Establishment of a Method Identifying the Varieties of Hulless Barley Based on Fluorescence Detection Technology

doi:10.13560/j.cnki.biotech.bull.1985.2020-1494

Abstract

Abstract:

A series of SSR core primers were screened to provide a DNA fingerprinting of hulless barley varieties,which may provide high-throughput detection method for the breeding and variety identification of hulless barley. First of all,24 hulless barley germplasms with large morphological differences were used,42 primers were screened from the original 198 SSR primers,which had clear amplification bands,rich polymorphism and stable amplification. These screened primers were labeled with fluorescent,and used for the PCR amplification of the total 226 cultivars. The amplified products were detected by capillary electrophoresis. Finally,28 pairs of SSR fluorescent primers were screened to establish an identification system for hulless barley variety based on the high-throughput fluorescent SSR marker. And the selected 28 primers were used to construct DNA fingerprinting for identifying the 226 cultivars of hulless barley. A total of 252 alleles were detected by 28 pairs of SSR primers,the effective number of alleles ranged from 5 to 16,and the average number of alleles was 9. The genetic diversity ranged from 0.48 to 0.86 and the average value was 0.68. The polymorphism information content（PIC）changed from 0.44 to 0.84 and the average value of PIC was 0.65. The genetic distance of 226 materials was 0-0.99. This set of primers distinguished the most of the test materials. Seven SSR primers（Bmag0211,Scssr07759,HVM62,EBmac0679,Scssr03907,Bmag0870,and EBmac0827）with good amplification and detection effect as well as distributed in different chromosomes were chosen from the 28 SSR primers,and they were used for constructing DNA fingerprinting spectrum. The corresponding reference cultivars to major allelic variations of 7 pairs of primers were selected to eliminate the errors caused by different platforms and experimental batches. These reference cultivars can be used as the basis for reading bands of ordinary polyacrylamide gel electrophoresis. In this study,a SSR high-throughput identification system was constructed in two detection platforms（fluorescent capillary electrophoresis platform and polyacrylamide gel electrophoresis platform）based on 28 pairs of primers.

Key words: hulless barley, SSR marker, core primers, fingerprinting, variety identification

GUAN Jun-jiao, YANG Xiao-hong, ZHANG Peng, HUANG Qing-mei, ZHANG Jian-hua. Establishment of a Method Identifying the Varieties of Hulless Barley Based on Fluorescence Detection Technology[J]. Biotechnology Bulletin, 2021, 37(11): 293-302.

