望春玉兰种特异InDel引物开发及在根皮真伪鉴定中的应用

doi:10.16420/j.issn.0513-353x.2024-0563

摘要/Abstract

摘要： 作为提取小白菊内酯的原料，收集望春玉兰（Magnolia biondii）根皮中可能混有玉兰（M. denudata）根皮。以望春玉兰和玉兰为研究材料，基于混池测序数据对其物种特异性InDel分子标记开展研究。结果显示，望春玉兰和玉兰的混池测序数据在过滤后分别获得了103.6 Gb和82.4 Gb的高质量序列，比对望春玉兰基因组后共检测到长度 ≥ 5 bp且在种间保守的InDel位点13 152个，并在其中10 627个位点成功设计了引物。从望春玉兰的19条染色体上随机选取2对引物进行合成，扩增结果显示，31对引物在玉兰中具有通用性，通用率达81.6%，其中有17对引物的扩增条带在望春玉兰和玉兰种间存在长度差异。通过对自然群体进一步扩增检测发现，引物Mbi010和Mbi069扩增出的条带存在种内保守、种间差异明显，在望春玉兰自然群体中的扩增条带分别为120 和146 bp，在玉兰自然群体中为115 和140 bp。利用上述2对引物分别对玉兰和望春玉兰混合根皮提取的DNA进行扩增，可分别扩增出两物种特异性片段，表明这两对引物可用于望春玉兰根皮真伪的鉴定。

关键词: 望春玉兰, 混池测序, 种特异性引物, 根皮, 分子鉴别

Abstract:

As raw materials for extracting parthenolide，the root barks of Magnolia biondii may be adulterated with that of M. denudata. Using these two species as research materials，species-specific InDel markers were identified based on pooled sequencing data. The filtered pooled sequencing data yieled 103.6 and 82.4 Gb of high-quality sequences for M. biondii and M. denudata，respectively. After alignment the filtered reads to the genome of M. biondii，a total of 13 152 interspecific-fixed InDel sites（≥ 5 bp）were detected，and primers were successfully designed for 10 627 of the identified InDels. Two pairs of primers were randomly selected from each of the 19 chromosomes of M. biondii for synthesis. Amplification results showed that 31 primer pairs（81.6%）exhibited universality in M. denudata，with 17 primer pairs displaying length polymorphism between the two species. Further examination by capillary electrophoresis using natural populations of M. biondii and M. denudata revealed that primers Mbi010 and Mbi069 produced species-specific bands with intraspecific conservation and marked interspecific differences. Primer Mbi010 could amplify 120 bp and 115 bp fragments in M. biondii and M. denudata，respectively，while Mbi069 generated 146 bp and 140 bp fragments in respective species. Using these two primer pairs to amplify DNA extracted from mixed root bark samples of M. biondii and M. denudata successfully identified species-specific fragments for each taxon，demonstrating their utility for authenticating of M. biondii root bark adulteration.

Key words: Magnolia biondii, Pool-seq, species-specific primers, root bark, molecular identification

戴晓港, 梁青青, 毕长伟, 魏铭辰, 刘景胜. 望春玉兰种特异InDel引物开发及在根皮真伪鉴定中的应用[J]. 园艺学报, 2025, 52(9): 2387-2394.

DAI Xiaogang, LIANG Qingqing, BI Changwei, WEI Mingchen, LIU Jingsheng. Development of Species-Specific InDel Primers for Magnolia biondii and Their Application in Root Bark Identification[J]. Acta Horticulturae Sinica, 2025, 52(9): 2387-2394.

