Development of Insertion-deletion Markers in Ananas comosus of Genome Based on Re-sequencing Data

doi:10.13560/j.cnki.biotech.bull.1985.2024-0728

Abstract

Abstract:

Objective The goal is to develop InDel molecular markers based on genome re-sequencing data, laying a theoretical foundation for innovative utilization of pineapple resources. Method Four representative pineapple accessions were selected for genome re-sequencing to identify InDel loci with insertion/deletion lengths > 30 bp. The InDel marker primers were designed based on the sequence information of these InDel loci, and those having clear amplification bands and favorable separation effects were selected for further analysis of genetic diversity. Result The 1 559 InDel loci with insertion/deletion length >30 bp and sequencing depths > 10 × were identified from re-sequencing data of four pineapple accessions. Based on the upstream and downstream sequences of these InDel loci, 372 pairs of InDel marker primers were designed across the entire genome, of which 264 pairs showed stable polymorphism, with a polymorphism ratio of 70.97%. The 50 of the 264 polymorphic InDel markers were assigned as core markers, further used for the genetic diversity analyzing of 58 pineapple accessions. A total of 103 alleles were identified, and the average effective numbers of alleles (Ne) for each marker are 1.816 5. The Shannon's Information index (I) ranged from 0.272 8 to 0.746 4, with an average value of 0.634 6. The polymorphism information content (PIC) valued from 0.132 9 to 0.425 1, with an average value of 0.342 2. While the variation range of Nei's gene diversity index (H) was 0.143 1 to 0.514 3, with an average value of 0.441 2. Through cluster analysis, 58 pineapple accessions were classified into 4 groups at a genetic distance of 0.75, with group IV can be further divided into 2 subgroups. The 58 pineapple accessions can also be divided into two stable populations through structure analysis, with the POP-1 population overlapping with the Ⅳ-2 subgroup obtained from cluster analysis, mainly composed of smooth cayenne germplasms. Conclusion The 50 core InDel markers developed in this study can accurately distinguish different pineapple accessions, which helps for deepening the analysis of pineapple genetic diversity, improving the construction of genetic map, and accelerating the process of molecular marker assisted breeding.

Key words: pineapple, re-sequencing, InDel markers, genetic diversity analysis

HE Han, LIU Chuan-he, YU Meng-fan, YUAN Meng-ping, WEI Yue-rong, YANG Min, KUANG Rui-bin, ZHOU Chen-ping, WU Xia-ming, XU Ze. Development of Insertion-deletion Markers in Ananas comosus of Genome Based on Re-sequencing Data[J]. Biotechnology Bulletin, 2025, 41(2): 65-76.

Figures/Tables 6

References 42

1	刘传和, 贺涵, 何秀古, 等. 我国菠萝品种结构与新品种自主选育推广 [J]. 中国热带农业, 2021(4): 13-15.
	Liu CH, He H, He XG, et al. Pineapple cultivar structure and extension of new independent breeding pineapple cultivars in China [J]. China Trop Agric, 2021(4): 13-15.
2	王健胜, 贺军虎, 陈华蕊, 等. 47份菠萝种质遗传多样性的RAPD分析 [J]. 热带作物学报, 2015, 36(8): 1392-1397.
	Wang JS, He JH, Chen HR, et al. Genetic diversity analysis of forty seven pineapple [Ananas comosus(L.) Merr] germplasm with RAPD marker [J]. Chin J Trop Crops, 2015, 36(8): 1392-1397.
3	Vanijajiva O. Assessment of genetic diversity and relationships in pineapple cultivars from Thailand using ISSR marker [J]. Int J Agric Technol, 2012, 8: 1829-1838.
4	陈香玲, 苏伟强, 刘业强, 等. 36份菠萝种质的遗传多样性SCoT分析 [J]. 西南农业学报, 2012, 25(2): 625-629.
	Chen XL, Su WQ, Liu YQ, et al. Analysis on genetic diversity of 36 pineapple collections by SCoT markers [J]. Southwest China J Agric Sci, 2012, 25(2): 625-629.
5	马帅鹏, 庞振才, 李静, 等. 分子标记在菠萝研究中的应用 [J]. 热带农业科学, 2014, 34(1): 54-58.
	Ma SP, Pang ZC, Li J, et al. Application of molecular markers on pineapple (Ananas comosus (L.) Merr.) [J]. Chin J Trop Agric, 2014, 34(1): 54-58.
6	Li DL, Jing MM, Dai XH, et al. Current status of pineapple breeding, industrial development, and genetics in China [J]. Euphytica, 2022, 218(6): 85.
7	Duval MF, Noyer JL, Perrier X, et al. Molecular diversity in pineapple assessed by RFLP markers [J]. Theor Appl Genet, 2001, 102(1): 83-90.
8	Kato CY, Nagai C, Moore PH, et al. Intra-specific DNA polymorphism in pineapple (Ananas comosus (L.) Merr.) assessed by AFLP markers [J]. Genet Resour Crop Evol, 2005, 51(8): 815-825.
9	刘卫国, 易干军, 刘岩, 等. 菠萝种质鉴定及亲缘关系的AFLP分析 [J]. 果树学报, 2008, 25(4): 516-520.
