InDel-85 marker related to pear flesh browning trait and its application

Abstract

The invention belongs to the technical field of pear molecular breeding, discloses an InDel‑85 marker related to a pear flesh browning trait, the InDel‑85 marker is located at the 17936028-17936232 bp position of chromosome 2 in a 'Mantianhong'×'Hongxiangsu' genetic linkage map, the nucleotide sequence of the InDel‑85 marker is shown in SEQ ID NO.85, the sequence is 205 bp in length, the forward primer of the InDel‑85 marker is 5'‑TTAGCCGTTTTCTTCATGTT‑3', and the reverse primer is 5'‑AGGCTCAAGAAAGGAGAGTT‑3'; the pear flesh browning individual plant is characterized by the insertion of the InDel‑85 marker sequence, and the pear flesh browning individual plant is characterized by the deletion of the InDel‑85 marker sequence. The InDel‑85 marker designed by the present invention can be used to identify whether the pear flesh is prone to browning, has good application value, and can be used for pear molecular breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of pear molecular breeding, and relates to an InDel-85 marker related to a pear flesh browning trait and an application thereof. Background Art

[0002] In recent years, fresh-cut fruits have been particularly popular with consumers for their freshness and convenience. However, the flesh of many pear varieties will quickly turn brown when exposed to air, resulting in poor appearance and destroyed nutrition, which greatly reduces consumers' desire to eat. Exploring the genes that control the browning of pear flesh and developing molecular markers related to pear flesh browning can accelerate the selection of pear varieties with browning resistance and reduce breeding costs. It is of great significance to extend the shelf life of fresh-cut pears, solve the restrictive factors of pear processing, and promote the diversified development of the pear industry. In terms of the physiological and molecular mechanism analysis of fruit browning, researchers found that flesh browning is a physiological and biochemical process in which phenolic substances in the flesh tissue are catalyzed by polyphenol oxidase to quinones under the action of oxygen, and then enzymatically browned to melanin, turning the fruit tissue brown. Chlorogenic acid is the main browning substrate of PPO. Boss et al. cloned the cDNA of polyphenoloxidase (PPO) from apple peel. Haruta et al. cloned two DNA fragments encoding apple PPO through PCR reaction and confirmed that the PPO in the fruit of Rosaceae plants is very similar. Guardo et al. located the genes related to the browning trait of apple flesh on the genome. Among these genes, PPO and PAL are most closely related to the browning of flesh. Gan Huifang used RNAi technology to introduce the apple PPO gene into pears and found that the interference effect was not as good as in apples, and the effect of inhibiting the browning of flesh was poor.

[0003] To date, the molecular mechanism that controls the browning of pear flesh still needs to be studied in depth, and there are no practical molecular markers required to assist in the screening of individual plants in hybrid populations that are not susceptible to browning, making it difficult to provide effective support for the breeding of new pear varieties that are resistant to browning. Summary of the invention

[0004] The purpose of the present invention is to provide an InDel-85 marker related to the browning trait of pear flesh, which can be used to identify whether the pear flesh is prone to browning, has good application value, and can be used for pear molecular breeding.

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

[0006] The invention provides an InDel-85 marker related to a pear flesh browning trait. The InDel-85 marker is located at the 17936028-17936232 bp position of chromosome 2 in a 'Mantianhong'×'Hongxiangsu' genetic linkage map. The nucleotide sequence of the InDel-85 marker is shown in SEQ ID NO.85, and the full length of the sequence is 205 bp. The forward primer of the InDel-85 marker is 5'-TTAGCCGTTTTCTTCATGTT-3', and the reverse primer is 5'-AGGCTCAAGAAAGGAGAGTT-3'. A pear flesh browning individual plant shows an insertion of the InDel-85 marker sequence, and a pear flesh browning individual plant shows a deletion of the InDel-85 marker sequence.

[0007] The invention also provides a kit containing a primer pair of InDel-85 markers related to the pear flesh browning trait.

[0008] The invention also provides application of the kit in identifying pear flesh browning varieties.

