基于KASP标记的葡萄品种鉴定

doi:10.3864/j.issn.0578-1752.2021.13.012

摘要/Abstract

摘要：

【目的】开发一组能够区分中国主栽葡萄品种的竞争性等位基因特异性PCR（KASP）分子标记,为中国葡萄品种保护、品种登记和市场维权等提供技术支撑。【方法】基于前人筛选的60个葡萄高多态性SNP位点,转化为KASP标记。分别使用23份代表性品种和76份主要栽培品种对转化成功的KASP标记进行初筛、复筛、验证,获得一组高质量的KASP标记,并用于构建76份葡萄品种的DNA指纹图谱。【结果】60个SNP位点有3个不具备基因组特异性,6个无法设计KASP-PCR引物,最终51个SNP成功转化为KASP标记,转化率达到89.47%。利用LGC-SNPline平台对23份代表性品种进行基因分型,51个KASP标记全部成功分型。基于次要等位基因频率（MAF）大于0.25、多态性信息含量（PIC）大于0.35、缺失率小于0.2、杂合率小于0.6等参数,初步筛选出27个优质KASP标记。使用构建指纹图谱的76份品种复筛出22个高质量KASP标记。22个KASP标记的缺失率均小于0.12,PIC均大于0.3,杂合率在0.4—0.6的标记占77.27%,MAF大于0.3的标记占95.45%。此外,利用这22个标记对同一品种不同树体提取的23份代表性品种的DNA扩增检测,前后两次检测分型结果稳定一致,表明这22个标记具有较好的重复性和稳定性。进一步将基于22个标记获得的76份葡萄品种分型结果转化为二元编码数据,得到76份葡萄品种的指纹图谱。经邻接聚类分析和群体结果分析,可将76份葡萄品种分为3类,并能正确区分二倍体和多倍体。仅用10个标记（VIT_15_18567587、VIT_6_4258638、VIT_8_3320936、VIT_12_22228357、VIT_11_19390306、VIT_16_17950801、VIT_18_11138668、VIT_12_739916、VIT_16_13454358、VIT_16_21202286）就能区分70份品种,其中有54份品种达到品种鉴定的标准（差异位点数≥2）。【结论】从60个葡萄SNP中成功转化51个KASP标记,并筛选出22个高质量KASP标记,构建了76份葡萄品种的SNP指纹图谱,首次验证了KASP技术在我国葡萄品种鉴定中的可行性。

关键词: 葡萄, KASP标记, 品种鉴定, 指纹图谱, 遗传多样性分析

Abstract:

【Objective】This study was aimed to develop a set of kompetitive allele specific PCR (KASP) markers that can be used to distinguish the main cultivated grape cultivars in China, so as to provide the technical support for domestic grape cultivar protection, cultivar registration and market right protection. 【Method】60 highly polymorphic SNP loci from previous studies in grapes were selected to design KASP markers. 23 representative grape cultivars and 76 main cultivated grape cultivars in China were used for preliminary screening, re-screening, and verification of the successfully transformed KASP markers. A set of high-quality KASP markers was further selected to establish the DNA fingerprint database for the 76 grape cultivars. 【Result】Among the 60 SNP loci, 3 were not genome-specific, 6 could not be used to design KASP-PCR markers, and 51 could be successfully transformed to KASP markers with a conversion rate of 89.47%. Next, 23 representative grape cultivars were successfully genotyped by using the 51 KASP markers on LGC-SNP line platform. Then, 27 KASP markers of high quality were selected successfully based on the minor allele frequency (MAF) greater than 0.25, polymorphism information content (PIC) greater than 0.35, deletion rate less than 0.2, and heterozygosity rate less than 0.6. Finally, 22 KASP markers were successfully re-screened in the 76 main cultivated grape cultivars. The missing rate of the 22 markers was less than 0.12, the PIC value of them was greater than 0.30, the heterozygosity rate of 0.40-0.60 was 77.27%, and the MAF of greater than 0.30 was 95.45%. In addition, the consistent genotype data were acquired with different trees from each of the 23 representative cultivars by using the 22KASP markers, indicating that these 22 KASP markers had good reproducibility and stability for grape cultivar identification. The SNP genotyping results of 76 grape cultivars based on 22 markers were converted to binary coding data, and the fingerprints of 76 grape cultivars were obtained. Based on neighbor-joining cluster analysis, the 76 grape cultivars could be divided into 3 populations. Diploid and polyploidy cultivars were also correctly identified. The 10 markers (VIT_15_18567587, VIT_6_4258638, VIT_8_3320936, VIT_12_22228357, VIT_11_19390306, VIT_16_17950801, VIT_18_11138668, VIT_12_739916, VIT_16_ 13454358, VIT_16_21202286) were enough to be able to distinguish 70 of the 76 cultivars, of which 54 cultivars met the criteria for variety identification (the number of different loci ≥2). 【Conclusion】From 60 grape SNPs, 51 KASP markers were successfully transformed, and 22 high-quality KASP markers were selected successfully for developing the fingerprints of the 76 main grape cultivars in China. It was the first time to verify the feasibility of KASP genotyping in the identification of grape cultivars in China.