图/表 5

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样品 Sample	原始测序序列 Raw read	过滤后序列 Clean read	高质量序列比例/% Clean reads rate	高质量碱基数量/Gb Clean read base	Q30/%	GC含量/% GC content
望春玉兰 Magnolia biondii	709 647 064	690 949 980	97.39	103.6	93.92	36.17
玉兰 Magnolia denudata	560 966 106	549 298 162	97.92	82.4	93.96	36.32

样品 Sample	原始测序序列 Raw read	过滤后序列 Clean read	高质量序列比例/% Clean reads rate	高质量碱基数量/Gb Clean read base	Q30/%	GC含量/% GC content
望春玉兰 Magnolia biondii	709 647 064	690 949 980	97.39	103.6	93.92	36.17
玉兰 Magnolia denudata	560 966 106	549 298 162	97.92	82.4	93.96	36.32

序号 Seq.	染色体 Chr.	引物名称 Primer name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer	片段大小/bp Fragment size	退火温度/℃ T_m
1	Chr01	Mbi002	TCACTTGATTTGACCGTTGA	ATCTGATGTCGCCGCAAG	104	57.6
2	Chr01	Mbi010	TTCAATCATCCCATTAGCTGC	GGGACCTTCATCCAAATCAA	123	60.0
3	Chr02	Mbi022	CAACGGTCGGAATTCAATCT	GCTCGACTCATAAACAGCCC	150	59.9
4	Chr02	Mbi030	CTCACCTCCATTACCCTCCA	TGGACTGGTTTGATGCGATA	137	59.9
5	Chr03	Mbi031	GCCCCAAAGATCTTAACCAG	TAAAGAGGCTGTTTGGTGCC	108	58.6
6	Chr03	Mbi032	CGGCCAGACAATCTAGGAAG	GGGCATCAAATCACAACCTT	109	59.8
7	Chr04	Mbi034	CTGGCCTCTTTCTTTGGAAG	TGGCTCACACAAAGCAAGTT	133	59.0
8	Chr04	Mbi037	GGATGGACGGTGTGGATAAG	ATGATGAGCAAGGCCAGAAT	101	60.1
9	Chr05	Mbi068	GACTATAAAGGCGCGTCAGC	GCCAGCTTCAAAACGTATGC	129	60.0
10	Chr05	Mbi069	GCATTTGGTTCGGCTGTAGT	TGGTGCTGTGGGTGTAAGTC	147	60.1
11	Chr06	Mbi076	CTTTTGGTTACTGGGCCATC	GGCCTCGAGTCGTACCATAG	133	59.4
12	Chr06	Mbi082	ACAAACGCCTACCGTTGAAA	CCAAAGTTTTCGATCAGGCT	127	60.5
13	Chr07	Mbi094	GCATTCCAAGAAAGAATGCC	TCCTCCCTTGTCATCAGCAT	101	59.6
14	Chr07	Mbi106	CAGCCTCATCCATGTGTGTT	GACCAAATTCCACATCTCGG	138	59.5