	Liu WG, Yi GJ, Liu Y, et al. Identification of pineapple germplasm and relationship analysis with AFLP [J]. J Fruit Sci, 2008, 25(4): 516-520.
10	Silva JM, Lima PRL, Souza FVD, et al. Genetic diversity and nonparametric statistics to identify possible ISSR marker association with fiber quality of pineapple [J]. An Acad Bras Cienc, 2019, 91(3): e20180749.
11	童和林, 冯素萍, 陈友, 等. 菠萝基因组SSR分子标记的开发 [J]. 果树学报, 2010, 27(4): 551-555.
	Tong HL, Feng SP, Chen Y, et al. Development of molecular markers from genomic-SSR of Pineapple (Ananas comosus) [J]. J Fruit Sci, 2010, 27(4): 551-555.
12	Shoda M, Urasaki N, Sakiyama S, et al. DNA profiling of pineapple cultivars in Japan discriminated by SSR markers [J]. Breed Sci, 2012, 62(4): 352-359.
13	Feng SP, Tong HL, Chen Y, et al. Development of pineapple microsatellite markers and germplasm genetic diversity analysis [J]. Biomed Res Int, 2013, 2013: 317912.
14	Lin YS, Kuan CS, Weng IS, et al. Cultivar identification and genetic relationship of pineapple (Ananas comosus) cultivars using SSR markers [J]. Genet Mol Res, 2015, 14(4): 15035-15043.
15	王健胜, 贺军虎, 陈华蕊, 等. 不同分子标记在菠萝中检测效率的比较 [J]. 湖北农业科学, 2015, 54(11): 2676-2679.
	Wang JS, He JH, Chen HR, et al. Comparison on the detection efficiency of different types of molecular markers in pineapple [J]. Hubei Agric Sci, 2015, 54(11): 2676-2679.
16	Carlier JD, Reis A, Duval MF, et al. Genetic maps of RAPD, AFLP and ISSR markers in Ananas bracteatus and A. comosus using the pseudo-testcross strategy [J]. Plant Breed, 2004, 123(2): 186-192.
17	Carlier JD, Sousa NH, Santo TE, et al. A genetic map of pineapple (Ananas comosus (L.) Merr.) including SCAR, CAPS, SSR and EST-SSR markers [J]. Mol Breed, 2012, 29(1): 245-260.
18	De Sousa NH, Carlier JD, Santo TE, et al. An integrated genetic map of pineapple (Ananas comosus (L.) Merr.) [J]. Sci Hortic, 2013, 157: 113-118.
19	Ming R, VanBuren R, Wai CM, et al. The pineapple genome and the evolution of CAM photosynthesis [J]. Nat Genet, 2015, 47(12): 1435-1442.
20	Chen LY, VanBuren R, Paris M, et al. The bracteatus pineapple genome and domestication of clonally propagated crops [J]. Nat Genet, 2019, 51(10): 1549-1558.
21	Nashima K, Shirasawa K, Isobe S, et al. Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing [J]. Plant J, 2022, 110(3): 720-734.
22	Feng JT, Zhang W, Chen CJ, et al. The pineapple reference genome: Telomere-to-telomere assembly, manually curated annotation, and comparative analysis [J]. J Integr Plant Biol, 2024, 66(10): 2208-2225.
23	Winfield M, Burridge A, Ordidge M, et al. Development of a minimal KASP marker panel for distinguishing genotypes in apple collections [J]. PLoS One, 2020, 15(11): e0242940.
24	黄小凤, 韦阳连, 袁叶, 等. 基于SNP分子标记的221份荔枝品种(品系)的遗传多样性分析及核心种质库构建 [J]. 植物资源与环境学报, 2022, 31(4): 74-84.
	Huang XF, Wei YL, Yuan Y, et al. Genetic diversity analysis and core collection construction of 221 cultivars(strains) of Litchi chinensis based on SNP molecular markers [J]. J Plant Resour Environ, 2022, 31(4): 74-84.
25	赵俊生, 杨晓燕, 曾祥有, 等. 利用SNP分子标记分析化橘红种质资源 [J]. 分子植物育种, 2016, 14(5): 1203-1211.
	Zhao JS, Yang XY, Zeng XY, et al. Analysis on germplasm resources of exocarpium citri grandis using SNP molecular markers [J]. Mol Plant Breed, 2016, 14(5): 1203-1211.
26	汤雨晴, 杨惠栋, 闫承璞, 等. 基于重测序的‘金兰柚’基因组InDel标记的开发及应用 [J]. 园艺学报, 2023, 50(1): 15-26.
	Tang YQ, Yang HD, Yan CP, et al. Development and application of Jinlan pummelo (Citrus maxima) InDel markers based on genome re-sequencing [J]. Acta Hortic Sin, 2023, 50(1): 15-26.
27	Zhou L, Matsumoto T, Tan HW, et al. Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm [J]. Hortic Res, 2015, 2: 15056.
28	高云飞, 林文秋, 吴青松, 等. 菠萝PARMS反应体系的建立 [J]. 热带作物学报, 2023, 44(2): 225-232.
	Gao YF, Lin WQ, Wu QS, et al. Establishment of PARMS reaction system for pineapple (Ananas comosus. L) [J]. Chin J Trop Crops, 2023, 44(2): 225-232.