Figures/Tables 7

References 22

[1]	徐菲, 党斌, 杨希娟, 等. 不同青稞品种的营养品质评价[J]. 麦类作物学报, 2016, 36(9): 1249-1257.
	Xu F, Dang B, Yang XJ, et al. Evaluation of nutritional quality of different hulless barleys[J]. Journal of Triticeae Crops, 2016, 36(9): 1249-1257.
[2]	刘欢, 张新全, 马啸, 等. 基于荧光检测技术的多花黑麦草 EST-SSR 指纹图谱的构建[J]. 中国农业科学, 2017, 50(3): 437-450.
	Liu H, Zhang XQ, Ma X, et al. Construction of EST-SSR fingerprinting based on fluorescence detection technology for Italian ryegrass[J]. Scientia Agricultura Sinica, 2017, 50(3): 437-450.
[3]	UPOV. Guidelines for DNA-profiling:Molecular markers selection and database construction[C]. Geneva:Office of the Union, 2010.
[4]	吴昆仑. 青稞种质资源的 SSR 标记遗传多样性分析[J]. 麦类作物学报, 2011, 31(6): 1030-1034.
	Wu KL. Genetic diversity analysis of hulless barley germplasm by SSR markers[J]. Journal of Triticeae Crops, 2011, 31(6): 1030-1034.
[5]	曾兴权, 王玉林, 徐齐君, 等. 利用SSR 引物分析西藏青稞种质资源的遗传多样性[J]. 麦类作物学报, 2013, 33(2): 260-267.
	Zeng XQ, Wang YL, Xu QJ, et al. Assessment of genetic diversity in Tibetan hulless barley germplasm(Hordeum vulgare L. var. nudum HK. F. )by SSR primers[J]. Journal of Triticeae Crops, 2013, 33(2): 260-267.
[6]	巴桑玉珍, 刘新春, 付国勇, 等. 青藏高原青稞耐寒种质资源基于 SSR 标记的遗传多样性及群体结构分析[J]. 麦类作物学报, 2017, 37(1): 40-47.
	Basang YZ, Liu XC, Fu GY, et al. Genetic diversity and population structure analysis of hulless barley with cold tolerance from the Qinghai-Tibetan Plateau revealed by SSR markers[J]. Journal of Triticeae Crops, 2017, 37(1): 40-47.
[7]	杨菁, 迟德钊, 吴昆仑, 等. 青海省栽培青稞 SSR 标记遗传多样性研究[J]. 安徽农业科学, 2010, 12(8): 4307-4309.
	Yang J, Chi DZ, Wu KL, et al. Genetic diversity of SSR markers in cultivated Horrdeum vulgare L. in Qinghai province[J]. Journal of Anhui Agricultural Science, 2010, 38(8): 4307-4309.
[8]	孟亚雄, 孟祎林, 汪军成, 等. 青稞遗传多样性及其农艺性状与SSR标记的关联分析[J]. 作物学报, 2016, 42(2): 180-189.
	Meng YX, Meng YL, Wang JC, et al. Genetic diversity and association analysis of agronomic characteristics with SSR markers in hulless barley[J]. Acta Agronomica Sinica, 2016, 42(2): 180-189. doi: 10.3724/SP.J.1006.2016.00180 URL
[9]	孟凡磊, 强小林, 佘奎军, 等. 西藏主要农区青稞品种的遗传多样性分析[J]. 作物学报, 2007, 33(11): 1910-1914.
	Meng FL, Qiang XL, She KJ, et al. Genetic diversity analysis among hulless barley varieties from the major agricultural areas of Tibet[J]. Acta Agronomica Sinica, 2007, 33(11): 1910-1914.
[10]	杨平, 刘仙俊, 刘新春, 等. 利用 SRAP 标记研究四川高原青稞育成品种的遗传多样性[J]. 遗传, 2008, 30(1): 115-122.
	Yang P, Liu XJ, Liu XC, et al. Genetic diversity analysis of the developed Qingke(hulless barley)varieties from the plateau regions of Sichuan Province in China revealed by SRAP markers[J]. Hereditas, 2008, 30(1): 115-122.
[11]	UPOV. Possible use of molecular markers in the examination of distinctness, uniformity and stability[C]. Geneva:Office of the Union, 2011.
[12]	王凤格, 杨扬, 易红梅, 等. 中国玉米审定品种标准 SSR 指纹库的构建[J]. 中国农业科学, 2017, 50(1): 1-14.
	Wang FG, Yang Y, Yi HM, et al. Construction of an SSR-based standard fingerprint database for corn variety authorized in China[J]. Scientia Agricultura Sinica, 2017, 50(1): 1-14.
[13]	唐浩, 余汉勇, 张新明, 等. 水稻新品种测试的标准品种DNA指纹图谱多样性分析[J]. 植物遗传资源学报, 2015, 16(1): 100-106.
	Tang H, Yu HY, Zhang XM, et al. Analysis on the diversity of DNA fingerprinting of the example varieties used for the test of rice new varieties[J]. Journal of Plant Genetic Resources, 2015, 16(1): 100-106.
[14]	郑永胜, 张晗, 王东建, 等. 基于荧光检测技术的小麦品种 SSR 鉴定体系的建立[J]. 中国农业科学, 2014, 47(19): 3725-3735.
	Zheng YS, Zhang H, Wang DJ, et al. Development of a wheat variety identification system based on fluorescently labeled SSR markers[J]. Scientia Agricultura Sinica, 2014, 47(19): 3725-3735.
[15]	陈亮, 郑宇宏, 范旭红, 等. 吉林省新育成大豆品种 SSR 指纹图谱身份证的构建[J]. 大豆科学, 2016, 35(6): 896-901.
	Chen L, Zheng YH, Fan XH, et al. Establishment of SSR fingerprint ID for new soybean varieties in Jilin[J]. Soybean Science, 2016, 35(6): 896-901.
[16]	中华人民共和国国家质量监督检验检疫总局. 中华人民共和国农业行业标准:NY/T 2224-2012 植物新品种特异性、一致性和稳定性测试指南大麦[S]. 北京: 中国标准出版社, 2012.
	General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Agricultural professional standard of the People’s Republic of China:NY/T 2224-2012 Guidelines for the conduct of tests for distinctness, uniformity and stability-Barley[S]. Beijing: China Zhijian Publishing House, 2012.
[17]	Porebski S, Bailey LG, Baum BR. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J]. Plant Mol Biol Rep, 1997, 15: 8-15. doi: 10.1007/BF02772108 URL
[18]	中华人民共和国农业部. 中华人民共和国农业行业标准: 植物品种鉴定 DNA分子标记法总则 NY/T 2594—2016[S]. 北京: 中国农业出版社, 2016.
	Ministry of Agriculture of the People’s Republic of China. Agriculture Standard of the People’s Republic of China: General guideline for identification of plant varieties using DNA markers. NY/T 2594—2016[S]. Beijing: Chinese Agriculture Press, 2016.
[19]	王凤格, 赵久然, 郭景伦, 等. 中国玉米新品种标准 DNA 指纹库构建研究的几点思考[J]. 植物学通报, 2005, 22(1): 121-128.
	Wang FG, Zhao JR, Guo JL, et al. Review of research into establishing a DNA fingerprinting database of new Chinese maize cultivars[J]. Chinese Bulletin of Botany, 2005, 22(1): 121-128.
[20]	赖勇, 王晋民, 任龙, 等. 大麦 SSR 标记遗传多样性及连锁不平衡分析[J]. 核农学报, 2016, 30(10): 1889-1897.
	Lai Y, Wang JM, Ren L, et al. Genetic diversity and linkage disequilibrium analysis of barley using SSR markers. Journal of Nuclear Agricultural Sciences, 2016, 30(10): 1889-1897.
[21]	郝晨阳, 王兰芬, 贾继增, 等. SSR 荧光标记和银染技术的比较分析[J]. 作物学报, 2005, 31(2): 144-149.
	Hao CY, Wang LF, Jia JZ, et al. Comparison of fluorescence and silver-staining detection systems of microsatellite markers[J]. Acta Agronomica Sinica, 2005, 31(2): 144-149.
[22]	李益, 马先锋, 唐浩, 等. 柑橘品种鉴定的 SSR 标记开发和指纹图谱库构建[J]. 中国农业科学, 2018, 51(15): 2969-2979.
	Li Y, Ma XF, Tang H, et al. SSR markers screening for identification of Citrus cultivar and construction of DNA fingerprinting Library[J]. Scientia Agricultura Sinica, 2018, 51(15): 2969-2979.