[0009] Preferably, the use of the above kit in the identification of pear flesh browning varieties, the specific identification method comprises the following steps:

[0010] a. Extracting genomic DNA of the pear material to be tested;

[0011] b. Using the primers labeled with InDel-85 to perform PCR amplification on the genomic DNA of the pear material to be tested;

[0012] c. The PCR product is tested by electrophoresis. If there are two bands, the pear flesh is prone to browning; if there is only one band, the pear flesh is not prone to browning.

[0013] The present invention also provides application of the InDel-85 marker related to the pear flesh browning trait in pear molecular marker-assisted breeding.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention first locates the QTL of the pear flesh browning trait and locates the main effect QTL site PFB. The SNP molecular marker closest to the PFB is Marker02874. The two-year positioning results show good repeatability, verifying the accuracy of the results. Then, 93 pairs of InDel markers are used to verify in offspring single plants with extreme traits (browning prone and non-browning prone). Finally, according to the phenotype and the electrophoresis results of the 22 pairs of primers, it is determined from the 22 pairs of InDel markers that the InDel-85 marker can be used to identify whether the pear flesh is prone to browning, has good application value, and can be used for pear molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the phenotypic identification result of the test material of the present invention.

[0017] Figure 2 This is the location map of the main effect QTL related to the pear flesh browning trait.

[0018] Figure 3 The mapping results show that 22 pairs of InDel markers were relocated on Chr2.

[0019] Figure 4 This is the electrophoresis result of the product amplified by the primer labeled InDel-85. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the 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 conventional methods unless otherwise specified.

[0021] Embodiment 1

[0022] 1. Phenotypic Identification

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

[0024] Table 1 Results of browning grade identification of F1 progeny population

[0025]

[0026]

[0027] 2. Construction of genetic map and QTL analysis

[0028] A high-density genetic map constructed from '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) was used to screen out the genotype data of the isolated population of the experimental material of the present invention.

[0029] Combined with the phenotypic identification results, MapQTL5 software was used to conduct QTL analysis on pear flesh browning. The LOD threshold of each linkage group was estimated based on the permutation test (number>1000), and then the interval mapping method was used to detect the position of markers related to the pear flesh browning trait on the linkage group to determine the presence and location of the QTL controlling the pear fruit browning trait. The results are shown in Figure 2. Figure 2 shown.

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

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

[0032] The molecular marker Marker02874 is located at the 176410bp of the genome Scaffold of 'Dangshan Pear' with accession number NW_008988236.1, and its base is G. From the positioning results of the two years in 2020 and 2021, it can be seen that the QTL positioning showed good repeatability, which verified the accuracy of the results. The QTL locus PFB is a major effect QTL.

[0033] 3. Fine positioning

[0034] The positioning results in 2020 and 2021 were reproducible, but the main effect QTL interval was large. In order to further locate more accurately, 93 pairs of InDel markers were designed in the located segments and screened and verified in the offspring of 8 easy-to-brown plants (offspring plant numbers 14, 112, 157, 183, 248, 265, 310 and 313) and 8 not easy-to-brown plants (offspring plant numbers 19, 50, 77, 97, 113, 263, 337 and 357). The 93 pairs of InDel marker sequences were designed according to the reference genome of 'Dangshan Pear', as shown in Table 2. PCR amplification conditions: pre-denaturation: 94℃2min; denaturation: 94℃30S; annealing: 55℃30S; extension 72℃1min; 30 cycles; 72℃10min. The results showed that 22 pairs of InDel markers showed trait separation in the two groups of extreme trait plants, and the phenotype and genotype consistency of the browning-prone plant group and the non-browning plant group were 75% and 87.5%, respectively. These 22 pairs of InDel markers were relocated 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, and InDel-93. According to the phenotype and the electrophoresis results of 22 pairs of primers, the InDel-85 marker was finally determined from the 22 pairs of InDel markers to be used to identify whether the pear flesh is prone to browning. Figure 4 The electrophoresis results of the amplification products using the InDel-85 labeled primer are shown.

