggtgg aagcatgagc catgatgtta ccaccgatag gcttaatttg aggaccggca 60 aagattgtag aaccatccac ttgtgcaacc acttgatttg tatcacaacg gccacgccaa 120 actgcgttca cagtacagag gggaaatccg gatcaataac aatgctaatc tagggggtcc 180 aagtatggtg ttgtgattag t 201 <210> 26 <211> 201 <212> DNA <213> Artificial Sequence (InDel-26) <220> <221> misc_feature <222> (111)..(137) <223> n is a, c, g, or t <400> 26 atgggtcaat aacctatgcg atggtcctat ggatgatcgt gtttgtggat gatataaact 60 aaatcccctt cttgccttca taacgtggat gttgaatgca gcattccttt nnnnnnnnnn 120 nnnnnnnnnn nnnnnnncat tcctgtggtt taaatctgtt caaactgcta acaaggaagc 180 tgaggagcaa gacaagaggg a 201 <210> 27 <211> 205 <212> DNA <213> Artificial Sequence (InDel-27) <400> 27 cgaaaagact tctcttttaa gacagagcag tttaaaaact gagatgggta agaaagcaag 60 gaatgaaaaa tctaacttaa ccatttcaat tgaacggact ccttcaagag taatcccatc 120 tggaaattca ccatccaagc cgccagagca gccggagaaa gcaaggaaga ttactgccgt 180 tgagatcaca caagttagtg aaggg 205 <210> 28 <211> 221 <212> DNA <213> Artificial Sequence (InDel-28) <400> 28 gcagcttact cccccgagct tggctgacgt tgtgcagcta ggcttgctac tggggtgggt 60 tgggcctcct gcccagggtt ttggggattg ggctgctgca aaggcagtga tgcatgcatc 120 tggcagtggc ctggcagcct tgtcatggct gttgggtcgt agcgacggta atggcgtggc 180 tccacgcttg tgggacttct cccgccattg cgttaagcct t 221 <210> 29 <211> 209 <212> DNA <213> Artificial Sequence (InDel-29) <400> 29 caatgctact caacactgac cacatcttct ccaacccacc caagtttcat tctttattta 60 caaattaatt tattgggtcg gattaaattg ttccgaagga caacaagaaa gaagaagaag 120 ttgcagaagc agaaatggtg gagatgaagg aggaagagtc tcagaaaatt gcagttttca 180 aaggcaggga aatattcggg tccaatgct 209 <210> 30 <211> 218 <212> DNA <213> Artificial sequence (InDel-30) <400> 30 taaagtgtaa aatgagattt ttggagtact aataacaatt tcctatatat attaaaccaa 60 aaatgaagta tttttcaaaa gcacaaaaga ataaaataaa ataaaataaa agaagaagag 120 aagaagatat taagcttaat tacatttctt ccattaaatt tgacatggat acattgtcaa 180 atcaatgtac ccatataagt ttaaaaaatg ggttagaa 218 <210> 31 <211> 206 <212> DNA <213> Artificial sequence (InDel-31) <400> 31 tgatatccac acactctatt ttacttttca tacacttttt taattttctg ctgttaggtt 60 ggatgaattg aaaaagatca acgaacataa attatcaagg agtgtgtgtg tgagaagtaa 120 aatggggtgt gtggatagca catccctttt gaaaatgttt tgaattcaaa atcatttaaa 180 agtttaataa gggtgtgagt ataaaa 206 <210> 32 <211> 213 <212> DNA <213> Artificial sequence (InDel-32) <400> 32 atgtatatgt gcacacacaa agtgtgtgag aaaatttttt aatcttgttt ttgaaataga 60 atgagagagg aagagactga tagagaatgt gggggtggga agtatttttt tttatttttt 120 ttaattagat atatgttatg ataggttttg aaaaaaaaaa aaaaaatttg attatgtgaa 180 attacatttt tgccctatat ttcttattca tgt 213 <210> 33 <211> 212 <212> DNA <213> Artificial sequence (InDel-33) <400> 33 aatggaacgc aaataaccat cgcctactca ccatccgcaa aactatacac cagaagtcaa 60 ataatcgaaa aaaaaatgag aacaaacaca tttatcccac aagataagca tggccccaat 120 cttccatgtc tccagtactc atgatctctt gccaactatc cctctgcacc tacttcaaaa 180 gacaacgaag ccctaatatt atcattaagg ta 212 <210> 34 <211> 208 <212> DNA <213> Artificial sequence (InDel-34) <400> 34 atttcaccat gtagtatgtg atccgatttg tttatttttc tcaaaacgaa aggttgacct 60 tgcaaaaact cactgaaatt aaattattta gcattttcta actagctata tgatcaaata 120 aattgataaa cataagcttt tgataaagaa tgcagatgct tgacacgata atcaaacaga 180 taagcgatta taatacaaca tgatcttg 208 <210> 35 <211> 209 <212> DNA <213> Artificial sequence (InDel - 35) <400> 35 gttccaaaag caattgaagt gctttctgca agaagcacta gttatatgct tcttctatga 60 agcattttag gtgtttttct agaatttact tgcatttaac taaactaaaa gaattggttc 120 aaaaaatatt ttaaccaaat acactttgaa tcattttaaa agtacattca aacgaagtag 180 tgttactttt aaaaaacact taaaaagtg 209 <210> 36 <211> 202 <212> DNA <213> Artificial sequence (InDel - 36) <400> 36 tattggtaca aggctttgga gaggtgtgat cagattgggt ggctatgata aggtatgcaa 60 cgtgcatttt taccttttag tcgtaaaggt ccatatgtta gtatatgtac ttatatatat 120 atattatgag ttttcttttc cagaaccata ttcttttctt gttacaatca catatatgat 180 ctgcctgctt accgtaggtg ac 202 <210> 37 <211> 202 <212> DNA <213> Artificial sequence (InDel-37) <400> 37 ttttgtgtta tctttttact gtttgatgta gttattgaat tgttaattaa gaatttaata 60 gaatgcatga aagttacgaa gtttataaat cagaatgaga ttcaaaagtt aatagaagtg 120 aaaatgttat aatagctcac tcgcaaatcg acgaacctcc tccatttgag ttcctccaaa 180 ctcagctctt gcaagaaaat gc 202 <210> 38 <211> 201 <212> DNA <213> Artificial sequence (InDel-38) <400> 38 atgaacaaca ggcaattcgg acgagccgac ggatgcaaga aatgtaacat ctaaatttac 60 atgtagtcag ttcaaagttc attacaaagc caaatggagc gaaaaaaaaa aagaaaaggt 120 aacgaaataa ggccgatttc tataagacgt gaagttgtga atcctacctc agctatccaa 180 tcctacctca tagctcacct g 201 <210> 39 <211> 201 <212> DNA <213> Artificial sequence (InDel-39) <220> <221> misc_feature <222> (109)..(146) <223> n is a, c, g, or t <400> 39 ctgtgactcg ccgagtcccc gccacctgtg agtttatcaa accctgcagg gtacactggc 60 ggaagcggtg gttaaagatt cagttatact ggtgcctggc gtgaaccgnn nnnnnnnnnn 120 nnnnnnnnnn nnnnnnnnnn nnnnnntgaa ccgccttttt ttcttcattc gttttcgaaa 180 cccgcgagct cggagagctc c 201 <210> 40 <211> 201 <212> DNA <213> Artificial sequence (InDel - 40) <400> 40 tgcagagttc ttcgccaggt ggataagaaa tatattgcag ttacagccgg tactacactt 60 gctgttattg accaggtctg tttgttgtat attttttcag gtcttttttt tttttttttt 120 tttttttttg agttttagtt tgggagttat gctcttatat aaatcagcag tttcttgtta 180 aataaggggc acctacagtt c 201 <210> 41 <211> 208 <212> DNA <213> Artificial sequence (InDel - 41) <400> 41 tgaccaggtc tgtttgttgt atattttttc aggctctttt tttttttttt tttttttttt 60 ttgagtttta gtttgggagt tatgctctta tataaatcag cagtaagtgt ttcttgttaa 120 ataaggggca cctacagttc acttatagat cacaacaaaa gtttgcatgt atctaatcac 180 gttctttact cacccaagag tggccttc 208 <210> 42 <211> 207 <212> DNA <213> Artificial sequence (InDel-42) <400> 42 catccatcct gagattgctc tcgcgcgaac tcgcttaact cagacaactt cttcagtttt 60 gtttctggaa actcatacaa gaacttccca gtgggtcacc caacccatcc tgagattgct 120 ctcgtgcgaa ctcgcttaac tcagacgaga caacttcttc agttttgttt ctggaaactc 180 atacaagaac ttcccagtgg gtcaccc 207 <210> 43 <211> 201 <212> DNA <213> Artificial sequence (InDel-43) <400> 43 tctggaaact