序号 No.	材料名称 Material name	来源 Origin	棱型 Type
1	长芒六棱露仁	江苏省农业科学院	多棱
2	藏83062	江苏省农业科学院	多棱
3	昆仑1号	青海省农林科学院	多棱
4	昆仑14号	青海省农林科学院	多棱
5	北青1号	青海省农林科学院	多棱
6	中甸紫青稞	云南地方品种	多棱
7	镇康软壳青稞	云南地方品种	多棱
8	禄劝裸黑大麦	云南地方品种	二棱
9	康青3号	四川省甘孜农科所	多棱
10	米大麦	云南地方品种	多棱
11	勾芒米大麦	云南地方品种	多棱
12	北青88	西北农林科技大学	多棱
13	阿青5号	青海省农林科学院	多棱
14	迪青3号	迪庆州农业科学院	多棱
15	黄长钩裸大麦	云南地方品种	多棱
16	紫光芒裸大麦	云南地方品种	二棱
17	开宝麦	云南地方品种	多棱
18	光芒鲜大麦	云南地方品种	二棱
19	早熟白青稞	云南地方品种	多棱
20	洋大麦	云南地方品种	多棱
21	早熟大麦	云南地方品种	多棱
22	云青3号	云南省农业科学院	多棱
23	Atahualpa	国际干旱农业研究中心	二棱
24	MADRE SELVA	国际干旱农业研究中心	二棱

