Development and Application of Insertion-Deletion（InDel）Markers in Gannan wild Fortunella hindsii Based on Whole Genome Re-sequencing Data

doi:10.16420/j.issn.0513-353x.2024-0357

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (2): 267-278.doi: 10.16420/j.issn.0513-353x.2024-0357

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles

Development and Application of Insertion-Deletion（InDel）Markers in Gannan wild Fortunella hindsii Based on Whole Genome Re-sequencing Data

LUO Sifang, YAN Xiang, XIE Lifang, SUN Jingxian, GUO Zijing, ZHANG Zuming^*(), CHEN Zhaoxing^*()

Institute of Citrus Science Research of Ganzhou，Gannan Academy of Sciences，Ganzhou，Jiangxi 341000，China

Received:2024-06-11 Revised:2024-09-23 Online:2025-02-25 Published:2025-02-23
Contact: ZHANG Zuming, CHEN Zhaoxing

Abstract

Abstract:

To clarify the InDel characteristics of Gannan（Southern Jiangxi Province）wild Fortunella hindsii，99 Gannan wild F. hindsii were selected for whole genome resequencing，and then InDel variant sites were screened to develop InDel markers. A total of 590.58 Mb valid fragment data was obtained，and 211 470 InDels were detected，including 98 043 insertions and 113 427 deletions. From 8 760 InDel markers with Insertion or Deletion base numbers greater than 10 180 polymorphic markers with uniform distribution on chromosomes were selected，and 58 pairs of InDel primers with good amplification bands were developed. The findings of 58 pairs of InDel markers demonstrated a similar grouping pattern with the clustering results obtained from whole genome resequencing. Additionally，the 99 Gannan wild F. count of core InDel marker sites and alleles present in each germplasm，a total of 29 Gannan wild F. hindsii were selected as the core germplasm. These germplasm samples had a genome coverage of 99.7%. Among them，Jiahu，Yukeng，and Caifang were recognised as the core germplasm regions. The DNA fingerprint was created by integrating the entire genome resequencing data with the core InDel marker sites. This fingerprint contains coding information such as plant number，genotype，mutation locus，and agronomic trait data. This study aimed to elucidate the InDel characteristics of Gannan wild F. hindsii. The identified InDel markers have potential applications in population structure analysis，core germplasm screening，and DNA fingerprint construction.

Key words: Fortunella hindsii, InDel marker, genetic diversity, core germplasm, DNA fingerprinting

LUO Sifang, YAN Xiang, XIE Lifang, SUN Jingxian, GUO Zijing, ZHANG Zuming, CHEN Zhaoxing. Development and Application of Insertion-Deletion（InDel）Markers in Gannan wild Fortunella hindsii Based on Whole Genome Re-sequencing Data[J]. Acta Horticulturae Sinica, 2025, 52(2): 267-278.

Figures/Tables 9

References 21

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序号 No.	采集地 Collection place	编号 ID	生境 Habitat	海拔/m Altitude
1 ~ 13	蔡坊 Caifang（CF）	CF001 ~ CF013	山谷 Valley	271.5
14 ~ 16	三百山 Sanbaishan（SBS）	SBS001 ~ SBS003	原始森林 Primary forest	417.8
17 ~ 20	张牯段zhangguduan（ZGD）	ZGD001 ~ ZGD004	人工移植，丘陵 Artificial plant，hills	315.4
21 ~ 23	里仁 Liren（LR）	LR001 ~ LR003	人工移植，盆景 Artificial plant，Bonsai	353.7
24 ~ 27	余坑Ⅰ Yukeng Ⅰ（YK Ⅰ）	YK Ⅰ 001 ~ YK Ⅰ 004	人工移植，山谷 Artificial plant，valley	277.8
28 ~ 36	余坑Ⅱ Yukeng Ⅱ（YK Ⅱ）	YK Ⅱ 001 ~ YK Ⅱ 009	人工移植，山谷 Artificial plant，valley	277.8
37 ~ 46	余坑Ⅲ Yukeng Ⅲ（YK Ⅲ）	YK Ⅲ 001 ~ YK Ⅲ 010	人工移植，山谷 Artificial plant，valley	277.8
47 ~ 48	余坑Ⅳ Yukeng Ⅳ（YK Ⅳ）	YK Ⅳ 001 ~ YK Ⅳ 002	人工移植，山谷 Artificial plant，valley	277.8
49 ~ 88	夹湖 Jiahu（JH）	JH001 ~ JH040	人工移植，丘陵 Artificial plant，hills	271.6
89 ~ 93	沙石 Shashi（SS）	SS001 ~ SS005	人工移植，山谷 Artificial plant，valley	149.8
94 ~ 96	崇义 Chongyi（CY）	CY001 ~ CY003	原始森林 Primary forest	360.1
97 ~ 99	于都 Yudu（YD）	YD001 ~ YD003	山林 Forest	343.2

