Development and Application of Abundance SNP Markers for Auricularia heimuer

doi:10.19586/j.2095-2341.2024.0120

Current Biotechnology ›› 2024, Vol. 14 ›› Issue (6): 892-901.DOI: 10.19586/j.2095-2341.2024.0120

• Special Forum on Edible and Medicinal Fungi Biotechnology • Previous Articles Next Articles

Development and Application of Abundance SNP Markers for Auricularia heimuer

Dinghong JIA¹(), Xun LIU¹, Bo WANG¹(), Xiaowei WANG²^,³, Tong LI², Lijing HUANG⁴

^1.Sichuan Institute of Edible Fungi，Sichuan Academy of Agricultural Sciences，Chengdu 610066，China
^2.Gansu Academy of Agricultural Sciences，Lanzhou 730070，China
^3.Tianshui Hongyahe Mushroom Planting Co. ，Ltd. ，Gansu Tianshui 741001，China
^4.School of Agriculture，Ludong University，Shandong Yantai 264001，China

Received:2024-07-02 Accepted:2024-08-14 Online:2024-11-25 Published:2024-12-27
Contact: Dinghong JIA,Bo WANG

黑木耳丰度SNP标记开发及应用

贾定洪¹(), 刘询¹, 王波¹(), 王晓巍²^,³, 李通², 黄礼璟⁴

^1.四川省农业科学院四川省食用菌研究所，成都 610066
^2.甘肃省农业科学院，兰州 730070
^3.天水红崖河菌业种植有限公司，甘肃天水 741001
^4.鲁东大学农学院，山东烟台 264001

通讯作者: 贾定洪,王波
作者简介:贾定洪E-mail: jdhdragon@163.com
贾定洪E-mail: jdhdragon@163.com
基金资助:
国家重点研发计划项目(2022YFD1602109)

Abstract

Abstract:

The genome of Auricularia heimuer Dai13782 was used as the reference genome， while the genomes of A. subglabra SS5， A. auricula-judae B14-8， A. cornea CCMJ2827， and A. cornea ACW001 from the NCBI database were utilized as the test data. The genome variation data of A. heimuer and its related species were analyzed using Bwa， Samtools and Bcftools. The genetic relationship matrix was obtained using Tassel 5 software， and the genetic relationships among five strains were visualized using the pheatmap package. VCFtools-0.1.16 software was used to filter and extract abundant single nucleotide polymorphisms （aSNPs）， specifically aSNP regions with a SNP count equal to or greater than 50 in a 300 bp sliding window. Subsequently， 47 407 pairs of primers were designed using Primer 3 software. E-PCR results showed that 1 020 pairs of primers met the experimental design， involving 8 aSNP regions. Each region retained 1 pair of specific primers， which were distributed in 6 contig regions of the reference genome. In this experiment， the commonly used ITS and LSU were employed as reference markers， and the genetic relationship of 13 A. heimuer strains was analyzed using aSNP markers. The results showed that the phylogenetic relationships revealed by aSNP markers were generally consistent with the results of ITS and LSU markers， and even better able to distinguish closely related A. heimuer strains. On the whole， it reflects that A. heimuer strains are relatively close to A. corne， and farthest from A. subglabra. This result is consistent with the genetic relationship revealed by the whole-genome SNP analysis， which also confirms the reliability of the results based on aSNP sequence analysis. The aSNP marker sequence interval developed in the experiment contains numerous mutation sites， which is expected to serve as a beneficial supplement to traditional target analysis sequences such as ITS， Rpb2， and Ef1α， providing assistance for the systematic development and genetic identification of A. heimuer and other germplasm resources.