catacaagaa cttcccagtg ggtcacccgt cctgagattg ctctcgtgcg 60 aactcgctta actcagacga gaacttccca gtgggtcacc cacccagtcc tgggattgct 120 cttgtgcgaa ctcgcttaac tcagacgaga acttcccagt gggtcaccca tcctgggatt 180 gctctagcac gaactcactt a 201 <210> 44 <211> 202 <212> DNA <213> Artificial sequence (InDel-44) <400> 44 tccctcgcag ctcgcatctc agtcgttctt tcacctcctc gagaaaatct tctataccac 60 ctttcctctc tagagtgttt gaagaacctg cccaagtctc ttcatggttg caaagtctat 120 caggaccagg tcttaagata actatccttg taggagcaga agatgcgtcg aatgactcga 180 atctcttctt actgttcatc ag 202 <210> 45 <211> 216 <212> DNA <213> Artificial sequence (InDel-45) <400> 45 gaataataga gcaataatta aacaggaatt caatctctca attgcatttc ctgcaacaga 60 aaagttcaga acctgaaaat gaagataagg agagagataa ggggagagag agagagagag 120 agagagagag agagagagat ggtaattcta atcccaaaat agttggggaa atttgcaaac 180 aaattttaga gaactcaaat tacaagcaac atgata 216 <210> 46 <211> 203 <212> DNA <213> Artificial sequence (InDel-46) <400> 46 atttaatata tttttttatt tttttatttt ttaattatta aaatatatat ttaaatatat 60 tttaatccaa ttggattagt aagtgacccc cgtatctagt caccgtcagt acttaacaaa 120 gaaacaaaca gaaaaactga cggaggtatg acattggcac aaattcataa gatgaggtat 180 gacattgtca ttttaaaaag atg 203 <210> 47 <211> 203 <212> DNA <213> Artificial sequence (InDel-47) <400> 47 tacactctga aggttagatg caattacaac ctcatacatc acctttaaaa tattttaatt 60 ttatatatgt acttgttatt tcatttcaat gtcatacatc gtttacattt tatgtcaact 120 tgactgttaa atgattgccg atgtgagtct aaatttatgt catacgaata aaaaagtagt 180 aaacaatata tggtatcatg taa 203 <210> 48 <211> 202 <212> DNA <213> Artificial sequence (InDel-48) <400> 48 tttaaaatat tttaatttta tatatgtact tgttatttca tttcaatgtc atacatcgtt 60 acattttatg tcaacttgac tgttaaatga ttgccgatgt gagtctaaat ttatgtcata 120 cgaataaaaa agtagtaaac aatatatggt atcatgtaaa atttagtaca aagcttcgac 180 aaaaatctaa taaaaaaata gt 202 <210> 49 <211> 201 <212> DNA <213> Artificial sequence (InDel-49) <400> 49 ttcaaaagtg tgaaaaataa gttattttta agtgtttatc aaatactttt tttagcttac 60 ttttttttat acctactttt tataaaagta tatcaatacc aaccagtact aaaatattat 120 cataggaagt gctgcaatat aataaagcaa aagccttggg cgcacctaat gcgcgtatgc 180 cgtgcataaa atgatgaaga a 201 <210> 50 <211> 202 <212> DNA <213> Artificial sequence (InDel-50) <400> 50 tacttacatg aagctgctca gcgatcttat tgccaggtcg aacgtaaacg tccttgaaca 60 agtcgatctc tgggaactta gaaccctcct aaaaaaaaaa aaccattagg aaaattttat 120 taatcaataa taaaaaataa attgtataaa atttcaaaat taatatactt ttacctgacg 180 tcgtgtctca agcctatacg aa 202 <210> 51 <211> 201 <212> DNA <213> Artificial sequence (InDel-51) <400> 51 actagtttgc agtttctacc acccaaactg attaagcaaa ggaaaaaaaa agtcgttgac 60 tttgcgtgac atatgtggcg tcgggcaaaa cagctttgcg cacgcacgtg cacttacgtc 120 gcgcaaagct gttttgcgtg acgccacata cgtcacgcaa agatgttttg cgcgacgtaa 180 gtgcacgtgc gtcgcgcaaa g 201 <210> 52 <211> 202 <212> DNA <213> Artificial sequence (InDel-52) <400> 52 cataaacagg tatacccata cccatacctg tttataaacg gttaaccata cacataaccg 60 catacctact tactcataat cgcgtatccg tttactttat