[0035] Table 2 Sequences of 93 pairs of InDel marker primers 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 browning-prone plants (the first 8 lanes) of the InDel-85 marker (SEQ ID NO.85) primer amplification products all have two bands, indicating that the browning-prone plants show the insertion of the InDel-85 marker sequence, and their pear flesh is prone to browning, while the electrophoresis results of the non-browning plants (the last 8 lanes) all have one band, indicating that the non-browning plants show the deletion of the InDel-85 marker sequence, and their pear flesh is not prone to browning.

[0048] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention. SEQUENCE LISTING <110> Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences <120> InDel-85 marker related to pear flesh browning trait 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 attaataat tactcgatag ataatgcttt tgtttgattt aattgctcat aaaatagaat 60 aatatagtac attaaataat ttacaattta ttttcatta aaaaataacg tagattcatg 120 aattttagaa gtttgactgt atattcacat aattcaagaa aaaaggaaaa attgggggtg 180 ttgggatagc agagccgtta gc 202 <210> 2 <211> 205 <212> DNA <213> InDel-02 <400> 2 cgtatcaagg tttgtaatta gatgtttgca aaataaagag ttatatagaa taagaaggtg 60 tattaagagt cgtagtatat gattgagaag tgttaagaag actcttctct aaaaaatgag 120 attctttgtg gaccttctat catatcatat ttttataaca atgttttaat gttttgtacgg 180 tttgaattgt aaagtgacag agagt 205 <210> 3 <211> 203 <212> DNA <213> InDel-03 <400> 3 aaacatatca ctcccacaaa ttaggaagca tattgttaac gtagatcctt tttgaccctc 60 aaaaaagtat aggcatggaa aaagaccgag taagctcaa gaagatcaag gtccaaagaa 120 ggaatggaaa tccaaagccc aatggaagta ggaagagtat ttccgagctt tatagctgat 180 agctcaccag cctagcttga cta 203 <210> 4 <211> 202 <212> DNA <213> InDel-04 <400> 4 taaaccgct acgaaattcc ataaaagtaa cgggcctcaa aaccaatttg cttcaggact 60 ttgagtcaac aatttgaatg ggcaggccca aagggaggga gtgtcctccg ggtccaaagt 120 ccaaaagcg cgtccttcta agagctctgc tgaaagaaaa gaatttagaa ctcgaagact 180 gagagagatg acgatagacg ac 202 <210> 5 <211> 201 <212> DNA <213> InDel-05 <400> 5 cggaccaagt tgttcaagca gttgcaagaa tagcaatccc acaaagaaat tttcacctg 60 aaaataccc agtcgcatat aaatgaaacc gtaagtaaac taactacatt gattgattca 120 gaatctgcat agacgaagaa gctaaatcac aaaattatta gagtgctgaa gctacacaat 180 attagaaac gtagcctaaa a 201 <210> 6 <211> 202 <212> DNA <213> 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> 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> 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 tatttcaca caccaaagtg tggggc 206 <210> 11 <211> 204 <212> DNA <213> 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> InDel-12 <400> 12 agctcaaact cgtctttgat caaaacttat gtttgttttt aaatgggagg gagggaacgt 60 gagaatgaga gcgggaatag aggttttgt ttttatttt ttatttttta ttttttaata 120 taaaaaatc acttgtagtt caagcatcat aaaaaacaac aaaatttaat tttatgaaat 180 tacattatct cacattattt tt 202 <210> 13 <211> 201 <212> DNA <213> InDel-13 <400> 13 tgatttagtg atttttcaaa attatcgga atttaaat ttttaggtta aaatgttcat 60 aaaattaatt taggataatc tacataaatt ttttttttaa aagttaattt aaaaaaaaaa 120 aagccttaat tcattctttg ataatctgga gctaaaattt taagccagaa gggttggagt 180 agaaaagctg ttttgggct a 201 <210> 14 <211> 203 <212> DNA <213> 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> 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> 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> InDel-17 <400> 17 gaaagctacc actccaacct tctctttga ccctgtctac tggtgtagca gcttgcaata 60 gtaaggtacc aactccatgc agcttctct tctctctct tctctcccc cacaaagtcc 120 ctctataaat ttcatcaatc caaccaaagg attcatcaaa ccaaagtctt caaatttcaa 180 gtctttttct ccaagaaacc acattatt 208 <210> 18 <211> 217 <212> DNA <213> InDel-18 <400> 