Key words: Auricularia heimuer, aSNP, VCFtools, germplasm resource

摘要：

以黑木耳（Auricularia heimuer Dai13782）的基因组作为参考框架，利用NCBI数据库中收录的皱木耳SS5、黑木耳B14-8、毛木耳CCMJ2827及毛木耳ACW001等菌株的基因组为测试数据，通过一系列生物信息学工具（如Bwa、Samtools、Bcftools）分析了黑木耳及近缘种共5个菌株之间的基因组变异情况。利用Tassel 5软件，构建了包含5个菌株在内的亲缘关系矩阵，利用R语言中的Pheatmap包，对这一矩阵进行了可视化处理，直观地展示了这5个菌株之间的亲缘关系。采用VCFtools-0.1.16软件，通过设定300 bp的滑动窗口，筛选出其中单核苷酸多态性（single nucleotide polymorphism, SNP）数量等于或大于50的丰度SNP（abundance SNP, aSNP）区域。利用Primer 3软件，针对这些aSNP区域设计了47 407对引物。经过e-PCR的严格筛选，发现1 020对引物符合实验设定，涉及8个aSNP区域，每个区域保留1对特异引物，分布在参考基因组的6个重叠群区域。以常用的ITS和LSU作为参考标记，以aSNP标记验证分析13个黑木耳菌株的遗传亲缘关系。结果显示，aSNP标记揭示的系统发育关系大体上与ITS、LSU标记结果一致，甚至能更细致地区分亲缘关系很近的黑木耳菌株。总体上反映出黑木耳菌株与毛木耳亲缘关系相对较近，与皱木耳关系最远，该结果与基于全基因组SNP揭示的亲缘关系结果基本一致，侧面证实了基于aSNP序列分析结果的可靠性。实验开发的aSNP标记序列区间含有丰富的变异位点，有望作为传统ITS、Rpb2、Ef1α等靶标分析序列的有益补充，为黑木耳等种质资源的系统发育及遗传鉴定提供助力。

关键词: 黑木耳, aSNP, VCFtools, 种质资源

CLC Number:

Dinghong JIA, Xun LIU, Bo WANG, Xiaowei WANG, Tong LI, Lijing HUANG. Development and Application of Abundance SNP Markers for Auricularia heimuer[J]. Current Biotechnology, 2024, 14(6): 892-901.

贾定洪, 刘询, 王波, 王晓巍, 李通, 黄礼璟. 黑木耳丰度SNP标记开发及应用[J]. 生物技术进展, 2024, 14(6): 892-901.