ttatttattt tttttactcg 120 tctacctatt tttttaacaa cttgaaaatt aaatagaaat ttgtcataat tttctttttt 180 ctaactatta aataccgtta ta 202 <210> 53 <211> 201 <212> DNA <213> Artificial sequence (InDel-53) <400> 53 attggacata tttcaagtta gtttatttac acataacaaa tttggaagga agaagttaat 60 ttaaatcaca tattaatgaa actctttttg tcaactataa ggggtgaaaa aacatttttg 120 tcttctatca caaaataaaa ttatggacaa taaagtaatt tgcacaaact aaattttata 180 cttgttttta aaagcatcat c 201 <210> 54 <211> 201 <212> DNA <213> Artificial sequence (InDel - 54) <400> 54 tattatctat tttttcttgt caatccggct ctggctttat aacaacaaag atatagatat 60 agagtcagcc aaattgatca gagcaatagt gtagatgtct taggcctccc gcaaaatcct 120 ctcaatcagt ccctaaaatc ttataatttg tcaaagaaat tattttcttt ttgggttatt 180 tctttagcaa gtgcaccaat t 201 <210> 55 <211> 201 <212> DNA <213> Artificial sequence (InDel - 55) <400> 55 accggaaata tagaggtgag cgtgagaaca caagcggttt caaggcttgc atttgcatct 60 atagccatat gcaagtacat ctatacatac attgtgtgtg tcaaaacaca gagagagaga 120 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga 180 gagagagaga gagagagaga g 201 <210> 56 <211> 201 <212> DNA <213> Artificial sequence (InDel-56) <400> 56 ttctcacttg tactgattct tatctcagtc atgtcgtgga ttgctagtac tggtacctgg 60 tatccattct tagaccaagt aacgttgcct tgcttgtacc agctgaggct gagatatatt 120 ctgtaaacaa gcatgaaaga tggttgtatt aatttactac acacaaccaa cttcaaaaag 180 agtaggaaga aagaatataa a 201 <210> 57 <211> 201 <212> DNA <213> Artificial sequence (InDel-57) <400> 57 gctaggatat ttgcatttca gaaaagagtg tctttaattt agagttacct cttgtgttct 60 tgttaaaagc catagctgca aaaccgactt catgcattaa tcccaaatca ttgtataaca 120 gggttgcatt ctacctcctg catcacaaat gttcgtagaa aacgaaaatc tttagaagaa 180 gaccaattgt ttcctcaatt a 201 <210> 58 <211> 209 <212> DNA <213> Artificial sequence (InDel-58) <400> 58 gttgtaagtg tgattaaaag tttataaccg gttttttttt tcatatttat tttcagctac 60 tgccgactta gtaaaatgct tgaccaaaga tttttttttt ttttttttgc gtaaaatgac 120 tcttctgtac tgaacatacg accgcaaagc ttttagattt ttttttggac cgagagagag 180 agagtgcctt gtttgggagc gcgaaattc 209 <210> 59 <211> 209 <212> DNA <213> Artificial sequence (InDel - 59) <400> 59 tcttgggata taatgctaca gttaccaatt tttggatacc aactgatgtg acaatttctt 60 atttttgtga atagataagg tccaccgccg gcgtacacat aaaactaata aacaccacca 120 cataaattgg tatccaaagt cgttttaata agttggcgac tctagcgtta ctctgggaca 180 ctaaagtaag aaggataaaa tgtttttat 209 <210> 60 <211> 201 <212> DNA <213> Artificial sequence (InDel - 60) <400> 60 gtcttctggg ttggaaaagt aacatttaca aactttgatt tgaattttga tgtcaactct 60 ggagcttttg atctctgtgc gtgctcctct ctctctctct cctctctcct ctctgtgtgt 120 attgtttctc tctctagaga gtagagagac ggtggagttt gggagtttat cctgatgtgg 180 tgaaggcaca aaaggacgga c 201 <210> 61 <211> 212 <212> DNA <213> Artificial sequence (InDel-61) <400> 61 aaaggacgga cactgagctg cagcaggggc aatgtggctt tgggcagaga gccagtgggt 60 gccttgttta tatttgaatt tagtgactaa attgccctca ccataagtct ggtcgtctgt 120 gtcagtctct ggaactgaaa cagtcagcat ccaaaggttt gagtttatga gtattgtact 180 ttacaaaagc ttattttgtt