序号 No.	采集地 Collection place	编号 ID	生境 Habitat	海拔/m Altitude
1 ~ 13	蔡坊 Caifang（CF）	CF001 ~ CF013	山谷 Valley	271.5
14 ~ 16	三百山 Sanbaishan（SBS）	SBS001 ~ SBS003	原始森林 Primary forest	417.8
17 ~ 20	张牯段zhangguduan（ZGD）	ZGD001 ~ ZGD004	人工移植，丘陵 Artificial plant，hills	315.4
21 ~ 23	里仁 Liren（LR）	LR001 ~ LR003	人工移植，盆景 Artificial plant，Bonsai	353.7
24 ~ 27	余坑Ⅰ Yukeng Ⅰ（YK Ⅰ）	YK Ⅰ 001 ~ YK Ⅰ 004	人工移植，山谷 Artificial plant，valley	277.8
28 ~ 36	余坑Ⅱ Yukeng Ⅱ（YK Ⅱ）	YK Ⅱ 001 ~ YK Ⅱ 009	人工移植，山谷 Artificial plant，valley	277.8
37 ~ 46	余坑Ⅲ Yukeng Ⅲ（YK Ⅲ）	YK Ⅲ 001 ~ YK Ⅲ 010	人工移植，山谷 Artificial plant，valley	277.8
47 ~ 48	余坑Ⅳ Yukeng Ⅳ（YK Ⅳ）	YK Ⅳ 001 ~ YK Ⅳ 002	人工移植，山谷 Artificial plant，valley	277.8
49 ~ 88	夹湖 Jiahu（JH）	JH001 ~ JH040	人工移植，丘陵 Artificial plant，hills	271.6
89 ~ 93	沙石 Shashi（SS）	SS001 ~ SS005	人工移植，山谷 Artificial plant，valley	149.8
94 ~ 96	崇义 Chongyi（CY）	CY001 ~ CY003	原始森林 Primary forest	360.1
97 ~ 99	于都 Yudu（YD）	YD001 ~ YD003	山林 Forest	343.2

染色体 Chromosome	插入 Insertion	缺失 Deletion	总计 Total	占比/% Proportion
Chr1	12 831	14 127	26 958	12.75
Chr2	13 704	14 709	28 413	13.44
Chr3	13 086	14 487	27 573	13.04
Chr4	6 321	11 316	17 637	8.34
Chr5	11 916	12 480	24 396	11.54
Chr6	8 223	11 244	19 467	9.20
Chr7	14 157	15 816	29 973	14.17
Chr8	8 598	9 360	17 958	8.49
Chr9	9 153	9 810	18 963	8.97
染色体骨架 Scaffold	54	78	132	0.06
总计Total	98 043	113 427	211 470	100.00

染色体 Chromosome	插入 Insertion	缺失 Deletion	总计 Total	占比/% Proportion
Chr1	12 831	14 127	26 958	12.75
Chr2	13 704	14 709	28 413	13.44
Chr3	13 086	14 487	27 573	13.04
Chr4	6 321	11 316	17 637	8.34
Chr5	11 916	12 480	24 396	11.54
Chr6	8 223	11 244	19 467	9.20
Chr7	14 157	15 816	29 973	14.17
Chr8	8 598	9 360	17 958	8.49
Chr9	9 153	9 810	18 963	8.97
染色体骨架 Scaffold	54	78	132	0.06
总计Total	98 043	113 427	211 470	100.00