序号 No.	材料名称 Material name	来源 Origin	棱型 Type
1	长芒六棱露仁	江苏省农业科学院	多棱
2	藏83062	江苏省农业科学院	多棱
3	昆仑1号	青海省农林科学院	多棱
4	昆仑14号	青海省农林科学院	多棱
5	北青1号	青海省农林科学院	多棱
6	中甸紫青稞	云南地方品种	多棱
7	镇康软壳青稞	云南地方品种	多棱
8	禄劝裸黑大麦	云南地方品种	二棱
9	康青3号	四川省甘孜农科所	多棱
10	米大麦	云南地方品种	多棱
11	勾芒米大麦	云南地方品种	多棱
12	北青88	西北农林科技大学	多棱
13	阿青5号	青海省农林科学院	多棱
14	迪青3号	迪庆州农业科学院	多棱
15	黄长钩裸大麦	云南地方品种	多棱
16	紫光芒裸大麦	云南地方品种	二棱
17	开宝麦	云南地方品种	多棱
18	光芒鲜大麦	云南地方品种	二棱
19	早熟白青稞	云南地方品种	多棱
20	洋大麦	云南地方品种	多棱
21	早熟大麦	云南地方品种	多棱
22	云青3号	云南省农业科学院	多棱
23	Atahualpa	国际干旱农业研究中心	二棱
24	MADRE SELVA	国际干旱农业研究中心	二棱