29	Wu DH, Wu HP, Wang CS, et al. Genome-wide InDel marker system for application in rice breeding and mapping studies [J]. Euphytica, 2013, 192(1): 131-143.
30	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining [J]. Mol Plant, 2023, 16(11): 1733-1742.
31	仝征, 彭存智, 常丽丽, 等. 一种高浓度琼脂糖电泳凝胶的制备方法及其应用: CN114397350A [P]. 2022-04-26.
	Tong Z, Peng CZ, Chang LL, et al ., Preparation and application of a high concentration agarose electrophoresis gel: CN114397350A [P]. 2022-04-26.
32	Letunic I, Bork P. Interactive tree of life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool [J]. Nucleic Acids Res, 2024, 52(W1): 78-82.
33	Li YL, Liu JX. StructureSelector: a web-based software to select and visualize the optimal number of clusters using multiple methods [J]. Mol Ecol Resour, 2018, 18(1): 176-177.
34	Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study [J]. Mol Ecol, 2005, 14(8): 2611-2620.
35	王日勇, 谢玲玲, 周火强, 等. 基于重测序‘墨地龙’冬瓜In Del标记的开发及纯度鉴定 [J]. 分子植物育种, 2021, 19(20): 6760-6766.
	Wang RY, Xie LL, Zhou HQ, et al. Development and purity identification of in del marker based on re-sequencing of 'modilong' wax gourd [J]. Mol Plant Breed, 2021, 19(20): 6760-6766.
36	杨洁, 赫佳, 王丹碧, 等. InDel标记的研究和应用进展 [J]. 生物多样性, 2016, 24(2): 237-243.
	Yang J, He J, Wang DB, et al. Progress in research and application of InDel markers [J]. Biodivers Sci, 2016, 24(2): 237-243.
37	陈正杰, 宛永璐, 钟文娟, 等. 基于大豆基因组重测序的InDel标记开发及应用 [J]. 植物遗传资源学报, 2021, 22(3): 815-833.
	Chen ZJ, Wan YL, Zhong WJ, et al. Development and application of soybean in del markers based on whole-genome resequencing datasets [J]. J Plant Genet Resour, 2021, 22(3): 815-833.
38	王健胜, 贺军虎, 陈华蕊, 等. 基于SSR标记的菠萝种质DNA指纹图谱构建 [J]. 热带作物学报, 2015, 36(7): 1274-1279.
	Wang JS, He JH, Chen HR, et al. Construction of DNA fingerprint for pineapple germplasm based on SSR marker [J]. Chin J Trop Crops, 2015, 36(7): 1274-1279.
39	窦美安, 邱文武, 吴青松, 等. 菠萝遗传多样性的SRAP分析 [J]. 果树学报, 2010, 27(6): 930-937.
	Dou MA, Qiu WW, Wu QS, et al. Genetic diversity analysis on pineapple by SRAP markers [J]. J Fruit Sci, 2010, 27(6): 930-937.
40	刘岩, 钟云, 孟祥春, 等. 菠萝新品种‘粤脆’ [J]. 果农之友, 2006(6): 14.
	Liu Y, Zhong Y, Meng XC, et al. A new pineapple variety 'Yuecui' [J]. Fruit Grow Friend, 2006(6): 14.
41	刘传和, 贺涵, 邵雪花, 等. 菠萝新品种‘粤彤’ [J]. 园艺学报, 2022, 49(9): 2053-2054.
	Liu CH, He H, Shao XH, et al. A new pineapple cultivar 'Yuetong' [J]. Acta Hortic Sin, 2022, 49(9): 2053-2054.
42	刘传和, 贺涵, 邵雪花, 等. 菠萝新品种‘粤甜’ [J]. 园艺学报, 2023, 50(9): 2059-2060.
	Liu CH, He H, Shao XH, et al. A new pineapple cultivar 'Yuetian' [J]. Acta Hortic Sin, 2023, 50(9): 2059-2060.