染色体 Chromosome	引物名称 Primer name	物理位置 Location	正向引物序列（5′-3′） Forward primer sequences	反向引物序列（5′-3′） Reverse primer sequences
Chr.1	Fh1-01	345 790	ATGCGCCCATATCAACAAAC	CTCGACCTCACAAGGGTCAT
	Fh1-03	1 028 626	TGCGAACTCTCAAAGAGCAA	CGCATGAAAAGTACCGGTGT
	Fh1-05	2 758 295	TTTAGCTCACGTGCAAGTCC	GCGACAAAATTCAACAATGC
	Fh1-08	6 542 069	ATCAATGCCGAAAGAAATCG	GCGGGAAAACCAAATGTTCT
	Fh1-14	12 876 172	TTACCTCTGTGGGCATGCTT	CGGCAATGGAACTGATTTCT
	Fh1-18	20 696 997	ATGTTCTTGGGCATCCTCAC	CAGCAAATGCAACCATCAAG
	Fh1-20	28 766 849	AAATTGTGATTGCTGGAGGC	GGAGGACAGCTTCCGTATTG
Chr.2	Fh2-01	109 117	GCCAAGCTCGACTTCACCTA	TCTCTTGTCGTGTTGGGTTG
	Fh2-03	5 553 966	TTCAACACACCAATTTTAAGAGATTT	TTCTGCTTCGTCCCTTCTTC
	Fh2-05	8 613 056	GCATTTCCCACCAATGTTTT	TTTGGAGCAGGATTTGATTTG
	Fh2-08	15 929 836	TGATGGAGTTGAAGCAGCAC	ACAACAAAAACAAGGCCAGC
	Fh2-11	21 318 214	CATGGGACTAAAATTGGGACC	CCAGGGTTCTTCTAGACGGT
	Fh2-16	28 640 384	AAATCCAACGGCGTACTTGT	TCAACGGATAACTTCTCGACG
	Fh2-18	30 563 177	GCATGCCTTTTGGTTTCATT	ATGCTCAAATCGCCTTCATT
Chr.3	Fh3-01	1 382 984	TTTGGGATATGAAATAATCTTCTATGA	TCTTTCTTTTCCCCTTTTTGG
	Fh3-04	4 927 541	ACGAGCAGAAGAGCTGAAGG	GCATGGCAAAGTAAGCGAAT
	Fh3-07	8 409 496	ATCGCTTAAATTGCTAGGGC	GCTCCAGCAATCCTTCTTTG
	Fh3-11	13 680 425	CAGGTTGCAATGTTGGATTC	GGGTGGACCCAAGAATTGAT
	Fh3-15	16 819 522	CAGCAGATGTCAGGAAATGG	TTGAGTCCAAACTCCAACCTG
	Fh3-17	21 775 047	TTCTGCCGTTTTTGTGTGAG	ATACGGCTATCGGCACAATC
	Fh3-19	26 439 136	AAGCTTTCACCAAATGCACC	TTCCCTGTAGAAGCATTGGC
Chr.4	Fh4-04	4 839 437	ACAAAACAAGAGAATGGCCG	CACGAAAAAGCAGAATGCAA
	Fh4-12	12 857 077	CTACCCACACACGAGAAGCA	AAATCGGAAAGGCCAAAAGT
	Fh4-15	14 192 085	CGTTGCCGGAAAGTTTTCTA	TTTGCAAAAGTTTTTGGGCT
	Fh4-19	19 886 665	TCATCCGAACACGGTATTGA	GATCGTGGTAAAGAGGGCAA
Chr.5	Fh5-03	3 603 056	CCTTGGGCTTTGTTGTTTGT	CCCGAGGCTCTTACAGGAAT
	Fh5-07	6 518 497	ATTTGTTGCTTGATCCGAGG	GGATTGGCTCTCCACTTTGT
	Fh5-11	11 510 323	CGGGTGGGTTTGGTATTAAC	TAAGCACCCATCCAACACAA