Key words: grape, KASP marker, cultivar identification, fingerprint, genetic diversity analysis

王富强,张建,温常龙,樊秀彩,张颖,孙磊,刘崇怀,姜建福. 基于KASP标记的葡萄品种鉴定[J]. 中国农业科学, 2021, 54(13): 2830-2842.

WANG FuQiang,ZHANG Jian,WEN ChangLong,FAN XiuCai,ZHANG Ying,SUN Lei,LIU ChongHuai,JIANG JianFu. Identification of Grape Cultivars Based on KASP Markers[J]. Scientia Agricultura Sinica, 2021, 54(13): 2830-2842.

图/表 9

表1

KASP标记初筛使用的23份葡萄代表品种信息"

编号 Code	品种 Cultivar	倍性 Ploidy	种性 Species	用途 Purpose	来源 Origin
1	赤霞珠 Cabernet Sauvignon	2x	欧亚种V. vinifera	酿酒Wine grape	法国France
2	霞多丽Chardonnary	2x	欧亚种V. vinifera	酿酒Wine grape	法国France
3	黑比诺Pinot Noir	2x	欧亚种V. vinifera	酿酒Wine grape	法国France
4	梅鹿辄Merlot	2x	欧亚种V. vinifera	酿酒Wine grape	法国France
5	紫秋Ziqiu	2x	刺葡萄V. davidii	酿酒Wine grape	中国China
6	北冰红 Beibinghong	2x	山欧杂种 V. amurensis-V. vinifera	酿酒Wine grape	中国China
7	无核白鸡心 Centennial Seedless	2x	欧亚种V. vinifera	制干Raisin grape	美国America
8	康可Concord	2x	美洲种V. labrusca	制汁Juice grape	美国America
9	贝达Beta	2x	美河杂种 V. labrusca-V. riparia	砧木Rootstock	美国America
10	抗砧3号 Kangzhen No.3	2x	冬河杂种 V. berlandieri-V. riparia	砧木Rootstock	中国China
11	SO4	2x	冬河杂种 V. berlandieri-V. riparia	砧木Rootstock	德国Germany
12	香妃Xiangfei	2x	欧亚种V. vinifera	鲜食Table grape	中国China
13	玫瑰香 Muscat Hamburg	2x	欧亚种V. vinifera	鲜食Table grape	英国England
14	红地球Red Globe	2x	欧亚种V. vinifera	鲜食Table grape	美国America
15	美人指 Manicure Finger	2x	欧亚种V. vinifera	鲜食Table grape	日本Japan
16	金手指Gold Finger	2x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	日本Japan
17	阳光玫瑰 Shine Muscat	2x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	日本Japan
18	夏黑Summer Black	3x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	日本Japan
19	月光无核 Yueguangwuhe	3x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	中国China
20	峰光Fengguang	4x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	中国China
21	京亚Jingya	4x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	中国China
22	巨玫瑰Jumeigui	4x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	中国China
23	巨峰Kyoho	4x	欧美杂种 V. vinifera-V. labrusca	鲜食Table grape	日本Japan

表1

表2

KASP标记复筛使用的76份葡萄品种信息"