标记编号 InDel tag	染色体位置 Location	上游引物 Forward primer	上游引物序列 Forward primer sequence（5'-3'）	下游引物 Reverse primer	下游引物序列 Reverse primer sequence（5'-3'）
P1-3	Chr1， 12031084	P1-3-F	TGGCTTCATGCTTCTTTTGCA	P1-3-R	TCAGACGCAGCCTGGTTG
P1-13	Chr1， 23303433	P1-13-F	GACTGCGCTCGGAAGTGA	P1-13-R	AGAAGGAGGAGGAAGGGGG
P2-1	Chr2， 2165847	P2-1-F	GCGGAGTGAACGATCCCT	P2-1-R	AGGAAAGTTTGGGTGCGTCT
P2-8	Chr2， 13525182	P2-8-F	TGGCAACATTCCCCTATGCA	P2-8-R	GGGGTCCTAAAGGGTCAAGC
P3-1	Chr3， 844091	P3-1-F	CCGAGAAAAGCGACAGCC	P3-1-R	TGCAGCGCAACTAAGAGGT
P3-4	Chr3， 1725841	P3-4-F	CCACAGGCCCCTCCATTG	P3-4-R	CCGCCAGACTGCTTTCGA
P4-4	Chr4， 5231326	P4-4-F	GGTCGAAGGCGCACTGTA	P4-4-R	CATGCCCGGCGAAGGATA
P4-9	Chr4， 13043220	P4-9-F	AGGAAAGAAGCAACAAGGCCT	P4-9-R	CCAAGGGGTGCGAACGTA
P5-7	Chr5， 1511655	P5-7-F	CTGCAAGGTGCTTCATTGAGT	P5-7-R	GGCATCCCTTGTCCAGGT
P5-9	Chr5， 2289873	P5-9-F	GGAATCCACAACCCCATTGC	P5-9-R	CAAGGGGTTGTGAAACTGCA
P6-4	Chr6， 10732786	P6-4-F	CCTGCGGGAGGATTCAGG	P6-4-R	GGCGGAATAAGGGCCTCG
P6-12	Chr6， 3392699	P6-12-F	GCAGCCAGCAGAACAACA	P6-12-R	GGCTTGAAGAACCCATCGC
P7-3	Chr7， 12588489	P7-3-F	GAAGCCCGACACGACCAG	P7-3-R	TGACCAGCACGAACGACC
P7-13	Chr7， 12954807	P7-13-F	CCCTTTGTCCTACAAATGGGG	P7-13-R	TCGCATGAACAGTGAGGCA
P8-2	Chr8， 9924768	P8-2-F	CGCCCACGCTCCTAAGAG	P8-2-R	GCACCATTCAACAAGCCACA
P8-4	Chr8， 12420683	P8-4-F	CGGTGAATTTGCTACACGGT	P8-4-R	TGTGGTAGCATAGATTGGCGA
P9-8	Chr9， 11439477	P9-8-F	CGGTGTATCGAGGTCGCC	P9-8-R	CCAGCGCCTTGAAGTCCA
P9-13	Chr9， 12410107	P9-13-F	AGTTCATTTGAAGTGGTGCCT	P9-13-R	TGTTTTGGGCCGATGAAAGA
P10-7	Chr10， 82948	P10-7-F	GGGCACAGCGACGTTACA	P10-7-R	GGGCCTGCCGTACACTTT
P10-15	Chr10， 3770865	P10-15-F	AAGATGTTTGGCCGTGCG	P10-15-R	CGCGCTGATGAGGAAAGC
P11-12	Chr11， 3139788	P11-12-F	TCAGATGCCCGAGGTCCA	P11-12-R	TGACATCCCGAATGCGCA
P11-15	Chr11， 9182497	P11-15-F	GTGGACGACAATGGCGGA	P11-15-R	TCTCAGCAACCATGTCGCA
P12-3	Chr12， 11355510	P12-3-F	AGGAGGCTTCGGAGTCGA	P12-3-R	CTGGGGTGTCACACTATCTGA
P12-7	Chr12， 3380797	P12-7-F	AGAAACCAAGGGCTCCGA	P12-7-R	TCGATCCGCATCACACGT
P13-5	Chr13， 1779898	P13-5-F	CACCGGGGCACGTCTAAA	P13-5-R	GGGCTTTGTGATTTTTGGGCT
P13-9	Chr13， 3803304	P13-9-F	GGGCATGCCGACCGAATA	P13-9-R	TGGTGCCCACTTGGTGAC
P14-2	Chr14， 1157124	P14-2-F	CTTCTTTGGCGGGGAGGG	P14-2-R	GTATGCAGTGGCCACCGT
P14-15	Chr14， 9737746	P14-15-F	TGGTTGCGTTGGTTGGGT	P14-15-R	CTCTGGAAGCCCAATCTCGA
P15-11	Chr15， 6253581	P15-11-F	GGGCACCGGACCAACTTT	P15-11-R	GGACGGCTAACAGGTTTCGA
P15-12	Chr15， 7964660	P15-12-F	GTGCCGTACCGTGCTAGG	P15-12-R	CTGGGCAGTTGGATCTTGGT
P16-6	Chr16， 1035082	P16-6-F	GTCGCGATTTCCCCACCA	P16-6-R	GCGTTTGATTGCTCAGCCA
P16-13	Chr16， 11032414	P16-13-F	CTGTACCTGTCGGTGGCG	P16-13-R	TGGCAGTTCAGCGACCAG
P17-7	Chr17， 2630836	P17-7-F	GCGCCGTAGTAGAGTTCCA	P17-7-R	TTCCCGTGTCACAGGCAC
P17-11	Chr17， 8060219	P17-11-F	TGAATGGGAAAGCAACACGC	P17-11-R	GCATCTACCGCACCCTTGA
P18-1	Chr18， 4131951	P18-1-F	CAGCGAGAGAGCGATGCA	P18-1-R	CAAGGGCGGTTGGTGGAT