	Fh5-14	19 373 991	AATTTGTTTGCCTGTACCCG	ATTCAGGCAGAGCTGGTTGT
	Fh5-18	25 493 920	AATCCATTAAACGATCGGCA	GTTTGTGGCAAATGAATCCC
	Fh5-20	28 017 235	TCACCGCAAAAACTCAACAA	ATGGCTCCCTCCTTTCATCT
Chr.6	Fh6-01	9 495	CCTGAATACCCCCGTCCTAT	CTCAGAACTGCTCCCCTCAC
	Fh6-06	6 836 044	GGAAATGTCTGAGCATGCAA	TTTTCCACCCACAACACAAA
	Fh6-10	10 384 324	TTTCATCCTCATCAAGTCCTCA	AGTGAAAACCGGACCACAAG
	Fh6-14	14 869 148	TAGTTTGAATTGCGAACCCC	TACACGCATGAGCTGAGAGC
	Fh6-17	18 459 550	AATTTTACCGGGCTCTCCTC	AACAACATAACTCGCTGCCA
	Fh6-19	21 169 068	CTGTTTTTCGGCGTTCAAA	CCTCCGATTGTGGTTCTGTT
Chr.7	Fh7-01	97 643	CCTTTGTGAGAGGGTAAGCG	GGCGCAATCATTTCTTTCAT
	Fh7-05	4 570 335	GGGCCAAAGAGTATTGATTTTT	CCACCACCAACTGACCTTTA
	Fh7-07	8 179 352	GATTCCTTCTCTCGTGTGGC	TGGAACTTGGAACAACACCA
	Fh7-09	11 357 338	AAACCGAAACCAAACCCAAT	TCTCTAATGAGGGTCGGGGT
	Fh7-10	14 093 476	ATTCAGGTCGATATACGGCG	ATCGTCCAAAATGGAGCAAC
	Fh7-11	19 572 019	CAGTACCACAGCAAGACGGA	ACTTGGTTAATCCCCGAACC
	Fh7-14	25 270 436	GGGCCAAGCTAGCACACTTA	CTTATTGTCGTAGGCCGCTC
	Fh7-16	28 329 053	GGCAATAGCCGTTCCATCTA	AATTTTCAGTTTGAGGCCGA
	Fh7-19	31 726 654	CGCCAACTCAGAGACATTGA	ATCGCTTGTGAAGCTTTGCT
	Fh7-20	32 198 988	AGGCCCATGTAGAAAGCTGA	TTGCACGTGCTTTTCCATAG
Chr.8	Fh8-03	537 740	TTGAGCAAAGATGGCGAATA	TGCCAAGAGCAACAGGAATA
	Fh8-06	5 852 004	GCAAGCATGTGCATTTAGGA	AATGGCTTCATGTCAAAGGG
	Fh8-09	8 990 917	GGAGGTTGCAAATATGGCTG	ACAAGGTGGCTGACCGTTAC
	Fh8-16	14 784 483	AATCACGCCAAGAAATCCAC	TCAACCGCACCTATAACCAA
	Fh8-19	19 111 826	TCAAACGTCCACGTTGTTCT	GGAGAAGGTCTCCTCATCCC
	Fh8-20	20 640 431	GTTGTGTTGCTCTGGGGTTT	CCACTGTGTATCTGGGTGGA
Chr.9	Fh9-01	2 559 855	TTTTGAACCATTGATTTCAAGC	TGCCCTTTTAAATCTCCAGTT
	Fh9-09	9 980 616	CATGCAGGCATACACAGTGA	GATGGGGCGAAAGAGTACAA
	Fh9-11	10 172 322	TACATAGGCATTTCCCCTGC	TTGGGAGATGCTTTCAGAGG
	Fh9-15	16 276 097	ATATGAGTTTCGGTGAGCCG	TCTGGATAGCCAACCAAGTAAAA
	Fh9-19	18 330 523	GACTCTCCCACTGGACCAAA	GTGCACATTATTCATTGGCG