序号 No.	位点 Locus	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	序号 No.	位点 Locus	引物序列 Primer sequences（5'-3'）	退火温度 Annealing temperature/℃
1	Bmag0211	F:ATTCATCGATCTTGTATTAGTCC R:ACATCATGTCGATCAAAGC	58	15	EBmac0701	F:ATGATGAGAACTCTTCACCC R:TGGCACTAAAGCAAAAGAC	55
2	Bmag0345	F:ATGAGGAATAACTCAACCAAA R:AATATATTTTCGATGTCGAGC	58	16	Bmac0310	F:CTACCTCTGAGATATCATGCC R:ATCTAGTGTGTGTTGCTTCCT	55
3	Bmag0579	F:CCTAGATAAGGAACATAGCCA R:CAAAGACCCTAACTCATGTTC	55	17	Bmag387	F:CGATGACCATTGTATTGAAG R:CTCATGTTGATGTGTGGTTAG	58
4	EBmac0501	F:ACTTAAGTGCCATGCAAAG R:AGGGACAAAAATGGCTAAG	58	18	Bmac0096	F:GCTATGGCGTACTATGTATGGTTG R:TCACGATGAGGTATGATCAAAGA	58
5	Scssr07759	F:GCAACTCCTCATCATCTCAGG R:CAACAGCCAGAAGGTCTACG	62	19	Scssr03907	F:CTCCCATCACACCATCTGTC R:GACATGGTTCCCTTCTTCTTC	60
6	Bmag0378	F:CTTTTGTTTCCGTAGCATCTA R:ATCCAACTATAGTAGCAAAGCC	58	20	Scssr10148	F:AAGCAGCAAAGCAAAGTACC R:TCATCAGCATCTGATCATCC	60
7	Bmag0711	F:GGAGAGTCACATATCAAGGAC R:CCACTCCTTCTCATACCTTTA	55	21	Bmac0018	F:GTCCTTTACGCATGAACCGT R:ACATACGCCAGACTCGTGTG	60
8	Bmag125	F:AATTAGCGAGAACAAAATCAC R:AGATAACGATGCACCACC	55	22	Bmag0009	F:AAGTGAAGCAAGCAAACAAACA R:ATCCTTCCATATTTTGATTAGGCA	58
9	Bmag0013	F:AAGGGGAATCAAAATGGGAG R:TCGAATAGGTCTCCGAAGAAA	58	23	Bmag0867	F:CCCCACACTGACCTACAG R:TTACATCTGCTAGATCGAAGC	55
10	EBmac705	F:GTGGAAAACTGAGTGAAACTC R:TTGAGGAGAAGTAATGACGAT	55	24	Bmag0870	F:AACCATAGGATTTGTACTAGTTTC R:TCATGACATCTCAAGAACG	55
11	HVM60	F:CAATGATGCGGTGAACTTTG R:CCTCGGATCTATGGGTCCTT	58	25	Bmac0064	F:CTGCAGGTTTCAGGAAGG R:AGATGCCCGCAAAGAGTT	58
12	HVM62	F:TCGCGACCAGACGAGAAG R:AGCTAGCCGACGACGCAC	60	26	HVCMA	F:GCCTCGGTTTGGACATATAAAG R:GTAAAGCAAATGTTGAGCAACG	62
13	EBmac0635	F:TGCTGCGATGATGAGAACT R:TAGGGTAGATCCGTCCCTATG	58	27	EBmac0827	F:CATGGTATTCAAACATACACG R:AAGGTCTTAAGGGGTGATG	55
14	EBmac0679	F:ATTGGAGCGGATTAGGAT R:CCCTATGTCATGTAGGAGATG	55	28	Scssr15864	F:GCATAAACGGGTGTAAGAGC R:CATCCAGTTCAGAGGATAGAGC	55

序号 No.	位点 Locus	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	序号 No.	位点 Locus	引物序列 Primer sequences（5'-3'）	退火温度 Annealing temperature/℃
1	Bmag0211	F:ATTCATCGATCTTGTATTAGTCC R:ACATCATGTCGATCAAAGC	58	15	EBmac0701	F:ATGATGAGAACTCTTCACCC R:TGGCACTAAAGCAAAAGAC	55
2	Bmag0345	F:ATGAGGAATAACTCAACCAAA R:AATATATTTTCGATGTCGAGC	58	16	Bmac0310	F:CTACCTCTGAGATATCATGCC R:ATCTAGTGTGTGTTGCTTCCT	55
3	Bmag0579	F:CCTAGATAAGGAACATAGCCA R:CAAAGACCCTAACTCATGTTC	55	17	Bmag387	F:CGATGACCATTGTATTGAAG R:CTCATGTTGATGTGTGGTTAG	58
4	EBmac0501	F:ACTTAAGTGCCATGCAAAG R:AGGGACAAAAATGGCTAAG	58	18	Bmac0096	F:GCTATGGCGTACTATGTATGGTTG R:TCACGATGAGGTATGATCAAAGA	58
5	Scssr07759	F:GCAACTCCTCATCATCTCAGG R:CAACAGCCAGAAGGTCTACG	62	19	Scssr03907	F:CTCCCATCACACCATCTGTC R:GACATGGTTCCCTTCTTCTTC	60
6	Bmag0378	F:CTTTTGTTTCCGTAGCATCTA R:ATCCAACTATAGTAGCAAAGCC	58	20	Scssr10148	F:AAGCAGCAAAGCAAAGTACC R:TCATCAGCATCTGATCATCC	60
7	Bmag0711	F:GGAGAGTCACATATCAAGGAC R:CCACTCCTTCTCATACCTTTA	55	21	Bmac0018	F:GTCCTTTACGCATGAACCGT R:ACATACGCCAGACTCGTGTG	60
8	Bmag125	F:AATTAGCGAGAACAAAATCAC R:AGATAACGATGCACCACC	55	22	Bmag0009	F:AAGTGAAGCAAGCAAACAAACA R:ATCCTTCCATATTTTGATTAGGCA	58
9	Bmag0013	F:AAGGGGAATCAAAATGGGAG R:TCGAATAGGTCTCCGAAGAAA	58	23	Bmag0867	F:CCCCACACTGACCTACAG R:TTACATCTGCTAGATCGAAGC	55
10	EBmac705	F:GTGGAAAACTGAGTGAAACTC R:TTGAGGAGAAGTAATGACGAT	55	24	Bmag0870	F:AACCATAGGATTTGTACTAGTTTC R:TCATGACATCTCAAGAACG	55
11	HVM60	F:CAATGATGCGGTGAACTTTG R:CCTCGGATCTATGGGTCCTT	58	25	Bmac0064	F:CTGCAGGTTTCAGGAAGG R:AGATGCCCGCAAAGAGTT	58
12	HVM62	F:TCGCGACCAGACGAGAAG R:AGCTAGCCGACGACGCAC	60	26	HVCMA	F:GCCTCGGTTTGGACATATAAAG R:GTAAAGCAAATGTTGAGCAACG	62
13	EBmac0635	F:TGCTGCGATGATGAGAACT R:TAGGGTAGATCCGTCCCTATG	58	27	EBmac0827	F:CATGGTATTCAAACATACACG R:AAGGTCTTAAGGGGTGATG	55
14	EBmac0679	F:ATTGGAGCGGATTAGGAT R:CCCTATGTCATGTAGGAGATG	55	28	Scssr15864	F:GCATAAACGGGTGTAAGAGC R:CATCCAGTTCAGAGGATAGAGC	55