编号 Code	品种 Cultivar	倍性 Ploidy	种性 Species	用途 Purpose	编号 Code	品种 Cultivar	倍性 Ploidy	种性 Species	用途 Purpose
G1	惠良刺葡萄 Huiliangciputao	2x	刺葡萄 V. davidii	制汁 Juice grape	G26	短枝玉玫瑰 Duanzhiyumeigui	2x	欧亚种 V. vinifera	鲜食 Table grape
G2	野酿2号 Yeniang No.2	2x	毛葡萄 V. heyneana	酿酒 Wine grape	G27	玉波黄地球 Yubohuangdiqiu	2x	欧亚种 V. vinifera	鲜食 Table grape
G3	媚丽 Meili	2x	欧亚种 V. vinifera	酿酒 Wine grape	G28	瑞都红玫 Ruiduhongmei	2x	欧亚种 V. vinifera	鲜食 Table grape
G4	泰美 Taimei	2x	欧亚种 V. vinifera	酿酒 Wine grape	G29	瑞都脆霞 Ruiducuixia	2x	欧亚种 V. vinifera	鲜食 Table grape
G5	MCS2	2x	欧亚种 V. vinifera	酿酒 Wine grape	G30	瑞都无核怡 Ruiduwuheyi	2x	欧亚种 V. vinifera	鲜食 Table grape
G6	玉玲珑 Yulinglong	2x	欧亚种 V. vinifera	酿酒 Wine grape	G31	瑞都香玉 Ruiduxiangyu	2x	欧亚种 V. vinifera	鲜食 Table grape
G7	碧香无核 Bixiangwuhe	2x	欧亚种 V. vinifera	鲜食 Table grape	G32	红特沙 Hongtesha	2x	欧亚种 V. vinifera	鲜食 Table grape
G8	早霞玫瑰 Zaoxiameigui	2x	欧亚种 V. vinifera	鲜食 Table grape	G33	晨香 Chenxiang	2x	欧亚种 V. vinifera	鲜食 Table grape
G9	里扎马特 Rizamat	2x	欧亚种 V. vinifera	鲜食 Table grape	G34	红艳香 Hongyanxiang	2x	欧亚种 V. vinifera	鲜食 Table grape
G10	脆红宝 Cuihongbao	2x	欧亚种 V. vinifera	鲜食 Table grape	G35	红艳无核 Hongyanwuhe	2x	欧亚种 V. vinifera	鲜食 Table grape
G11	翠香宝 Cuixiangbao	2x	欧亚种 V. vinifera	鲜食 Table grape	G36	科玉无籽 Keyuwuzi	2x	欧亚种 V. vinifera	鲜食 Table grape
G12	早康宝 Zaokangbao	2x	欧亚种 V. vinifera	鲜食 Table grape	G37	郑美 Zhengmei	2x	欧亚种 V. vinifera	鲜食 Table grape
G13	丽红宝 Lihongbao	2x	欧亚种 V. vinifera	鲜食 Table grape	G38	华葡早玉 Huapuzaoyu	2x	欧亚种 V. vinifera	鲜食 Table grape
G14	秋黑宝 Qiuheibao	2x	欧亚种 V. vinifera	鲜食 Table grape	G39	瑞都早红 Ruiduzaohong	2x	欧亚种 V. vinifera	鲜食 Table grape
G15	无核翠宝 Wuhecuibao	2x	欧亚种 V. vinifera	鲜食 Table grape	G40	瑞都科美 Ruidukemei	2x	欧亚种 V. vinifera	鲜食 Table grape
G16	云楚无核 Yuchuwuhe	2x	欧亚种 V. vinifera	鲜食 Table grape	G41	赤霞珠 Cabernet Sauvignon	2x	欧亚种 V. vinifera	鲜食 Table grape
G17	卓越黑香蜜 Zhuoyueheixiangmi	2x	欧亚种 V. vinifera	鲜食 Table grape	G42	霞多丽 Chardonnary	2x	欧亚种 V. vinifera	鲜食 Table grape
G18	卓越玫瑰 Zhuoyuemeigui	2x	欧亚种 V. vinifera	鲜食 Table grape	G43	红地球 Red Globe	2x	欧亚种 V. vinifera	鲜食 Table grape
G19	岳秀无核 Yuexiuwuhe	2x	欧亚种 V. vinifera	鲜食 Table grape	G44	玫瑰香 Muscat Hamburg	2x	欧亚种 V. vinifera	鲜食 Table grape
G20	华葡翠玉 Huapucuiyu	2x	欧亚种 V. vinifera	鲜食 Table grape	G45	美人指 Manicure Finger	2x	欧亚种 V. vinifera	鲜食 Table grape
G21	华葡紫峰 Huapuzifeng	2x	欧亚种 V. vinifera	鲜食 Table grape	G46	香妃 Xiangfei	2x	欧亚种 V. vinifera	鲜食 Table grape
G22	爱格丽 Ecolly	2x	欧亚种 V. vinifera	鲜食 Table grape	G47	红宝石无核 Ruby Seedless	2x	欧亚种 V. vinifera	鲜食 Table grape