Development and Application of Insertion-Deletion（InDel）Markers in Gannan wild Fortunella hindsii Based on Whole Genome Re-sequencing Data

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标记名称 Marker name	主等位基因频率 Major allele frquency	基因型数 Genotype number	等位基因数 Number of allele	基因多样性指数 Gene diversity index	期望杂合度 Expected heterozygosity	多态性信息含量 Polymorphic information content
Fh1-01	0.7345	3	2	0.3900	0.0345	0.3140
Fh1-03	0.8048	3	2	0.3319	0.2123	0.3054
Fh1-05	0.6563	2	1	0.5036	0.3889	0.4460
Fh1-08	0.4452	4	2	0.7375	0.6027	0.0709
Fh1-14	0.5856	6	4	0.4853	0.2808	0.3676
Fh1-18	0.5822	2	1	0.5498	0.4589	0.4723
Fh1-20	0.7466	3	1	0.3784	0.3151	0.3068
Fh2-01	0.4041	5	2	0.7663	0.2877	0.3400
Fh2-03	0.4349	12	8	0.6885	0.3973	0.6344
Fh2-05	0.5651	4	2	0.4915	0.4315	0.3707
Fh2-08	0.7363	7	3	0.3883	0.0342	0.3129
Fh2-11	0.4212	3	1	0.6663	0.4932	0.3988
Fh2-16	0.5616	3	1	0.4924	0.3151	0.3712
Fh2-18	0.4555	4	2	0.6767	0.2945	0.4225
Fh3-01	0.6746	5	3	0.3553	0.4621	0.2922
Fh3-04	0.5868	6	4	0.4962	0.2966	0.4443
Fh3-07	0.7600	2	1	0.2340	0.1438	0.2111
Fh3-11	0.4754	5	3	0.6334	0.4496	0.5872
Fh3-15	0.4509	3	2	0.4062	0.4067	0.3748
Fh3-17	0.5445	7	4	0.4021	0.3589	0.4018
Fh3-19	0.5313	13	8	0.4171	0.3388	0.4442
Fh4-04	0.5151	15	10	0.4302	0.1772	0.4532
Fh4-12	0.5768	3	2	0.3830	0.3587	0.4446
Fh4-15	0.4465	6	4	0.5226	0.5389	0.0585
Fh4-19	0.2613	4	2	0.7128	0.7570	0.4830
Fh5-03	0.3845	6	3	0.5077	0.4286	0.5068
Fh5-07	0.3815	3	1	0.5602	0.6103	0.0612
Fh5-11	0.5257	5	2	0.4208	0.4636	0.4248
Fh5-14	0.2253	6	2	0.7362	0.4356	0.0837
Fh5-18	0.2523	14	6	0.6729	0.5474	0.0737
Fh5-20	0.3665	4	2	0.5128	0.5823	0.2856
Fh6-01	0.5167	6	3	0.4289	0.1769	0.4306
Fh6-06	0.2403	3	1	0.5351	0.4950	0.2626
Fh6-10	0.4855	16	10	0.3937	0.3133	0.3458
Fh6-14	0.3925	3	1	0.5436	0.2922	0.5851
Fh6-17	0.6675	19	12	0.2823	0.4633	0.2706
Fh6-19	0.5797	2	1	0.3968	0.2944	0.4154
Fh7-01	0.7529	6	2	0.1837	0.1385	0.1933
Fh7-05	0.4683	6	3	0.5084	0.4413	0.3515
Fh7-07	0.4438	7	3	0.3996	0.3985	0.3492
Fh7-09	0.5374	9	6	0.4865	0.3506	0.3750
Fh7-10	0.5242	6	3	0.5049	0.3305	0.4153
Fh7-11	0.6372	20	9	0.3473	0.2086	0.2424
Fh7-14	0.6989	6	4	0.3657	0.1885	0.2828
Fh7-16	0.5686	4	1	0.3819	0.0269	0.2913
Fh7-19	0.3834	16	8	0.3238	0.2084	0.2831
Fh7-20	0.5066	3	2	0.4955	0.3886	0.4170
Fh8-03	0.5036	14	11	0.7294	0.6067	0.1676
Fh8-06	0.6478	7	4	0.4772	0.2783	0.3424
Fh8-09	0.3960	6	4	0.5417	0.4600	0.4421
Fh8-16	0.4268	3	2	0.3703	0.3133	0.2845
Fh8-19	0.5570	2	1	0.7582	0.2853	0.2970
Fh8-20	0.7282	8	5	0.6804	0.3971	0.2648
Fh9-01	0.4131	14	8	0.4834	0.4320	0.3453
Fh9-09	0.5535	3	1	0.3802	0.0266	0.2903
Fh9-11	0.4474	4	3	0.6582	0.4851	0.1626
Fh9-15	0.7609	15	9	0.4843	0.3070	0.3458
Fh9-19	0.6609	3	2	0.6686	0.2864	0.2851
平均值 Mean	0.5274	6.5345	3.6207	0.4959	0.3534	0.3363