18 agaatcccaa aatttattga ggtttcaaaa cttgaattat cctcgggtga ctaatttcac 60 ttcgaaagat gggacccaat tagttgaaa ataagcctag tctagacact ttccaacgac 120 actgtctaag tactggtttg gtattgatat atgcttttat aaaaaaataa gcataaaaaa 180 aaaaactgag ctaaaaaaaa tgtttggtaa acattta 217 <210> 19 <211> 217 <212> DNA <213> InDel-19 <400> 19 tttcgtcgt attgtaaaac tggttcaaca ttgcaggatt tctcttctct acgaaaaaat 60 aacctttaaa tcgcgcggct aaaagaacaa tttagatggt gcgaggcaat ggctttcaca 120 ctaaaggaga tttcaagtt gtgcatgtag ttcttgtatg gaaggcatgt gtatgatgcc 180 ttgtacctct tccgctgaat actcgtcctt cagcagg 217 <210> 20 <211> 203 <212> DNA <213> 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 atgctctcc act 203 <210> 21 <211> 219 <212> DNA <213> 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 aaattcaaa gatactgccc gctatcatta aagaggaca 219 <210> 22 <211> 203 <212> DNA <213> 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> InDel-23 <400> 23 atggctccag attcggaac gccgggtttg cttgccgcca tttctcaatc ctcttcaact 60 cacaacaaag tcggttcata caacggttgc agtcgccgtc gccgtcgccg tcacgtcatc 120 cacaattcgc ctcctctcct ccgccctct ttctcccagt cgagtttttt ctcagtcctt 180 cgctccctct tccgacgata aagatttaaa cgc 213 <210> 24 <211> 201 <212> DNA <213> InDel-24 <400> 24 ttttttttaa tttatacc tttattttaa aaacattatt tgaatttatt taaattttat 60 aatttgtggg atattgatca atgcataatg tgcagagttt ttttatttt attttttttt 120 aaaaataat attatctaaa ttgaggggga gggtgggctt agcctcgtaa tagactagca 180 ataatgtggt ttaaattcac t 201 <210> 25 <211> 201 <212> DNA <213> 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 ttgtgatg t 201 <210> 26 <211> 201 <212> DNA <213> 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 gcattcctttt nnnnnnnnn 120 nnnnnnnnn nnnnncat tcctgtggtt taaatctgtt caaactgcta acaaggaagc 180 tgaggagcaa gacaagagggg a 201 <210> 27 <211> 205 <212> DNA <213> InDel-27 <400> 27 cgaaaagact tctctttaa 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> 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> InDel-29 <400> 29 caatgctact caacactgac cacatcttct caacccacc 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> InDel-30 <400> 30 taaagtgtaa aatgagattt ttggagtact aataacaatt tcctatatat attaaaccaa 60 aaatgaagta tttttcaaaa gcacaaaaga ataaaataaa ataaaataaa agaagaagag 120 aagaagatat tagcttaat tacatttctt ccattaaatt tgacatggat acattgtcaa 180 atcaatgtac ccatataagt ttaaaaaatg ggttagaa 218 <210> 31 <211> 206 <212> DNA <213> 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> InDel-32 <400> 32 atgtatatgt gcacacacaa agtgtgtgag aaaattttt aatcttgttt ttgaaataga 60 atgagagagg aagagactga tagagaatgt gggggtggga agtatttttt tttatttttt 120 ttaattagat atatgttatg ataggttttg aaaaaaaaa aaaaaatttg attatgtgaa 180 attacattt tgccctatat ttcttattca tgt 213 <210> 33 <211> 212 <212> DNA <213> 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> InDel-34 <400> 34 atttcaccat gtagtatgtg atccgatttg tttatttttc tcaaaacgaa aggttgacct 60 tgcaaaaact cactgaaatt aaattta gcattttcta actagctata tgatcaaata 120 aattgaaa cataagcttt tgataaagaa tgcagatgct tgacacgata atcaaacaga 180 tagcgatta tatacaaca tgatcttg 208 <210> 35 <211> 209 <212> DNA <213> 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> InDel-36 <400> 36 tattggtaca aggctttgga gaggtgtgat cagattgggt ggctatgata aggtatgcaa 60 cgtgcatttt taccttttag tcgtaaaggt ccatatgtta gtatatgtac ttatatatat 120 atattatgag ttttctttc cagaaccata ttctttttctt gttacaatca catatatgat 180 ctgcctgctt accgtaggtg ac 202 <210> 37 <211> 202 <212> DNA <213> InDel-37 <400> 37 ttttgtgtta tctttttact gtttgatgta gttattgaat tgttaattaa gaatttaata 60 gaatgcatga aagttacgaa gtttataaat cagaatgaga ttcaaaagtt aatagaagtg 120 aaaatgttat atagctcac tcgcaaatcg acgaacctcc tccatttgag ttcctccaaa 