Figures/Tables 9

References 32

1	SUN X, YANG C, MA Y, et al.. Research progress of Auricularia heimuer on cultivation physiology and molecular biology[J/OL]. Front. Microbiol., 2022, 13: 1048249[2024-07-25]. .
2	崔玉玲. 黑木耳种质资源遗传特性及优良菌株筛选研究[D]. 长春:吉林农业大学, 2023.
3	尹显达. 黑木耳菌糠相关成分检测及其资源化开发技术创新[D]. 保定: 河北大学, 2023.
4	员瑗. 中国野生黑木耳遗传多样性及全基因组信息分析[D]. 北京: 北京林业大学, 2018.
5	曹建刚, 毛仪楠, 陈再民, 等. 不同木屑栽培黑木耳配方对比试验[J]. 食用菌, 2024, 46(2): 37-39.
	CAO J G, MAO Y N, CHEN Z M, et al.. Comparative experiment on formula of Auricularia auricula cultivated with different sawdust[J]. Edible Fungi, 2024, 46(2): 37-39.
6	李富春, 陈晓文, 张蓉, 等. 光伏钢架塑料大棚袋料栽培黑木耳品种筛选[J]. 寒旱农业科学, 2023, 2(11): 1026-1030.
	LI F C, CHEN X W, ZHANG R, et al. Screening Auricularia auricula varieties used for bagged cultivation in steel-framed photovoltaic greenhouses[J]. J. Cold-Arid. Agric. Sci., 2023, 2(11): 1026-1030.
7	杨迪. 玉米芯栽培黑木耳的配方筛选及胞外酶活性研究[D]. 长春:吉林农业大学, 2023.
8	姜冬洋, 苏林贺, 陈亚东, 等. 富硒黑木耳菌丝体硒多糖的提取与抗氧化活性[J]. 菌物学报, 2024, 43(2): 115-128.
	JIANG D Y, SU L H, CHEN Y D, et al. Extraction and antioxidant activities of selenium polysaccharide from selenium-enriched Auricularia heimuer mycelia[J]. Mycosystema, 2024, 43(2): 115-128.
9	李博文, 毛雪, 姜威, 等. 黑木耳降血糖的研究进展及趋势分析[J]. 中国农学通报, 2024, 40(2):143-151.
	LI B W, MAO X, JIANG W, et al. Research progress and trend analysis of hypoglycemia of Auricularia auricula [J]. Chin. Agri. Sci. Bull., 2024, 40(2): 143-151.
10	马银鹏.黑木耳黑色素液体发酵制备及其抗氧化作用[D].哈尔滨:东北林业大学,2023.
11	ZONG X, ZHANG H, ZHU L, et al.. Auricularia auricula polysaccharides attenuate obesity in mice through gut commensal Papillibacter cinnamivorans [J]. J. Adv. Res., 2023, 52: 203-218.
12	李伶俐, 路新彦, 蒋俊, 等. 黑木耳‘林黑1号’‍[J]. 园艺学报, 2023, 50(S2):103-104.
	LI L L, LU X Y, JIANG J, et al.. Auricularia auricula ‘Linhei 1’[J]. Acta Hortic. Sinica, 2023, 50(S2): 103-104.
13	于海洋, 盛春鸽, 史磊, 等. 5个野生黑木耳菌株的驯化栽培及评价研究[J]. 北方园艺, 2024,14:123-128.
	YU H Y, SHENG C G, SHI L, et al. Domestication cultivation and evaluation of five wild Auricularia heimuer strains[J]. North. Hortic., 2024, 14: 123-128.
14	马庆芳,张丕奇,戴肖东,等.黑木耳Au185菌株一个SCAR标记的建立[J].菌物研究,2009,7(2):104-108+115.
	MA Q F, ZHANG P Q, DAI X D, et al.. A scar marker for Auricularia auricula strain Au185[J]. J. Fungal Res., 2009, 7(2): 104-108+115.
15	赵丽, 陈艳秋. 黑木耳栽培菌株亲缘关系的RAPD分析[J]. 食用菌, 2009, 31(4):14-15.
	ZHAO L, CHEN Y Q. RAPD analysis of genetic relationship of cultivated strains of Auricularia auricula [J]. Edible Fungi, 2009, 31(4): 14-15.
16	李红,张敏,屈麟,等.基于ISSR-PCR分子标记的黑木耳遗传多样性分析[J].园艺与种苗,2019,39(6):1-3+23.
	LI H, ZHANG M, QU L, et al.. Analyse on genetic diversity of Auricularia auricular strains with ISSR-PCR molecular marker[J]. Hortic. Seed, 2019, 39(6): 1-3+23.
17	史灵燕,刘保卫,顾新颖,等.生化和ISSR分子标记在黑木耳品种鉴定中的应用[J].北方园艺,2019,9:136-141.
	SHI L Y, LIU B W, GU X Y, et al.. Application of biochemical and ISSR molecular markers in identification of Auricularia auricular[J]. North. Hortic., 2019, 9: 136-141.
18	王立枫, 许修宏, 刘华晶. 黑龙江地区野生黑木耳菌种的ISSR指纹分析[J]. 东北农业大学学报, 2011, 42(2): 109-114.