G23	玉波一号 Yubo 1 hao	2x	欧亚种 V. vinifera	鲜食 Table grape	G48	无核白鸡心 Centennial Seedless	2x	欧亚种 V. vinifera	制干 Raisin grape
G24	玉波二号 Yubo 2 hao	2x	欧亚种 V. vinifera	鲜食 Table grape	G49	北冰红 Beibinghong	2x	山欧杂种 V. amurensis-V. vinifera	酿酒 Wine grape
G25	玉珍香 Yuzhenxiang	2x	欧亚种 V. vinifera	鲜食 Table grape	G50	凌丰红 Lingfenghong	2x	山欧杂种 V. amurensis-V. vinifera	酿酒 Wine grape
编号 Code	品种 Cultivar	倍性 Ploidy	种性 Species	用途 Purpose	编号 Code	品种 Cultivar	倍性 Ploidy	种性 Species	用途 Purpose
G51	凌砧1号 Lingzhen No.1	2x	山欧杂种 V. amurensis-V. vinifera	砧木 Rootstock	G64	申烁 Shenshuo	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G52	庆丰 Qingfeng	2x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G65	申奕 Shenli	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G53	着色香 Zhuosexiang	2x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G66	华葡玫瑰 Huapumeigui	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G54	碧玉香 Biyuxiang	2x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G67	华葡黑峰 Huapuheifeng	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G55	郑艳无核 Zhengyanwuhe	2x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G68	瑞峰 Ruifeng	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G56	金手指 Gold Finger	2x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G69	瑞紫香 Ruizixiang	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G57	阳光玫瑰 Shine Muscat	2x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G70	红蜜香 Hongmixiang	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G58	春香无核 Chunxiangwuhe	3x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G71	华葡黄玉 Huapuhuangyu	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G59	烟葡一号 Yanpu 1 hao	3x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G72	华葡瑰香 Huapuguixiang	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G60	润堡早夏 Ruibaozaoxia	3x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G73	巨峰 Kyoho	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G61	天工墨玉 Tiangongmoyu	3x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G74	巨玫瑰 Jumeigui	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G62	夏黑 Summer Black	3x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G75	天工玉液 Tiangongyuye	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape
G63	申丽 Shenli	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape	G76	天工翠玉 Tiangongcuiyu	4x	欧美杂种 V. vinifera-V. labrusca	鲜食 Table grape