P18-13	Chr18， 5367546	P18-13-F	TGCAGTCGCATATGCAGCT	P18-13-R	ACTCTGGCGGGAAGTCGA
P19-1	Chr19， 8770836	P19-1-F	CCGAAACAGCAACTCAAATGC	P19-1-R	GCCTCACCTTGTGGTTGC
P19-7	Chr19， 9071773	P19-7-F	GAACGGGAGGAGCGATCG	P19-7-R	TCCTCGTATCACCGATCCCT
P20-1	Chr20， 4990506	P20-1-F	TGAATGCCAATCCTTGCACC	P20-1-R	CTGAGAGCATCACAGCTTGTG
P20-9	Chr20， 7316522	P20-9-F	TGCAGGCAATGATGGAGCA	P20-9-R	CACCTTAATGAAGCCGCAGC
P21-3	Chr21， 10154987	P21-3-F	TCGGGTTTTGACAAGGGAGT	P21-3-R	GAGTTTCTCATTGAGTGGGCA
P21-8	Chr21， 8270960	P21-8-F	TGCTGAGCTAAACCTTAGGCA	P21-8-R	CGTGGGTCAATGCTTTCCC
P22-14	Chr22， 73132	P22-14-F	GCGCAATCTCTTCCGACC	P22-14-R	GCAACCACAGCAGCCAAC
P22-15	Chr22， 1287004	P22-15-F	TGGCAATGAGTTGTGATTGGT	P22-15-R	TCGGAAAAACACTGAGCTGA
P23-7	Chr23， 3066097	P23-7-F	CGAACGGTGAAACAGGGGA	P23-7-R	GCAGGTAGTAAAGTTGCTCGC
P23-15	Chr23， 3085762	P23-15-F	ACTAGAACGAGTCCGGGCA	P23-15-R	AGCGCCAGTCGAAAGAACA
P24-7	Chr24， 40948	P24-7-F	GGGTCCCGACGTAGGAGT	P24-7-R	ACGAGATCTGGTCCGGCT
P24-8	Chr24， 6182284	P24-8-F	CGGGGGAGTATGTGACACC	P24-8-R	GGTTGATGCAGCACGCTG
P25-5	Chr25， 2263248	P25-5-F	GCACATTTCACTTCGAGTCGA	P25-5-R	GAAGGTGGAATGGACCCCA
P25-10	Chr25， 2380378	P25-10-F	CTCGACAACTGCTGCGGA	P25-10-R	AGGTGGATGAGACACGTAACT

标记编号 InDel tag	染色体位置 Location	上游引物 Forward primer	上游引物序列 Forward primer sequence（5'-3'）	下游引物 Reverse primer	下游引物序列 Reverse primer sequence（5'-3'）
P1-3	Chr1， 12031084	P1-3-F	TGGCTTCATGCTTCTTTTGCA	P1-3-R	TCAGACGCAGCCTGGTTG
P1-13	Chr1， 23303433	P1-13-F	GACTGCGCTCGGAAGTGA	P1-13-R	AGAAGGAGGAGGAAGGGGG
P2-1	Chr2， 2165847	P2-1-F	GCGGAGTGAACGATCCCT	P2-1-R	AGGAAAGTTTGGGTGCGTCT
P2-8	Chr2， 13525182	P2-8-F	TGGCAACATTCCCCTATGCA	P2-8-R	GGGGTCCTAAAGGGTCAAGC
P3-1	Chr3， 844091	P3-1-F	CCGAGAAAAGCGACAGCC	P3-1-R	TGCAGCGCAACTAAGAGGT
P3-4	Chr3， 1725841	P3-4-F	CCACAGGCCCCTCCATTG	P3-4-R	CCGCCAGACTGCTTTCGA
P4-4	Chr4， 5231326	P4-4-F	GGTCGAAGGCGCACTGTA	P4-4-R	CATGCCCGGCGAAGGATA
P4-9	Chr4， 13043220	P4-9-F	AGGAAAGAAGCAACAAGGCCT	P4-9-R	CCAAGGGGTGCGAACGTA
P5-7	Chr5， 1511655	P5-7-F	CTGCAAGGTGCTTCATTGAGT	P5-7-R	GGCATCCCTTGTCCAGGT
P5-9	Chr5， 2289873	P5-9-F	GGAATCCACAACCCCATTGC	P5-9-R	CAAGGGGTTGTGAAACTGCA
P6-4	Chr6， 10732786	P6-4-F	CCTGCGGGAGGATTCAGG	P6-4-R	GGCGGAATAAGGGCCTCG
P6-12	Chr6， 3392699	P6-12-F	GCAGCCAGCAGAACAACA	P6-12-R	GGCTTGAAGAACCCATCGC
P7-3	Chr7， 12588489	P7-3-F	GAAGCCCGACACGACCAG	P7-3-R	TGACCAGCACGAACGACC
P7-13	Chr7， 12954807	P7-13-F	CCCTTTGTCCTACAAATGGGG	P7-13-R	TCGCATGAACAGTGAGGCA
P8-2	Chr8， 9924768	P8-2-F	CGCCCACGCTCCTAAGAG	P8-2-R	GCACCATTCAACAAGCCACA
P8-4	Chr8， 12420683	P8-4-F	CGGTGAATTTGCTACACGGT	P8-4-R	TGTGGTAGCATAGATTGGCGA
P9-8	Chr9， 11439477	P9-8-F	CGGTGTATCGAGGTCGCC	P9-8-R	CCAGCGCCTTGAAGTCCA
P9-13	Chr9， 12410107	P9-13-F	AGTTCATTTGAAGTGGTGCCT	P9-13-R	TGTTTTGGGCCGATGAAAGA
P10-7	Chr10， 82948	P10-7-F	GGGCACAGCGACGTTACA	P10-7-R	GGGCCTGCCGTACACTTT
P10-15	Chr10， 3770865	P10-15-F	AAGATGTTTGGCCGTGCG	P10-15-R	CGCGCTGATGAGGAAAGC