	WANG L F, XU X H, LIU H J. ISSR fingerprint analysis of wild strains of Auricularia auricula in Heilongjiang Province[J]. J. North. Agric. Uni., 2011, 42(2):109-114.
19	史灵燕,张云龙,刘保卫,等.基于SRAP分子标记的24个黑木耳栽培菌株遗传分析[J].食用菌,2019,41(6):20-23+33.
	SHI L Y, ZHANG Y L, LIU B W, et al.. Genetic analysis of 24 strains of Auricularia auricula based on SRAP molecular markers[J]. Edible Fungi, 2019, 41(6): 20-23+33.
20	陶朋飞,许修宏,刘华晶.黑龙江省野生黑木耳菌株遗传多样性分析[J].中国农学通报,2011,27(22):182-186.
	TAO P F, XU X H, LIU H J. Genetic diversity analysis of wild Auricularia auriculfrom Heilongjiang Province[J]. Chin. Agric. Sci. Bull., 2011, 27(22): 182-186.
21	路新彦,刘昆,蒋俊,等.7个黑木耳菌株遗传差异性分析[J].中国食用菌,2017,36(1):52-55.
	LU X Y, LIU K, JIANG J, et al.. Study on genetic polymorphism of 7 Auricularia auricula strains[J]. Edible Fungi China, 2017, 36(1): 52-55.
22	施树, 蒋厚阳, 罗章. 西藏不同地区黑木耳多样性分析[J]. 中国林副特产, 2017(3):11-14.
	SHI S, JIANG H Y, LUO Z. Analysis of genetic diversity and relationship of black fungus from different regions of Tibet[J]. Forest Product Special. China, 2017(3): 11-14.
23	李晶晶, 姚方杰, 鲁丽鑫. 黑木耳自交菌株菌丝体的遗传多样性分析[J]. 吉林农业大学学报, 2024,46(4):566-572
	LI J J, YAO F J, LU L X. Genetic diversity analysis of the mycelium of selfed strains of Auricularia heimuer [J]. J. Jilin Agric. Univ., 2024, 46(4): 566-572.
24	徐安然,付永平,王延锋,等.黑木耳部分种质资源SSR分子身份证的构建[J].农业生物技术学报,2017,25(12):1930-1939.
	XU A R, FU Y P, WANG Y F, et al.. Establishment of a SSR molecular ID system for some Auricularia heimuer germplasm resources[J]. J. Agric. Biotechnol., 2017, 25(12): 1930-1939.
25	申淑慧,戴习林.基于生长和抗逆功能基因SNP分子标记的凡纳滨对虾野生及选育群体遗传多样性分析[J].南方农业学报,2020,51(11):2836-2845.
	SHEN S H, DAI X L. Genetic diversity analysis in wild and selected populations of Penaeus vannamei based on SNP markers in growth and stress resistance genes[J]. J. South. Agric., 2020, 51(11): 2836-2845.
26	闫国跃. 基于转录组SSR、SNP标记的苦玄参遗传多样性及有效成分关联位点分析[D]. 北京:中央民族大学, 2020.
27	贾定洪, 王波, 何晓兰, 等. 基于基因组重测序发掘金针菇InDel、SV及丰度SNP标记[J]. 菌物学报, 2024,43(7):42-53.
	JIA D H, WANG B, HE X L, et al.. Exploitation of InDel, SV and abundance SNP markers in Flammulina filiformis based on genome resequencing[J]. Mycosystema, 2024, 43(7): 42-53.
28	钱修伟. 基于宏基因组的鼠疫耶尔森菌快速溯源技术研究[D]. 合肥:安徽医科大学, 2023.
29	ZHANG L C, LI N, LIU X, et al.. A genome-wide association study of limb bone length using a Large White × Minzhu intercross population[J/OL]. Genet. Sel. Evol., 2014, 46: 56[2024-07-25]. .
30	ZHENG X, MA Y, BAI Y, et al.. Identification and validation of immunotherapy for four novel clusters of colorectal cancer based on the tumor microenvironment[J/OL]. Front. Immunol., 2022, 13: 984480[2024-07-25]. .
31	任静, 何晓莹, 原小燕, 等. 芸薹属SSR标记e-PCR与云南省芥菜型油菜种质遗传多样性分析[J]. 分子植物育种, 2023, 21(16): 5348-5364.
	REN J, HE X Y, YUAN X Y, et al.. Genetic diversity analysis of Brassica by SSR marker e-PCR and Brassica juncea germplasm in Yunnan Province[J]. Mol. Plant Breed., 2023, 21(16): 5348-5364.
32	杨晟, 尹誉蓉, 王长彪, 等. 基于e-PCR的小麦、山羊草属SSR电子指纹图谱的开发[J]. 麦类作物学报, 2023, 43(5):551-561.
	YANG S, YIN Y R, WANG Z B, et al.. Development of the wheat SSR electronic fingerprint based on e-PCR[J]. J. Triticeae Crops, 2023, 43(5): 551-561.