表2

附表1

成功设计的51个KASP引物序列"

名称 Name	变异类型Alleles type	引物序列 Sequence of primer (5′-3′)
VIT_1_729514	A/G	F: GAAGGTGACCAAGTTCATGCTGCTGAAGGTTTCTTGAAAAAGTACTGAT F: GAAGGTCGGAGTCAACGGATTCTGAAGGTTTCTTGAAAAAGTACTGAC R: CACAGGTTGGACAAGCAAAGGGAATT
VIT_1_5948674	A/G	F: GAAGGTGACCAAGTTCATGCTATGGTTGAGGAAAGAATATTGGAAGCT F: GAAGGTCGGAGTCAACGGATTGGTTGAGGAAAGAATATTGGAAGCC R: CCGCTAGTCTTTCTAACCATGAGCTA
VIT_1_22828604	A/T	F: GAAGGTGACCAAGTTCATGCTCTACTATTATCTCTCTCGCTTATTGCTA F: GAAGGTCGGAGTCAACGGATTCTACTATTATCTCTCTCGCTTATTGCTT R: GGCAAATCCCAAAGAACTATGAATGGAA
VIT_2_5141894	A/G	F: GAAGGTGACCAAGTTCATGCTTGCGGCAAGAAGGTCCCAACT F: GAAGGTCGGAGTCAACGGATTGCGGCAAGAAGGTCCCAACC R: GTAGTGAGAACTAGAGCGGAGAACTAT
VIT_2_6474327	T/A	F: GAAGGTGACCAAGTTCATGCTCTCACCATTAATTGGGGTGAAGATTAA F: GAAGGTCGGAGTCAACGGATTCTCACCATTAATTGGGGTGAAGATTAT R: AAAGTAATCTCCATTTTGGTGTGATAACCTT
VIT_2_17198115	G/C	F: GAAGGTGACCAAGTTCATGCTTCCTCCGCTTCTCCGCCC F: GAAGGTCGGAGTCAACGGATTTCCTCCGCTTCTCCGCCG R: AAAATCTTGCGGGCGTAGTCGAGAT
VIT_3_1348328	C/T	F: GAAGGTGACCAAGTTCATGCTGATAAGACGGAGTGGCTGGAC F: GAAGGTCGGAGTCAACGGATTGGATAAGACGGAGTGGCTGGAT R: CCTGGCCGAACACCACGTGCTT
VIT_3_3676120	A/C	F: GAAGGTGACCAAGTTCATGCTAATTTAGCCAATTTCTATCTTTTGCCGTTT F: GAAGGTCGGAGTCAACGGATTTTTAGCCAATTTCTATCTTTTGCCGTTG R: ATCTACAAGGCAGGAAGGAAAGAGTTAT
VIT_3_5724485	T/C	F: GAAGGTGACCAAGTTCATGCTCCCTTTTTGGTGTAACATCCTTCCA F: GAAGGTCGGAGTCAACGGATTCCTTTTTGGTGTAACATCCTTCCG R: GATAGGAATAGCCTCGTAGAATTTTATGTAAAA
VIT_4_680574	C/T	F: GAAGGTGACCAAGTTCATGCTGGAGCTTTGATCCTGAAACCGAG F: GAAGGTCGGAGTCAACGGATTTAGGAGCTTTGATCCTGAAACCGAA R: GCGTGCAGCTATCTCTTGAAGCAAAAA
VIT_4_6409234	C/T	F: GAAGGTGACCAAGTTCATGCTATTATCCCAAAGGGAAAATAAAAACTTTCC F: GAAGGTCGGAGTCAACGGATTGATTATCCCAAAGGGAAAATAAAAACTTTCT R: GTTCTCACAGGACAAAGCATCTTGGTT
VIT_4_21849155	A/G	F: GAAGGTGACCAAGTTCATGCTCACTGGTAATAGATAGAATTAAACACATCT F: GAAGGTCGGAGTCAACGGATTACTGGTAATAGATAGAATTAAACACATCC R: CTTCCTAGGTGGAAGCAGCTGTATTAA
VIT_5_1785979	C/T	F: GAAGGTGACCAAGTTCATGCTGGTCCCTCACCTATTACTCCAG F: GAAGGTCGGAGTCAACGGATTCGGTCCCTCACCTATTACTCCAA R: TTTCGGACATGGAAAGCTTGAGTTTCTT
VIT_5_5773320	C/T	F: GAAGGTGACCAAGTTCATGCTGGCTGTATCCTGTCTTGAAGCTC F: GAAGGTCGGAGTCAACGGATTTGGCTGTATCCTGTCTTGAAGCTT R: GATCCAAGGTCACGAGCTGTTTATGAT
VIT_5_6744629	A/G	F: GAAGGTGACCAAGTTCATGCTTGAGTCAATCTCGCCGAATATGAGT F: GAAGGTCGGAGTCAACGGATTAGTCAATCTCGCCGAATATGAGC R: AAACTCAAGATTGGACAGCAATATCCATATT
VIT_6_327200	A/G	F: GAAGGTGACCAAGTTCATGCTAAAAGCCTTTGGAGCATCTCCAGAA F: GAAGGTCGGAGTCAACGGATTAGCCTTTGGAGCATCTCCAGAG R: GAAGGTTTTTGAAAATGGATCAGTTGCCAA
VIT_6_4258638	C/T	F: GAAGGTGACCAAGTTCATGCTTGTCCAGAGATCCTGTTTTCTCG F: GAAGGTCGGAGTCAACGGATTACTGTCCAGAGATCCTGTTTTCTCA R: CTTTCAGCAGGCAGCAATGGAAAGTT