P11-12	Chr11， 3139788	P11-12-F	TCAGATGCCCGAGGTCCA	P11-12-R	TGACATCCCGAATGCGCA
P11-15	Chr11， 9182497	P11-15-F	GTGGACGACAATGGCGGA	P11-15-R	TCTCAGCAACCATGTCGCA
P12-3	Chr12， 11355510	P12-3-F	AGGAGGCTTCGGAGTCGA	P12-3-R	CTGGGGTGTCACACTATCTGA
P12-7	Chr12， 3380797	P12-7-F	AGAAACCAAGGGCTCCGA	P12-7-R	TCGATCCGCATCACACGT
P13-5	Chr13， 1779898	P13-5-F	CACCGGGGCACGTCTAAA	P13-5-R	GGGCTTTGTGATTTTTGGGCT
P13-9	Chr13， 3803304	P13-9-F	GGGCATGCCGACCGAATA	P13-9-R	TGGTGCCCACTTGGTGAC
P14-2	Chr14， 1157124	P14-2-F	CTTCTTTGGCGGGGAGGG	P14-2-R	GTATGCAGTGGCCACCGT
P14-15	Chr14， 9737746	P14-15-F	TGGTTGCGTTGGTTGGGT	P14-15-R	CTCTGGAAGCCCAATCTCGA
P15-11	Chr15， 6253581	P15-11-F	GGGCACCGGACCAACTTT	P15-11-R	GGACGGCTAACAGGTTTCGA
P15-12	Chr15， 7964660	P15-12-F	GTGCCGTACCGTGCTAGG	P15-12-R	CTGGGCAGTTGGATCTTGGT
P16-6	Chr16， 1035082	P16-6-F	GTCGCGATTTCCCCACCA	P16-6-R	GCGTTTGATTGCTCAGCCA
P16-13	Chr16， 11032414	P16-13-F	CTGTACCTGTCGGTGGCG	P16-13-R	TGGCAGTTCAGCGACCAG
P17-7	Chr17， 2630836	P17-7-F	GCGCCGTAGTAGAGTTCCA	P17-7-R	TTCCCGTGTCACAGGCAC
P17-11	Chr17， 8060219	P17-11-F	TGAATGGGAAAGCAACACGC	P17-11-R	GCATCTACCGCACCCTTGA
P18-1	Chr18， 4131951	P18-1-F	CAGCGAGAGAGCGATGCA	P18-1-R	CAAGGGCGGTTGGTGGAT
P18-13	Chr18， 5367546	P18-13-F	TGCAGTCGCATATGCAGCT	P18-13-R	ACTCTGGCGGGAAGTCGA
P19-1	Chr19， 8770836	P19-1-F	CCGAAACAGCAACTCAAATGC	P19-1-R	GCCTCACCTTGTGGTTGC
P19-7	Chr19， 9071773	P19-7-F	GAACGGGAGGAGCGATCG	P19-7-R	TCCTCGTATCACCGATCCCT
P20-1	Chr20， 4990506	P20-1-F	TGAATGCCAATCCTTGCACC	P20-1-R	CTGAGAGCATCACAGCTTGTG
P20-9	Chr20， 7316522	P20-9-F	TGCAGGCAATGATGGAGCA	P20-9-R	CACCTTAATGAAGCCGCAGC
P21-3	Chr21， 10154987	P21-3-F	TCGGGTTTTGACAAGGGAGT	P21-3-R	GAGTTTCTCATTGAGTGGGCA
P21-8	Chr21， 8270960	P21-8-F	TGCTGAGCTAAACCTTAGGCA	P21-8-R	CGTGGGTCAATGCTTTCCC
P22-14	Chr22， 73132	P22-14-F	GCGCAATCTCTTCCGACC	P22-14-R	GCAACCACAGCAGCCAAC
P22-15	Chr22， 1287004	P22-15-F	TGGCAATGAGTTGTGATTGGT	P22-15-R	TCGGAAAAACACTGAGCTGA
P23-7	Chr23， 3066097	P23-7-F	CGAACGGTGAAACAGGGGA	P23-7-R	GCAGGTAGTAAAGTTGCTCGC
P23-15	Chr23， 3085762	P23-15-F	ACTAGAACGAGTCCGGGCA	P23-15-R	AGCGCCAGTCGAAAGAACA
P24-7	Chr24， 40948	P24-7-F	GGGTCCCGACGTAGGAGT	P24-7-R	ACGAGATCTGGTCCGGCT
P24-8	Chr24， 6182284	P24-8-F	CGGGGGAGTATGTGACACC	P24-8-R	GGTTGATGCAGCACGCTG
P25-5	Chr25， 2263248	P25-5-F	GCACATTTCACTTCGAGTCGA	P25-5-R	GAAGGTGGAATGGACCCCA
P25-10	Chr25， 2380378	P25-10-F	CTCGACAACTGCTGCGGA	P25-10-R	AGGTGGATGAGACACGTAACT

标记编号 InDel tag	有效等位位点数（Ne） Effective number of alleles	Shannon's多样性指数（I） Shannon's information index	多态性信息含量（PIC） Polymorphic information content	Nei's基因多样性指数（H） Nei's gene diversity index
P1-3	1.999 4	0.693 0	0.374 9	0.499 9
P1-13	1.167 0	0.272 8	0.132 9	0.143 1
P2-1	1.768 2	0.626 1	0.340 1	0.434 5
P2-8	1.806 2	0.638 5	0.346 7	0.446 3
P3-1	1.555 6	0.542 6	0.293 4	0.357 2
P3-4	1.904 3	0.667 8	0.362 1	0.474 9
P4-4	1.768 2	0.626 1	0.340 1	0.434 5
P4-9	1.686 6	0.597 1	0.324 2	0.407 1