组装版本号	物种名称	菌株	基因组大小/Mb	组装水平
GCA_002092955.1	黑木耳（A. auricula-judae）	B14-8	43.57	Scaffold
GCA_030578095.1	毛木耳（A. cornea）	ACW001	78.72	Scaffold
GCA_002287115.1	黑木耳（A. heimuer）	Dai 13782	49.76	Scaffold
GCA_008368385.1	毛木耳（A. cornea）	CCMJ2827	78.50	Contig
GCA_000265015.1	皱木耳（A. subglabra）	TFB-10046 SS5	74.92	Scaffold

组装版本号	物种名称	菌株	基因组大小/Mb	组装水平
GCA_002092955.1	黑木耳（A. auricula-judae）	B14-8	43.57	Scaffold
GCA_030578095.1	毛木耳（A. cornea）	ACW001	78.72	Scaffold
GCA_002287115.1	黑木耳（A. heimuer）	Dai 13782	49.76	Scaffold
GCA_008368385.1	毛木耳（A. cornea）	CCMJ2827	78.50	Contig
GCA_000265015.1	皱木耳（A. subglabra）	TFB-10046 SS5	74.92	Scaffold

引物名称	上游序列（5′→3′）	下游序列（5′→3′）
ITS4/5	5′-TCCTCCGCTTATTGATATGC-3′	5′-GGAAGTAAAAGTCGTAACAAGG-3′
Rpb2	5′-TGGGGKWTGGTYTGYCCTGC-3′	5′-CCCATRGCTTGYTTRCCCAT-3′
IGS	5′-TATGTCCCGCATGTGTTAGT-3′	5′-GCGTCTATAAGGCGTAACTA-3′
LSU	5′-ACCCGCTGAACTTAAGC-3′	5′-TACTACCACCAAGATCT-3′

引物名称	上游序列（5′→3′）	下游序列（5′→3′）
ITS4/5	5′-TCCTCCGCTTATTGATATGC-3′	5′-GGAAGTAAAAGTCGTAACAAGG-3′
Rpb2	5′-TGGGGKWTGGTYTGYCCTGC-3′	5′-CCCATRGCTTGYTTRCCCAT-3′
IGS	5′-TATGTCCCGCATGTGTTAGT-3′	5′-GCGTCTATAAGGCGTAACTA-3′
LSU	5′-ACCCGCTGAACTTAAGC-3′	5′-TACTACCACCAAGATCT-3′

	皱木耳 SS5	黑木耳B14-8	毛木耳CCMJ2827	毛木耳ACW001
皱木耳SS5	0.017 141	-0.001 727	-0.009 575	-0.005 840
黑木耳B14-8	-0.001 727	0.003 728	-0.001 102	-0.000 900
黑木耳Dai13782	0.000 000	0.000 000	0.000 000	0.000 000
毛木耳CCMJ2827	-0.009 575	-0.001 102	0.013 200	-0.002 523
毛木耳ACW001	-0.005 840	-0.000 900	-0.002 523	0.009 262