15	Chr08	Mbi114	GGGTTGCAATAGGACTGAGC	CTGCAGAAGTTGGTCCTGCT	138	59.7
16	Chr08	Mbi115	AGCCATGGGGAAAAATTCAT	CTAATCAATCCATGCTGGGG	137	60.5
17	Chr09	Mbi116	GGCTCAGTTTCTCCTTACCG	ACCCACCATCCTCTTCCTCT	107	58.9
18	Chr09	Mbi117	ACACAAATGCCCATACATGC	GTGTGGCCCACCAAATAAAC	110	59.2
19	Chr10	Mbi121	CCATTAAGACGGTTCGGCTA	CGGACTTCTCGTTTCCAAAA	149	60.0
20	Chr10	Mbi123	TAGGGCCACTGAAATGGAAG	GTTTCCAACAGGATTGCACC	142	60.0
21	Chr11	Mbi134	TCCAAATGTGTGGAGGATAGC	TTATTCCAGTGATGCAGTAGCA	146	59.9
22	Chr11	Mbi137	GTTGGTACCTAGCAGACCGC	TGGGACCCTATGAAGCTTTT	149	59.7
23	Chr12	Mbi141	TCTTTTCTATCGGTGCGTCC	GCAAGCAAGATTCAACCCAT	107	60.2
24	Chr12	Mbi142	AACCTAAACCCGATCCTGAAA	AGGTTATAGGTTATAGGTTTGGGAA	100	59.8
25	Chr13	Mbi144	ACTTTCGCTTCCCTCACCTT	TAATGGGCAATCACTTGTGG	140	60.2
26	Chr13	Mbi153	AACGGTCCAACCATCTCAAG	CCTCCACGTGTGGTCTACCT	132	59.9
27	Chr14	Mbi154	AGCATGTGGTGTTCAGTGGA	CCCTACAAAACACCGTAGGAA	149	60.1
28	Chr14	Mbi159	TCCTAAATGGTGAAAAGCAAA	TCAGAGAGGAACCTTGTGGC	126	57.4
29	Chr15	Mbi164	GAGATCCAGGCCATCAAATG	GATCAGGTGGGGTCCTCTTT	108	60.4
30	Chr15	Mbi169	TCTGAGCTTTCCACTCGTGA	CCAGCCATTGATTTGTTTTG	114	59.7
31	Chr16	Mbi184	GGCCATTCTACTCCGAACTG	GGGGTGGTTATGAGTTCTGC	137	59.6
32	Chr16	Mbi195	GATTCGACCGCTGGAGTTAC	TCTCCTAGAGGATGGCCAAA	124	59.7
33	Chr17	Mbi196	GGCGAGCATGACTCAATATG	AGGGGGTTTTATGAGTCGCT	130	59.2
34	Chr17	Mbi202	TTGTATTTGGCCGGATCAAC	CATCTTGGGTTGTTAGTTGGG	146	60.7
35	Chr18	Mbi204	ACCACTGTTGTTTGGGCTGT	TTGAAGCTGGTGGAACTGTG	132	60.4
36	Chr18	Mbi210	ACCAGGACCACACCTTTCAG	CACGTGTCGAAACGCTACTC	129	60.0
37	Chr19	Mbi216	GGCTACATGAGACTCGACCC	TGTGCACTGCTTAGCAAATTC	118	59.6
38	Chr19	Mbi218	GAAAACTTCCTAGGGCCCAC	CTAATTGAACGGCCACCATT	141	59.9