gaaaaaacta at 212 <210> 62 <211> 217 <212> DNA <213> Artificial sequence (InDel-62) <400> 62 ctacccattt caagggtgtg aatcatgtga tgtcctttgg aaatttagac ataaacataa 60 agtaaattcc tttttctttc ctttctattt tttagaagag attctctccg gatctctccc 120 actaagtcca caaaattcag tgatccgagt ccttaaaatt tgatccaaca actaaaagca 180 agaggtctct ttaaaagtta taataatttt agacatc 217 <210> 63 <211> 215 <212> DNA <213> Artificial sequence (InDel-63) <400> 63 tggttaccga ctagtacact gtagatccat gtgtagcaga tttttttgaa acatatgcag 60 cagaccaacg gcataaaaaa ttgtacgaac tcacttggaa cgaagtagta ctaggagtac 120 tccatacgtt caccccatac cactacaatt gagaaaacag gattttaacg aaaaatttct 180 agtactgttc attttaagaa aaaactacat tttta 215 <210> 64 <211> 203 <212> DNA <213> Artificial sequence (InDel-64) <400> 64 aaataccgtg agcctctcac aaagttcagt agggcactga gtttgttaat ccagcctcgt 60 ttgtggaatt gtggcaattt tttattgaat gaccctgaaa gtttaattta gtttgctttc 120 gtttgatcga cgtattgact agtgtttacg ttgtacttga atctttatca ggagctactg 180 aaaggggaat cagaggggct gtt 203 <210> 65 <211> 201 <212> DNA <213> Artificial sequence (InDel-65) <400> 65 gtgcatctat aaaaaaagat ggggggacca tcttaagtaa aatcgtgcaa ttaaaatgat 60 gttgggaaca tatttgtctt cttcctcgcg tgaggccaac ctgcaacaaa agatttctcg 120 attccggcct ttacgtataa gcccgcatag ctctcaccta tctccgtcca aatctacaga 180 gttctgccta aaaattgagg g 201 <210> 66 <211> 202 <212> DNA <213> Artificial Sequence (InDel-66) <400> 66 cctaggcgga tgcctactcc ggctaccctc agggccggcc ctccatcttc cagtgtgaag 60 gcagctgtga agaccaatac catattggca caagtgtagt gggagttgct gttactgctg 120 cggtggtgac cctgagttac ttatacgaag tttgtaaaaa tgttgtcatt ctaataattt 180 catcattaga tgtcggtcat gt 202 <210> 67 <211> 224 <212> DNA <213> Artificial Sequence (InDel-67) <400> 67 atgcaaaagt ttcgtccaag acatgaagac tctcgtttag ggcagtaacg tacctatccc 60 tcgtttgtat atacccgtct ctctcaatct ttctcgttac atgattattt aaagtgaaaa 120 cttgggtcac cacagcatgt atatgtatta tgttcaacac ttcattatat aatctagatt 180 ggttttattg ttcaaaactt tgaactcgct aatgaatatt tcca 224 <210> 68 <211> 201 <212> DNA <213> Artificial sequence (InDel-68) <400> 68 ttatgttcaa cacttcatta tataatctag attggtttta ttgttcaaaa ctttgaactc 60 gctaatgaat atttccataa tttccctcaa acgattatct ttgatggcta ccccggcaac 120 cagggaggcc aaccgcgctg gtctcagcaa aggccccggg agttctggcc gtcatgaaaa 180 aacagcaaag ggaagcatga g 201 <210> 69 <211> 204 <212> DNA <213> Artificial sequence (InDel-69) <400> 69 tggaaggctt tggattgaag agaaagaaag cctctgagga gagaagaaga ttggaggaga 60 gggagaggga gagggagagg gagagggaga gagagagaga gggagggaag ggaagggaag 120 ggaagaggat gaagaatctg tgtgacttgt ggggtaatat gagtaaaata tagggattcg 180 aatttttgac cgttgggggt tacc 204 <210> 70 <211> 202 <212> DNA <213> Artificial sequence (InDel-70) <400> 70 acgccatcac aaattttgaa tagttataca aaacatcagc tgcggcttgc tttttctcgt 60 caatctatat aaacaaagcc aatggagagc aaattagagt aagtcgtttc ggcagttaag 120 aagtatctca tcaaacttcc aacaccaaaa accagttgat ttaaaacata aacgaaagta 180 acaaattcaa acaataaaaa cc 202 <210> 71 <211> 201 <212> DNA <213> Artificial sequence (InDel-71) <400> 71 caatctatat aaacaaagcc aatggagagc aaattagagt aagtcgtttc ggcagttaag 60 aagtatctca tcaaacttcc aacaccaaaa accagttgat taaaacataa acgaaagtaa 120 caaattcaaa caataaaaac ctctttccgc cagcagtaca caaactattc aagacattcg 180 aacttgaaaa ggtaccaatg g 201 <210> 72 <211> 203 <212> DNA <213> Artificial sequence (InDel-72) <400> 72 aatttaataa cttttcagat tcgattagga cttccaagca tgattaccaa tattttaaac 60 aaatgggctg tcgactactt tagaaataag taaaaaatat atttttcact gattagacta 120 gaatttccac cttaaaattg acatcttgtt cgtcttaaaa ttgcaacgac ccaattgaaa 180 tcttacccat tactttcaac tgc 203 <210> 73 <211> 201 <212> DNA <213> Artificial sequence (InDel-73) <400> 73 aatctataat ccctattagc aagttcaaaa ttgtacccga tctcacaaac caatcaaaac 60 taaacaatgg agtgagggtg tgcgaaaatg ccgctctcga catggggcta aaaatatcaa 120 ttcatgtgca gaaagtttta aatttttaga catgaaaatg attgggaagt ttagcaatgt 180 tgctgcttgg atatatagtt g 201 <210> 74 <211> 208 <212> DNA <213> Artificial sequence (InDel-74) <400> 74 tcacccatct cctataaaaa aataaaaaat aaaaaaaaag gtttttttga gaaacatgaa 60 tagagctgtt tctacttttg ttattttaca ctcataacac cttttttttt tttttttttt 120 tcatttacta aatatatttt agccccaatt ttttttttct ttctgaaaaa ctgcttttaa 180 aaaaaattta gtgggggtag ctgctgct 208 <210> 75 <211> 201 <212> DNA <213> Artificial sequence (InDel-75) <400> 75 aatttgataa ttcatcatct agaacgtagg atttgtgatc caactcacca gtcactagtt 60 atcacttatc atgtatgttt ggagtgtttt tttttttttt tttttttttt ttaatctaaa 120 ctacacgctc tactgctaat aagattgtgc gtttaaaagc actcgattct ttacgtacga 180 ggaatatttt atctttgtaa a 201 <210> 76 <211> 217 <212> DNA <213> Artificial Sequence (InDel - 76) <400> 76 tgcttttggc cttttcaagt aaacttttct tttgttttac cttctgaaat gaatgcgact 60 agctagctag ctacctcacc tgcatgtact ttggttcaat cgagagagag agagagacga 120 gagagagaga gagatctgcc atctcaagtg agaagtcaca ctcgcacatg tatgatgcat 180 gcagatcacc ttcaacatat ctcaactcaa atcatgt 217 <210> 77 <211> 207 <212> DNA <213> Artificial Sequence (InDel - 77) <400> 77 cactgagttc gccaaagtga caaaagaggt aggggtgagc attgtgtcta aagttaaaag 60 gaagagaaga ttgattcttg gctgcgtgat atgcatgaac aagctaggac tcttgattta 120 tagtcacatg atcggtcaaa cctccaatat tttcatagaa tacgatccaa ataaatgatc 180 tttctagtat tacagacatt tgttaat 207 <210> 78 <211> 201 <212> DNA <213> Artificial sequence (InDel-78) <400> 78 gtcaaactcc cttatttgtt acaaccaatc gctacaacag tataatacaa gtgtagtaat 60 tataaattcc attcccaaaa gaatcttaat gcaaaccaaa cccagggaca ggacattggt 120 gtctaaagca tccaaagcta aggtcatgtt ttccattttt ttaattttct aaattgataa 180 tcaggtcaat atacaaaaca a 201 <210> 79 <211> 202 <212> DNA <213> Artificial sequence (InDel-79) <400> 79 cgcacacatg tgagagagtc aatcattgaa tgaggtaatt gaaacgccat taatttgaat 60 acaaagagga ccaccgaacc acacaggctg cacccacgag cataagagca ggaggtgctt 120 aacagattcc gaatgagcaa ggcaaatagg acagcacgga tttcaacaca cctcatccta 180 aacaggttct cctgagtagc aa 202 <210> 80 <211> 203 <212> DNA <213> Artificial Sequence (InDel-80) <400> 80 ctgatctcta cgtcctacgc tctgtgtcaa tgatcgaagc caccacccgt gtcctgtcaa 60 