位点 Locus	染色体 Chromosome	等位基因数 Allele number	基因多样性指数 Gene diversity	杂合性 Heterozygosity	多态信息量 PIC	特异等位基因 Unique gene
Bmag0211	1H	8	0.68	0.01	0.62	1
Bmag0345	1H	5	0.75	0.00	0.71	0
Bmag0579	1H	9	0.69	0.06	0.63	0
EBmac0501	1H	10	0.45	0.02	0.44	2
Scssr07759	2H	6	0.70	0.13	0.64	1
Bmag0378	2H	6	0.55	0.02	0.45	2
Bmag0711	2H	11	0.77	0.14	0.74	0
Bmag125	2H	11	0.66	0.12	0.62	0
Bmag0013	3H	16	0.86	0.09	0.84	3
EBmac705	3H	11	0.72	0.19	0.68	3
HVM60	3H	10	0.72	0.09	0.69	3
HVM62	3H	11	0.78	0.18	0.75	3
EBmac0635	4H	8	0.64	0.07	0.62	0
EBmac0679	4H	11	0.72	0.15	0.70	1
EBmac0701	4H	9	0.66	0.10	0.64	1
Bmac0310	4H	12	0.83	0.14	0.81	0
Bmag0387	5H	10	0.79	0.08	0.76	3
Bmac0096	5H	10	0.71	0.07	0.68	2
Scssr03907	5H	13	0.78	0.11	0.75	3
Scssr10148	5H	9	0.80	0.13	0.77	1
Bmac0018	6H	5	0.53	0.12	0.50	0
Bmag0009	6H	5	0.56	0.10	0.51	0
Bmag0867	6H	8	0.50	0.12	0.45	2
Bmag0870	6H	7	0.82	0.15	0.80	0
Bmac0064	7H	9	0.54	0.08	0.50	1
HVCMA	7H	6	0.63	0.16	0.58	1
EBmac0827	7H	10	0.75	0.13	0.73	1
Scssr15864	7H	9	0.48	0.15	0.46	3
平均（Average）	-	9	0.68	0.10	0.65	1.32