180 ctcagctctt gcaagaaaat gc 202 <210> 38 <211> 201 <212> DNA <213> 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> 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 nnnnnnnnn 120 nnnnnnnnn nnnnnnnnn nnnnnntgaa ccgcctttttt ttcttcattc gttttcgaaa 180 cccgcgagct cggagagctc c 201 <210> 40 <211> 201 <212> DNA <213> InDel-40 <400> 40 tgcagagttc ttcgccaggt ggataagaaa tatattgcag ttacagccgg tactacactt 60 gctgttattg accaggtctg tttgttgtat atttttcag gtcttttttt tttttttttt 120 ttttttttg agttttagtt tgggagttat gctcttatat aaatcagcag tttcttgtta 180 aataaggggc acctacagtt c 201 <210> 41 <211> 208 <212> DNA <213> InDel-41 <400> 41 tgaccaggtc tgtttgttgt atattttttc aggctcttt 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> InDel-42 <400> 42 catccatcct gagattgctc tcgcgcgaac tcgcttaact cagacaactt cttcagtttt 60 gttctggaa actcatacaa gaacttccca gtgggtcacc caacccatcc tgagattgct 120 ctcgtgcgaa ctcgcttaac tcagacgaga caacttctttc agttttgttt ctggaaactc 180 atacaagaac ttcccagtgg gtcaccc 207 <210> 43 <211> 201 <212> DNA <213> InDel-43 <400> 43 tctggaaact catacaagaa cttcccagtg ggtcaccgt 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> InDel-44 <400> 44 tccctcgcag ctcgcatctc agtcgttctt tcacctcctc gagaaaatct tctataccac 60 ctttcctc tagagtgttt gaagaacctg cccaagtctc ttcatggttg caaagtctat 120 caggaccagg tcttaagata actatccttg taggagcaga agatgcgtcg aatgactcga 180 atctcttctt actgttcatc ag 202 <210> 45 <211> 216 <212> DNA <213> 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> InDel-46 <400> 46 attaatata ttttttatt ttttatttt ttaattatta aaatatat 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> InDel-47 <400> 47 tacactctga aggttagatg caattacaac ctcatacatc acctttaaaa tattttaatt 60 ttatatgt acttgttatt tcatttcaat gtcatacatc gtttacattt tatgtcaact 120 tgactgttaa atgattgccg atgtgagtct aaatttatgt catacgaata aaaaagtagt 180 aaacaata tggtatcatg taa 203 <210> 48 <211> 202 <212> DNA <213> 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> 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 aagaataa a 201 <210> 57 <211> 201 <212> DNA <213> 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> InDel-58 <400> 58 gttgtaagtg tgattaaaag tttataaccg gtttttttt tcatatttatt 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> 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> InDel-60 <400> 60 gtcttctggg ttggaaaagt aacatttaca aactttgatt tgaattttga tgtcaactct 60 ggagctttg atctctgtgc gtgctcctct cctctctct cctctctct ctctgtgtgt 120 attgtttc tctctagaga gtagagagac ggtggagttt gggagtttat cctgatgtgg 180 tgaaggcaca aaaggacgga c 201 <210> 61 <211> 212 <212> DNA <213> 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> deletion (InDel-62) <400> 62 ctacccattt caagggtgtg aatcatgtga tgtcctttgg aaatttagac ataaacataa 60 agtaaattcc ttttctttc ctttctattt tttagaagag attctctccg gatctctccc 120 actaagtcca caaaattcag tgatccgagt cttaaaatt tgatccaaca actaaaagca 180 agaggtctct ttaaaagtta tataatttt agacatc 217 <210> 63 <211> 215 <212> DNA <213> InDel-63 <400> 63 tggttaccga ctagtacact gtagatccat gtgtagcaga ttttttgaa acatatgcag 60 cagaccaacg gcataaaaaa ttgtacgaac tcacttggaa cgaagtagta ctaggagtac 120 tccatacgtt caccccatac cactacaatt gagaaaacag gattttaacg aaaaatttct 180 agtactgttc atttaagaa aaaactacat tttta 215 <210> 64 <211> 203 <212> DNA <213> InDel-64 <400> 64 aaataccgtg agcctctcac aaagttcagt agggcactga gtttgttaat ccagcctcgt 60 ttgtggaatt gtggcaattt tttattgaat gaccctgaaa gtttaattta gtttgctttc 120 gttgatcga cgtattgact agtgttacg ttgtacttga atctttatca ggagctactg 180 aaaggggaat cagaggggct gtt 203 <210> 65 <211> 201 <212> DNA <213> 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> InDel-68 <400> 68 tttgttcaa 