gctcaacccc atctctagga gtcggatgac cattcaggat gcccaggaaa cccatttttc 120 aacccacaga ttgatcagat tcctgcttaa aacttgccat ctcaacccac gagtgataaa 180 atcccgtccc tccaacaagc tag 203 <210> 81 <211> 215 <212> DNA <213> Artificial Sequence (InDel-81) <400> 81 caactcagtg tgaatggacc aaagcttacc catttacttt ttgctgatga cacgcttgtg 60 ttcctgaaag ctactcctga caattgccgg aatatgtcaa acttgctgaa agcttttact 120 gtcgggcttc tggtcagcaa gttagcttac aaaaatcaac ggtctacttc agcgccaaca 180 cccctatggc aatggcccaa gagctcggtg tcaac 215 <210> 82 <211> 211 <212> DNA <213> Artificial Sequence (InDel-82) <400> 82 gtccccacag tgaaaaatga ttgtttgatc tatagtttct gctcaagtgg ccccgcaatg 60 aatgccccca ctcaattgtt ttttggtgga taaaaaaagt tacattaata aacgtatcgc 120 tagtttactt gcttgtgata ttaattagca ttaattcttt tagttggcca atgttgaaag 180 agcattttca accatgaaat ttctgaagaa t 211 <210> 83 <211> 201 <212> DNA <213> Artificial sequence (InDel-83) <400> 83 atattcgacg tttatgtaat aagtaccaat taaacatcat ccattcaaaa caagaaatta 60 ctgaactaaa cagccaaatg aaattacgtg ttttgttaat cgtttgacaa gagaacatta 120 gttacgatct ggagagacca cttgaagatt tcggggtgtg actactatgt ttttgtctgt 180 taggaaattt gaaagaagag a 201 <210> 84 <211> 202 <212> DNA <213> Artificial sequence (InDel-84) <400> 84 gatggcattg gactccagag gtttcatgtc gacgtagata gcctttcctt tcagagcaca 60 agcttaaggt gtttggagtt gaatgatagc agcatcgata tcgacatcga aaattgggaa 120 gatatacatg aggaatctat tagagaaaaa gaactgaaca tgtaatacat ctttcttctg 180 tgtgtgcatt ttgtaagagt tt 202 <210> 85 <211> 205 <212> DNA <213> Artificial sequence (InDel-85) <400> 85 agttcttcat attcttagcc gttttcttca tgtttgaaac tgggaaacct cttttcatat 60 ttaaattctt tttaggaaaa atcgctttcc tttttttttt ttttattttt ctaactctcc 120 tttcttgagc ctaaagttag aacattctag aagtggaata ttcagttatg ttgtcgagtt 180 taatcataac atgtaaaatt gtaaa 205 <210> 86 <211> 201 <212> DNA <213> Artificial sequence (InDel-86) <400> 86 gcaccaaaaa atccgtagac aaagaaggga agtagtggaa tatgaggaga tcaaaagcca 60 agacttaccc taactccact cttggccaac cctctactcc tccatactta cacagttgaa 120 ccaagtctcc aaccaaaatg tggctccact tctcactagt ttctcactcc cctctctctc 180 tctctctctc tctctctctc t 201 <210> 87 <211> 206 <212> DNA <213> Artificial sequence (InDel-87) <400> 87 gttatctaat ctaatattta tatgtaataa tgttttatat ttaaaaattg attttgttat 60 gcaataatgt tctatattta aaaattgttt ttgttagcat tttaaatttc attgtgtact 120 ccttataaac atattttttt ttcctttgta acaccttaaa aatggtgttc ttttttcaat 180 tttattatat aataatgtta tgtata 206 <210> 88 <211> 201 <212> DNA <213> Artificial sequence (InDel-88) <400> 88 gtcaaacaat ttgcaaattc tcctaatatc tttatagtaa agacatgcaa gattgtgttt 60 ttatactaga aacatgcaca atcatttttt tttaaatgtt atgacaactt tcaaagagat 120 acaagtaaat gaaaatttaa ggagacttgc ataaactcaa tgttaactca acaaatatat 180 gcgaaaaaat cataacttct t 201 <210> 89 <211> 201 <212> DNA <213> Artificial sequence (InDel-89) <400> 89 tgtctaatca cccctcaact ttcatgctat tcattaactt cctaattttt ttcaagccat 60 attgattaaa ttctagaaaa atgatttcta cacttattta cttttttttt attttttttt 120 acacacctgt taatttatgt