种质 Germplasm	样品数 Number of accessions	观测杂合度 Observed heterozygosity	期望杂合度 Expected heterozygosity	Nei 多样性指数 Nei’s diversity index	香农维纳指数 Shanon-Wiener index	多态信息含量 PIC
所有All	99	0.010 ~ 1.000（0.244）	0.095 ~ 0.500（0.304）	0.096 ~ 0.505（0.306）	0.199 ~ 0.693（0.470）	0.090 ~ 0.375（0.248）
核心Core	29	0.034 ~ 1.000（0.264）	0.095 ~ 0.500（0.320）	0.097 ~ 0.517（0.326）	0.199 ~ 0.693（0.490）	0.090 ~ 0.375（0.260）

编号 ID	叶片Leaf			果实Fruit				种子Seed
编号 ID	长度/cm Length	宽度/cm Width	长宽比 Length-width ratio	横径/mm Transverse diameter	纵径/mm Vertical diameter	横纵比 Horizontal ratio	单果质量/g Single fruit weight	数量 Number	粒质量/g Grain weight
CF03	6.00	3.00	2.000	10.00	9.00	1.111	0.43	2.67	0.100
CF04	6.60	3.20	2.063	8.00	6.00	1.333	0.36	2.95	0.073
CF06	11.50	4.00	2.875	10.00	10.00	1.000	0.50	1.80	0.122
CF07	10.00	3.00	3.333	10.00	8.00	1.250	0.50	1.67	0.120
SBS02	9.20	2.80	3.286	6.00	4.00	1.500	0.05	1.00	0.300
ZGD03	6.15	2.55	2.431	11.49	10.92	1.057	0.81	2.67	0.087
ZGD04	6.23	2.47	2.535	11.59	10.22	1.136	0.77	1.83	0.013
YK13	6.28	2.93	2.202	11.08	8.89	1.251	0.70	0
YK19	4.82	2.32	2.088	11.29	9.39	1.202	0.67	0
YK25	6.10	2.75	2.229	11.88	11.59	1.026	1.18	0.67	0.250
JH01	6.23	2.97	2.100	22.55	23.36	0.970	6.58	3.17	0.134
JH02	6.87	2.38	2.891	19.70	17.59	1.121	3.66	4.67	0.140
JH11	5.38	2.13	2.530	20.73	20.14	1.030	5.02	5.33	0.133
JH15	5.58	2.53	2.417	19.42	19.80	0.981	3.64	3.83	0.098
JH16	6.03	2.33	2.583	22.40	21.95	1.021	5.76	6.17	0.143
JH17	6.63	2.53	2.632	18.48	16.86	1.098	3.43	5.50	0.102
JH18	6.20	2.22	2.792	19.55	17.34	1.129	3.68	4.20	0.089
JH19	5.36	1.96	2.736	19.81	18.24	1.089	3.95	4.00	0.161
JH22	6.17	3.02	2.061	10.25	10.59	0.967	0.57	3.00	0.073
JH23	5.82	2.87	2.049	12.48	14.27	0.876	1.18	1.83	0.127
JH24	4.68	2.47	1.894	12.14	10.73	1.132	0.92	3.67	0.154
JH26	5.50	2.12	2.606	20.48	19.08	1.075	4.80	6.40	0.028
JH36	5.57	2.78	2.012	12.58	11.27	1.118	0.92	3.33	0.103
JH37	5.25	2.60	2.047	11.12	11.41	0.977	0.72	2.67	0.095
SS02	6.73	2.07	3.247	10.09	9.73	1.033	0.53	2.50	0.089
SS04	3.55	1.88	1.930	8.74	9.41	0.935	0.40	1.33	0.046
SS05	6.75	3.42	1.994	9.11	9.36	0.973	0.55	1.60	0.083
CY01	5.77	2.93	1.977	9.67	7.69	1.257	1.20	2.33	0.062
YD03	4.57	2.82	1.651	10.33	9.55	1.082	1.26	1.67	0.047

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