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> InDel-69 <400> 69 tggaaggctt tggattgaag agaaagaaag cctctgagga gagaagaaga ttggaggaga 60 gggagaggga gagggagagg gagaggga gagagagaga gggaggggaag ggaagggaag 120 ggaagaggat gaagaatctg tgtgacttgt ggggtaaatat gagtaaaata tagggattcg 180 aatttttgac cgttgggggt tacc 204 <210> 70 <211> 202 <212> DNA <213> InDel-70 <400> 70 acgccatcac aaatttgaa tagtataca aaacatcagc tgcggcttgc ttttctcgt 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> InDel-73 <400> 73 aatctataat ccctattagc aagttcaaaa ttgtaccga 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> InDel-74 <400> 74 tcacccatct cctataaaaa aataaaaaat aaaaaaaaag gttttttga gaaacatgaa 60 taggctgtt tctacttttg ttattttaca ctcataacac ctttttttttt tttttttttt 120 tcatttacta aatatttt agccccaatt ttttttttct ttctgaaaaa ctgcttttaa 180 aaaaaattta gtgggggtag ctgctgct 208 <210> 75 <211> 201 <212> DNA <213> 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> InDel-78 <400> 78 gtcaaactcc cttatttgtt acaaccaatc gctacaacag tataatacaa gtgtagtaat 60 tataaattcc attcccaaaa gaatcttaat gcaaaccaaa cccagggaca ggacattggt 120 gtctaaagca tccaaagcta aggtcatgtt ttccattttt ttaatttct aaattgataa 180 tcaggtcaat atacaaaaca a 201 <210> 79 <211> 202 <212> DNA <213> 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> InDel-85 <400> 85 agttcttcat attcttagcc gttttcttca tgtttgaaac tgggaaacct cttttcatat 60 ttaaattct tttaggaaaa atcgctttcc tttttttt tttattttt ctaactctcc 120 tttcttgagc ctaaagttag aacattctag aagtggaata ttcagttatg ttgtcgagtt 180 taatcataac atgtaaaatt gtaaa 205 <210> 86 <211> 201 <212> DNA <213> InDel-86 <400> 86 gcaccaaaaa atccgtagac aaagaaggga agtagtggaa tatgaggaga tcaaaagcca 60 agacttaccc taactccact cttggccaac cctctactcc tccatactta cacagttgaa 120 ccaagtctcc aaccaaaatg tgctccact tctcactagt ttctcactcc cctctctc 180 tctctctc tctctcttc t 201 <210> 87 <211> 206 <212> DNA <213> InDel-87 <400> 87 gttatctaat ctaatattta tatgtaataa tgttttatat ttaaaaattg atttgttat 60 gcaataatgt tctatattta aaaattgttt ttgttagcat tttaaatttc attgtgtact 120 ccttataaac atatttttt ttcctttgta acaccttaaa aatggtgttc ttttttcaat 180 tttattat aataatgtta tgtata 206 <210> 88 <211> 201 <212> DNA <213> InDel-88 <400> 88 gtcaaacaat ttgcaaattc tcctaatatc tttatagtaa agacatgcaa gattgtgttt 60 ttactaga aacatgcaca atcattttt tttaaatgtt atgacaactt tcaaagagat 120 acaagtaaat gaaaatttaa ggagacttgc ataaactcaa tgttaactca acaaatatat 180 gcgaaaaaat cataacttct t 201 <210> 89 <211> 201 <212> DNA <213> InDel-89 <400> 89 tgtctaatca cccctcaact ttcatgctat tcattaactt cctaattttt ttcaagccat 60 attgattaaa ttctagaaaa atgattcta cacttattta ctttttttt attttttttt 120 acacacctgt taatttatgt ttctttgtat taattaggct tatctaattc gaaggcaaaa 180 ggtaaagatg catagagcag a 201 <210> 90 <211> 203 <212> DNA <213> InDel-90 <400> 90 gggccgccat ctctccacca ccgacgatca aacgacgccg cttgctttcg aatacggaaa 60 catcacgtat cggattagtg catttggaca gtaaggactt ttcctcctct catttttacc 120 ttaaagttca tatattttg gtgttgaaat tgatggggtt tttgtaattg ttgattttt 180 cctgcgttcc tgaattttta cag 203 <210> 91 <211> 224 <212> DNA <213> InDel-91 <400> 91 ggcatttgt tttggaggta gaatttacac ggcgattttt ggtatattat tcgtagttat 60 agcaacatgg ctttgtacgt aagattttag ttgtgactac tgaccacaga ccacagacca 120 cagaatcttt ttttttgtgt tacaataata aatgattttt agtttttggt ttattctttg 180 gaatcgagac ccttctagat cttgcaatcc agagacgatt gatt 224 <210> 92 <211> 212 <212> DNA <213> 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> InDel-93 <400> 93 gcgcttgctt atacctcta agccagtatt aaaagtgatc tccatatcct ggtcattgtc 60 ttcaccgtct ccttctgacg catcttcatc tccttctcct cctttctcct tctccattc 120 cagacaggag taaagcacgg tacatatctc gttttttact cttttatta gtcttgtcct 180 ctgcagcatc atcactctcc tcc 203