Development and Application of Abundance SNP Markers for Auricularia heimuer

黑木耳丰度SNP标记开发及应用

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 32

Related Articles 2

Recommended Articles

Metrics

Comments

引物名称	引物序列(5'→3')	aSNP锚定区SNP数量				ePCR-SNP数量				参考基因组ePCR产物长度/bp
引物名称	引物序列(5'→3')	序列重叠群	起始位点	区域编号	SNP数量	皱木耳SS5	黑木耳B14-8	毛木耳CCMJ	毛木耳ACW001	参考基因组ePCR产物长度/bp
NEKD01000001.1:2330400-2331300_2	5'-TGCCGCCATCTGTAGTAAGGATC-3'/5'-CTGAACTGCGACCAGATGTTG-3'	NEKD01000001.1	2 330 700	1	54	101	0	95	95	636
NEKD01000001.1:2384700-2385600_1	5'-GGTTGTTCTCCTCCTCCTCCATC-3'/5'-TTGTAGACGTACTCGGTCGAACC-3'	NEKD01000001.1	2 385 000	2	56	119	0	118	116	642
NEKD01000007.1:430800-431700_9	5'-TCCAGGACAAGAAGTGGTTCGAC-3'/5'-AATGCGACTGAAATCGTGAGCTG-3'	NEKD01000007.1	431 100	3	53	136	1	134	134	811
NEKD01000038.1:287100-288000_4	5'-TTCAGAGCGTACTTGGTCAGGAC-3'/5'-CATCACCACTCCTGACGGATCAC-3'	NEKD01000038.1	287 400	4	50	84	11	77	74	646
NEKD01000007.1:764400-765300_18	5'-ATTTGCCATTCTTGGGGTTCTCG-3'/5'-TTCCCCTCCTCGACTGTTTTTCC-3'	NEKD01000007.1	764 700	5	51	105	4	87	87	594
NEKD01000006.1:1829100-1830000_3	5'-CCAATGCAATGTCCGTCCTCAAG-3'/5'-GAATATCTGCGCCAGAAGCTCTG-3'	NEKD01000006.1	1 829 400	6	52	97	0	90	93	542
NEKD01000003.1:1054500-1055400_6	5'-GTGCTCACGATCTTTGTCAACCC-3'/5'-CGTAGTCCAGCTTCATATGCACG-3'	NEKD01000003.1	1 054 800	7	55	87	12	74	74	638
NEKD01000005.1:157500-158400_18	5'-GAGCGGCCGTACTTCTTGTAGTG-3'/5'-ACGTCATCCCGTACTTCAAGGG-3'	NEKD01000005.1	157 800	8	55	80	25	78	78	454

	序列长度（bp)及SNP数量（个）							菌株来源
aSNP编号及传统标记序列	1	3	4	6	7	ITS	LSU	菌株来源
ePCR及测序比对序列	581	740	565	493	566	611	1 366	/
皱木耳SS5	104	131	83	97	84	/	/	NCBI
黑木耳B14-8	0	0	11	0	12	4	0	NCBI
毛木耳CCMJ2827	98	130	75	90	70	50	19	NCBI
毛木耳ACW001	98	130	72	93	70	50	19	NCBI
HmF	4	1	4	3	12	4	1	四川省食用菌研究所
Hm159	4	2	10	2	10	5	1	四川省食用菌研究所
HmDS	4	0	1	4	12	4	1	四川省食用菌研究所
Hm231	4	0	1	3	12	4	4	四川省食用菌研究所
Hm168	4	1	1	3	12	4	2	四川省食用菌研究所
Hm10	4	3	1	3	12	4	4	四川省食用菌研究所
Hm29	4	2	12	2	10	5	0	四川省食用菌研究所
HmC	2	2	5	3	12	4	2	四川省食用菌研究所
HmDSH	4	1	5	3	12	4	2	四川省食用菌研究所
Hm2	4	0	0	3	12	4	3	四川省食用菌研究所
HmXB	4	0	10	2	10	5	0	四川省食用菌研究所
Hm216	4	4	65	9	11	2	2	四川省食用菌研究所
Hm233	4	1	6	3	12	4	3	四川省食用菌研究所

[1]	Jingjing YANG, Kewei NING, Mingjuan WANG, Binghui TANG, Junling CAI, Aiping LIU, Xiaodong XIE. The Evaluation Method for Drought Resistance During the Germination Period of Upland Cotton and Screening of High-quality Germplasm Resources in the Shihezi Reclamation Area [J]. Current Biotechnology, 2025, 15(3): 486-494.
[2]	Wenling ZHAI, Caiyun LIU, Ying LIU, Bisheng FU, Jin CAI, Wei GUO, Qiaofeng ZHANG, Jizhong WU. Phenotypic and Molecular Identification of New Wheat Germplasm Resistant to Fusarium Head Blight [J]. Current Biotechnology, 2021, 11(5): 581-589.