ttctttgtat taattaggct tatctaattc gaaggcaaaa 180 ggtaaagatg catagagcag a 201 <210> 90 <211> 203 <212> DNA <213> Artificial sequence (InDel-90) <400> 90 gggccgccat ctctccacca ccgacgatca aacgacgccg cttgctttcg aatacggaaa 60 catcacgtat cggattagtg catttggaca gtaaggactt ttcctcctct catttttacc 120 ttaaagttca tatatttttg gtgttgaaat tgatggggtt tttgtaattg ttgatttttt 180 cctgcgttcc tgaattttta cag 203 <210> 91 <211> 224 <212> DNA <213> Artificial sequence (InDel-91) <400> 91 ggcattttgt tttggaggta gaatttacac ggcgattttt ggtatattat tcgtagttat 60 agcaacatgg ctttgtacgt aagattttag ttgtgactac tgaccacaga ccacagacca 120 cagaatcttt ttttttttgt tacaataata aatgattttt agtttttggt ttattctttg 180 gaatcgagac ccttctagat cttgcaatcc agagacgatt gatt 224 <210> 92 <211> 212 <212> DNA <213> Artificial sequence (InDel-92) <400> 92 cttttctagg atgcgcttgc ttatatcctc taagccagta ttaaaagtga tctccatatc 60 ctggtcattg tcttcaccgt ctccttctga cgcatcttca tctccttctc cttccttctc 120 ctccttctcc ttctccattt ccagacagga gtaaagcacg gtacatatct cgttttttac 180 tctttttatt agtcttgtcc tctgcagcat ca 212 <210> 93 <211> 203 <212> DNA <213> Artificial sequence (InDel-93) <400> 93 gcgcttgctt atatcctcta agccagtatt aaaagtgatc tccatatcct ggtcattgtc 60 ttcaccgtct ccttctgacg catcttcatc tccttctcct cctttctcct tctccatttc 120 cagacaggag taaagcacgg tacatatctc gttttttact ctttttatta gtcttgtcct 180 ctgcagcatc atcactctcc tcc 203

Claims

1. InDel-90 marker related to the browning trait of pear pulp, characterized in that, The InDel-90 marker is located at the position of 17958988-17959190 bp on chromosome 2 in the genetic linkage map of 'Mantianhong' × 'Hongxiangsu'. The nucleotide sequence of the InDel-90 marker is as shown in SEQ ID NO.90, with a full length of 203 bp. The forward primer of the InDel-90 marker is 5'-GAAACATCACGTATCGGATT-3', and the reverse primer is 5'-TCAACAATTACAAAAACCCC-3'. Single plants with easily browning pear pulp show an insertion of the InDel-90 marker sequence, while single plants with not easily browning pear pulp show a deletion of the InDel-90 marker sequence.

2. A kit containing the primer pair of the InDel-90 marker related to the pear pulp browning trait described in claim 1.

3. Use of the kit described in claim 2 in the identification of pear pulp browning varieties.

4. The application according to claim 3, wherein The specific identification method includes the following steps: a. Extract the genomic DNA of the pear material to be detected; b. Perform PCR amplification on the genomic DNA of the pear material to be detected using the primer pair of the InDel-90 marker; c. Detect the PCR product by electrophoresis. If there are two bands, the pear pulp is easily browned; if there is one band, the pear pulp is not easily browned.

5. Use of the detection reagent of the InDel-90 marker related to the pear pulp browning trait described in claim 1 in pear molecular marker-assisted breeding.

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