Claims

1. InDel-85 marker related to pear flesh browning trait, characterized in that: The InDel-85 marker is located at the 17936028~17936232bp position of chromosome 2 in the genetic linkage map of 'Mantianhong'×'Hongxiangsu', the nucleotide sequence of the InDel-85 marker is shown in SEQ ID NO.85, the full length of the sequence is 205bp, the forward primer of the InDel-85 marker is 5'-TTAGCCGTTTTCTTCATGTT-3', and the reverse primer is 5'-AGGCTCAAGAAAGGAGAGTT-3'; the pear flesh of the individual plant that is easy to brown shows the insertion of the InDel-85 marker sequence, and the pear flesh of the individual plant that is not easy to brown shows the deletion of the InDel-85 marker sequence.

2. A kit comprising a primer pair labeled InDel-85 related to the pear flesh browning trait according to claim 1.

3. Use of the kit according to claim 2 in identifying pear flesh browning varieties.

4. The use according to claim 3, characterized in that: The specific identification method includes the following steps: a. Extracting genomic DNA of the pear material to be tested; b. Using the primers labeled with InDel-85 to perform PCR amplification on the genomic DNA of the pear material to be tested; c. The PCR product is tested by electrophoresis. If there are two bands, the pear flesh is prone to browning. If there is only one band, the pear flesh is not prone to browning.

5. Use of the detection reagent for the InDel-85 marker associated with the pear flesh browning trait according to claim 1 in pear molecular marker-assisted breeding.

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