序号 Seq.	染色体 Chr.	引物名称 Primer name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer	片段大小/bp Fragment size	退火温度/℃ T_m
1	Chr01	Mbi002	TCACTTGATTTGACCGTTGA	ATCTGATGTCGCCGCAAG	104	57.6
2	Chr01	Mbi010	TTCAATCATCCCATTAGCTGC	GGGACCTTCATCCAAATCAA	123	60.0
3	Chr02	Mbi022	CAACGGTCGGAATTCAATCT	GCTCGACTCATAAACAGCCC	150	59.9
4	Chr02	Mbi030	CTCACCTCCATTACCCTCCA	TGGACTGGTTTGATGCGATA	137	59.9
5	Chr03	Mbi031	GCCCCAAAGATCTTAACCAG	TAAAGAGGCTGTTTGGTGCC	108	58.6
6	Chr03	Mbi032	CGGCCAGACAATCTAGGAAG	GGGCATCAAATCACAACCTT	109	59.8
7	Chr04	Mbi034	CTGGCCTCTTTCTTTGGAAG	TGGCTCACACAAAGCAAGTT	133	59.0
8	Chr04	Mbi037	GGATGGACGGTGTGGATAAG	ATGATGAGCAAGGCCAGAAT	101	60.1
9	Chr05	Mbi068	GACTATAAAGGCGCGTCAGC	GCCAGCTTCAAAACGTATGC	129	60.0
10	Chr05	Mbi069	GCATTTGGTTCGGCTGTAGT	TGGTGCTGTGGGTGTAAGTC	147	60.1
11	Chr06	Mbi076	CTTTTGGTTACTGGGCCATC	GGCCTCGAGTCGTACCATAG	133	59.4
12	Chr06	Mbi082	ACAAACGCCTACCGTTGAAA	CCAAAGTTTTCGATCAGGCT	127	60.5
13	Chr07	Mbi094	GCATTCCAAGAAAGAATGCC	TCCTCCCTTGTCATCAGCAT	101	59.6
14	Chr07	Mbi106	CAGCCTCATCCATGTGTGTT	GACCAAATTCCACATCTCGG	138	59.5
15	Chr08	Mbi114	GGGTTGCAATAGGACTGAGC	CTGCAGAAGTTGGTCCTGCT	138	59.7
16	Chr08	Mbi115	AGCCATGGGGAAAAATTCAT	CTAATCAATCCATGCTGGGG	137	60.5
17	Chr09	Mbi116	GGCTCAGTTTCTCCTTACCG	ACCCACCATCCTCTTCCTCT	107	58.9
18	Chr09	Mbi117	ACACAAATGCCCATACATGC	GTGTGGCCCACCAAATAAAC	110	59.2
19	Chr10	Mbi121	CCATTAAGACGGTTCGGCTA	CGGACTTCTCGTTTCCAAAA	149	60.0
20	Chr10	Mbi123	TAGGGCCACTGAAATGGAAG	GTTTCCAACAGGATTGCACC	142	60.0
21	Chr11	Mbi134	TCCAAATGTGTGGAGGATAGC	TTATTCCAGTGATGCAGTAGCA	146	59.9
22	Chr11	Mbi137	GTTGGTACCTAGCAGACCGC	TGGGACCCTATGAAGCTTTT	149	59.7
23	Chr12	Mbi141	TCTTTTCTATCGGTGCGTCC	GCAAGCAAGATTCAACCCAT	107	60.2
24	Chr12	Mbi142	AACCTAAACCCGATCCTGAAA	AGGTTATAGGTTATAGGTTTGGGAA	100	59.8
25	Chr13	Mbi144	ACTTTCGCTTCCCTCACCTT	TAATGGGCAATCACTTGTGG	140	60.2
26	Chr13	Mbi153	AACGGTCCAACCATCTCAAG	CCTCCACGTGTGGTCTACCT	132	59.9
27	Chr14	Mbi154	AGCATGTGGTGTTCAGTGGA	CCCTACAAAACACCGTAGGAA	149	60.1
28	Chr14	Mbi159	TCCTAAATGGTGAAAAGCAAA	TCAGAGAGGAACCTTGTGGC	126	57.4
29	Chr15	Mbi164	GAGATCCAGGCCATCAAATG	GATCAGGTGGGGTCCTCTTT	108	60.4
30	Chr15	Mbi169	TCTGAGCTTTCCACTCGTGA	CCAGCCATTGATTTGTTTTG	114	59.7
31	Chr16	Mbi184	GGCCATTCTACTCCGAACTG	GGGGTGGTTATGAGTTCTGC	137	59.6
32	Chr16	Mbi195	GATTCGACCGCTGGAGTTAC	TCTCCTAGAGGATGGCCAAA	124	59.7
33	Chr17	Mbi196	GGCGAGCATGACTCAATATG	AGGGGGTTTTATGAGTCGCT	130	59.2
34	Chr17	Mbi202	TTGTATTTGGCCGGATCAAC	CATCTTGGGTTGTTAGTTGGG	146	60.7
35	Chr18	Mbi204	ACCACTGTTGTTTGGGCTGT	TTGAAGCTGGTGGAACTGTG	132	60.4
36	Chr18	Mbi210	ACCAGGACCACACCTTTCAG	CACGTGTCGAAACGCTACTC	129	60.0
37	Chr19	Mbi216	GGCTACATGAGACTCGACCC	TGTGCACTGCTTAGCAAATTC	118	59.6
38	Chr19	Mbi218	GAAAACTTCCTAGGGCCCAC	CTAATTGAACGGCCACCATT	141	59.9