P5-7	1.978 8	0.687 8	0.372 3	0.494 6
P5-9	1.806 2	0.638 5	0.346 7	0.446 3
P6-4	1.917 9	0.671 6	0.364 1	0.478 6
P6-12	1.622 0	0.571 6	0.309 9	0.383 5
P7-3	2.000 0	0.693 1	0.375 0	0.500 0
P7-13	1.999 4	0.693 0	0.374 9	0.499 9
P8-2	1.904 3	0.667 8	0.362 1	0.474 9
P8-4	1.858 6	0.654 6	0.355 3	0.462 0
P9-8	1.997 6	0.692 6	0.374 7	0.499 4
P9-13	1.858 6	0.654 6	0.355 3	0.462 0
P10-7	2.058 8	0.825 1	0.425 1	0.514 3
P10-15	1.768 2	0.626 1	0.340 1	0.434 5
P11-12	1.999 4	0.693 0	0.374 9	0.499 9
P11-15	1.622 0	0.571 6	0.309 9	0.383 5
P12-3	1.997 6	0.692 6	0.374 7	0.499 4
P12-7	1.787 5	0.632 5	0.343 5	0.440 5
P13-5	1.421 5	0.473 0	0.252 6	0.296 5
P13-9	1.686 6	0.597 1	0.324 2	0.407 1
P14-2	1.643 8	0.580 5	0.315 0	0.391 6
P14-15	1.978 8	0.687 8	0.372 3	0.494 6
P15-11	1.990 5	0.690 8	0.373 8	0.497 6
P15-12	1.999 4	0.693 0	0.374 9	0.499 9
P16-6	1.962 7	0.683 6	0.370 2	0.490 5
P16-13	1.600 0	0.562 3	0.304 7	0.375 0
P17-7	1.169 9	0.313 9	0.138 5	0.145 2
P17-11	1.942 3	0.678 2	0.367 5	0.485 1
P18-1	1.874 6	0.659 3	0.357 7	0.466 6
P18-13	1.748 4	0.619 4	0.336 4	0.428 1
P19-1	1.824 3	0.644 2	0.349 8	0.451 8
P19-7	1.841 8	0.649 6	0.352 6	0.457 0
P20-1	1.889 9	0.663 7	0.360 0	0.470 9
P20-9	1.889 9	0.663 7	0.360 0	0.470 9
P21-3	1.577 9	0.552 7	0.299 2	0.366 2
P21-8	1.904 3	0.667 8	0.362 1	0.474 9
P22-14	1.917 9	0.671 6	0.364 1	0.478 6
P22-15	1.990 5	0.690 8	0.373 8	0.497 6
P23-7	1.643 8	0.580 5	0.315 0	0.391 6
P23-15	1.665 3	0.589 0	0.319 7	0.399 5
P24-7	1.981 7	0.746 4	0.388 6	0.495 4
P24-8	1.904 3	0.667 8	0.362 1	0.474 9
P25-5	1.997 6	0.692 6	0.374 7	0.499 4
P25-10	1.942 3	0.678 2	0.367 5	0.485 1
平均值	1.816 4	0.634 6	0.342 2	0.441 2

标记编号 InDel tag	有效等位位点数（Ne） Effective number of alleles	Shannon's多样性指数（I） Shannon's information index	多态性信息含量（PIC） Polymorphic information content	Nei's基因多样性指数（H） Nei's gene diversity index
P1-3	1.999 4	0.693 0	0.374 9	0.499 9
P1-13	1.167 0	0.272 8	0.132 9	0.143 1
P2-1	1.768 2	0.626 1	0.340 1	0.434 5
P2-8	1.806 2	0.638 5	0.346 7	0.446 3
P3-1	1.555 6	0.542 6	0.293 4	0.357 2
P3-4	1.904 3	0.667 8	0.362 1	0.474 9
P4-4	1.768 2	0.626 1	0.340 1	0.434 5
P4-9	1.686 6	0.597 1	0.324 2	0.407 1
P5-7	1.978 8	0.687 8	0.372 3	0.494 6
P5-9	1.806 2	0.638 5	0.346 7	0.446 3
P6-4	1.917 9	0.671 6	0.364 1	0.478 6
P6-12	1.622 0	0.571 6	0.309 9	0.383 5
P7-3	2.000 0	0.693 1	0.375 0	0.500 0
P7-13	1.999 4	0.693 0	0.374 9	0.499 9
P8-2	1.904 3	0.667 8	0.362 1	0.474 9
P8-4	1.858 6	0.654 6	0.355 3	0.462 0
P9-8	1.997 6	0.692 6	0.374 7	0.499 4
P9-13	1.858 6	0.654 6	0.355 3	0.462 0
P10-7	2.058 8	0.825 1	0.425 1	0.514 3
P10-15	1.768 2	0.626 1	0.340 1	0.434 5
P11-12	1.999 4	0.693 0	0.374 9	0.499 9
P11-15	1.622 0	0.571 6	0.309 9	0.383 5
P12-3	1.997 6	0.692 6	0.374 7	0.499 4
P12-7	1.787 5	0.632 5	0.343 5	0.440 5
P13-5	1.421 5	0.473 0	0.252 6	0.296 5
P13-9	1.686 6	0.597 1	0.324 2	0.407 1
P14-2	1.643 8	0.580 5	0.315 0	0.391 6
P14-15	1.978 8	0.687 8	0.372 3	0.494 6
P15-11	1.990 5	0.690 8	0.373 8	0.497 6
P15-12	1.999 4	0.693 0	0.374 9	0.499 9
P16-6	1.962 7	0.683 6	0.370 2	0.490 5
P16-13	1.600 0	0.562 3	0.304 7	0.375 0
P17-7	1.169 9	0.313 9	0.138 5	0.145 2
P17-11	1.942 3	0.678 2	0.367 5	0.485 1
P18-1	1.874 6	0.659 3	0.357 7	0.466 6
P18-13	1.748 4	0.619 4	0.336 4	0.428 1
P19-1	1.824 3	0.644 2	0.349 8	0.451 8
P19-7	1.841 8	0.649 6	0.352 6	0.457 0
P20-1	1.889 9	0.663 7	0.360 0	0.470 9
P20-9	1.889 9	0.663 7	0.360 0	0.470 9
P21-3	1.577 9	0.552 7	0.299 2	0.366 2
P21-8	1.904 3	0.667 8	0.362 1	0.474 9
P22-14	1.917 9	0.671 6	0.364 1	0.478 6
P22-15	1.990 5	0.690 8	0.373 8	0.497 6
P23-7	1.643 8	0.580 5	0.315 0	0.391 6
P23-15	1.665 3	0.589 0	0.319 7	0.399 5
P24-7	1.981 7	0.746 4	0.388 6	0.495 4
P24-8	1.904 3	0.667 8	0.362 1	0.474 9
P25-5	1.997 6	0.692 6	0.374 7	0.499 4
P25-10	1.942 3	0.678 2	0.367 5	0.485 1
平均值	1.816 4	0.634 6	0.342 2	0.441 2

[1]	LIU Chuan-he, HE Han, SHAO Xue-hua, HE Xiu-gu. Analysis of Differential Metabolites and Bacterial Community Structure in the Soils of a Pineapple Orchard under Different Mulching Treatments [J]. Biotechnology Bulletin, 2024, 40(7): 247-258.
[2]	LI Qing, SHI Yu-he, ZHU Jue, LI Xiao-ling, HOU Chao-wen, TONG Qiao-zhen. Genetic Diversity Analysis and DNA Fingerprint Construction of Atractylodes macrocephala Germplasm Resources Based on SCoT Molecular Markers [J]. Biotechnology Bulletin, 2024, 40(11): 142-151.
[3]	LIU Chuan-he, HE Han, HE Xiu-gu, CHEN Xin, LIU Kai, SHAO Xue-hua, LAI Duo, QIN Jian, ZHUANG Qing-li, KUANG Shi-zi, XIAO Wei-qiang. Physiological and Metabolitic Mechanisms of Different Pineapple Cultivars Responding to Low Temperature Stress [J]. Biotechnology Bulletin, 2023, 39(10): 219-230.
[4]	LIU Chuan-he, HE Han, HE Xiu-gu, LAI Qiu-qin, LIU Kai, SHAO Xue-hua, LAI Duo, KUANG Shi-zi, XIAO Wei-qiang. Unveiling the Mechanisms of Pineapple Responding to Anti-chilling by Gauze Covering in Winter via Transcriptome and Metabolome Profiling [J]. Biotechnology Bulletin, 2022, 38(11): 58-69.
[5]	LIU Chuan-he, HE Han, HE Xiu-gu, LIU Kai, SHAO Xue-hua, LAI Duo, KUANG Shi-zi, XIAO Wei-qiang. Analysis of Differential Metabolites and Bacterial Community Structure in Soils of a Pineapple Orchard in Different Continuous-cropping Years [J]. Biotechnology Bulletin, 2021, 37(8): 162-175.
[6]	WANG Yan-li, YANG Yi-ming, FAN Shu-tian, ZHAO Ying, XU Pei-lei, LU Wen-peng, LI Chang-yu. Genetic Diversity Analysis of 73 Vitis amurensis and Its Hybrids Offsprings Based on SSR Molecular Markers [J]. Biotechnology Bulletin, 2021, 37(1): 189-197.
[7]	WANG Ping, WANG Chun-yu, ZHANG Li-xia, CONG Ling, ZHU Zhen-xing, LU Xiao-chun. Development of SSR Molecular Markers with Sorghum Polymorphism Using Re-sequencing [J]. Biotechnology Bulletin, 2019, 35(11): 217-223.