VIT_6_17593092	A/T	F: GAAGGTGACCAAGTTCATGCTCTGCATTCGTTCACCTGTCAACTTA F: GAAGGTCGGAGTCAACGGATTCTGCATTCGTTCACCTGTCAACTTT R: ATCTGCATCTTCGAGCTTGTCCTTAATT
VIT_7_1388822	A/G	F: GAAGGTGACCAAGTTCATGCTCGTCCGGATGCATTGCGCCT F: GAAGGTCGGAGTCAACGGATTGTCCGGATGCATTGCGCCC R: GGCAAACGCTGATTGGCTGGAGTA
VIT_7_18046355	A/G	F: GAAGGTGACCAAGTTCATGCTAAATGTGGCTGCAGTTGAGAAGACT F: GAAGGTCGGAGTCAACGGATTTGTGGCTGCAGTTGAGAAGACC R: CATACCACAGGATTAACTGATACATCCATA
VIT_8_3320936	C/T	F: GAAGGTGACCAAGTTCATGCTTGGAGGGTAAAAATGAACTCAATTTGAC F: GAAGGTCGGAGTCAACGGATTATTGGAGGGTAAAAATGAACTCAATTTGAT R: CCTTTCTGATGATAGAAGCAGTGGGAA
VIT_8_13401437	A/C	F: GAAGGTGACCAAGTTCATGCTCTGGTTCCAATCCCTCATCCGTA F: GAAGGTCGGAGTCAACGGATTTGGTTCCAATCCCTCATCCGTC R: AAGGAGACTAAGATAAAGGTGTAGTATACAT
VIT_9_3123999	A/T	F: GAAGGTGACCAAGTTCATGCTTGTCTTGACTCATCATATTTGACAGCA F: GAAGGTCGGAGTCAACGGATTTGTCTTGACTCATCATATTTGACAGCT R: GGTGTGAAGACGATAATGGGTCCAAT
VIT_9_21409416	C/T	F: GAAGGTGACCAAGTTCATGCTTCCAGAAACCAGCTAGTGTGGC F: GAAGGTCGGAGTCAACGGATTATTCCAGAAACCAGCTAGTGTGGT R: CGGAAAACTTCCATACCGCGTGAAA
VIT_10_5489212	C/T	F: GAAGGTGACCAAGTTCATGCTTGCTTCTCCACGAAGGCTGTC F: GAAGGTCGGAGTCAACGGATTCTGCTTCTCCACGAAGGCTGTT R: GCAACGTCTCCTACGACACGGAA
VIT_11_311765	A/C	F: GAAGGTGACCAAGTTCATGCTTGGGGGAAGTAGTTGGTTGCCA F: GAAGGTCGGAGTCAACGGATTGGGGGAAGTAGTTGGTTGCCC R: GAATTAGAGACCCAGAGAGAAGGGAAA
VIT_11_5406647	A/C	F: GAAGGTGACCAAGTTCATGCTTATCAGAATGTTGGATTTTGAAATTGAATGTTA F: GAAGGTCGGAGTCAACGGATTTCAGAATGTTGGATTTTGAAATTGAATGTTC R: TGTTACGGTTTTGCTAACTCAAAATCCTTAT
VIT_11_19390306	G/A	F: GAAGGTGACCAAGTTCATGCTGTTCAAGCTGGGGGAGAATATATAC F: GAAGGTCGGAGTCAACGGATTGGTTCAAGCTGGGGGAGAATATATAT R: GTCGGAGTCCATGTATCGCCGTTA
VIT_12_739916	C/T	F: GAAGGTGACCAAGTTCATGCTGCTGAACACACTTTTTCCAAGTTCG F: GAAGGTCGGAGTCAACGGATTAAGCTGAACACACTTTTTCCAAGTTCA R: GGTTTGGGAGGGGACAAAGATCTAATT
VIT_12_22228357	A/C	F: GAAGGTGACCAAGTTCATGCTCAACTTCACGGTGACCGAATTCATA F: GAAGGTCGGAGTCAACGGATTAACTTCACGGTGACCGAATTCATC R: GGTCGACGGCAGCCACAGGTTT
VIT_13_21618145	A/T	F: GAAGGTGACCAAGTTCATGCTTCGAGTTGCTTAGATAAACACATTAATCT F: GAAGGTCGGAGTCAACGGATTTCGAGTTGCTTAGATAAACACATTAATCA R: CATCATGTGATTCTATGCAAAAATTATTAGAATTAA
VIT_14_4947068	T/G	F: GAAGGTGACCAAGTTCATGCTTATTCCTACCTGCGTTTCGTCGA F: GAAGGTCGGAGTCAACGGATTATTCCTACCTGCGTTTCGTCGC R: CTGTGGCCCGGTGGGGCTT
VIT_14_5687725	A/T	F: GAAGGTGACCAAGTTCATGCTATGATGCTTTGAATAGAATAAATGAGAACAAT F: GAAGGTCGGAGTCAACGGATTATGATGCTTTGAATAGAATAAATGAGAACAAA R: AACTTGCAACATGACAACCACAAACCAA
VIT_14_23135445	A/T	F: GAAGGTGACCAAGTTCATGCTAATTAGCAGGGAGGGGTGGCAA F: GAAGGTCGGAGTCAACGGATTAATTAGCAGGGAGGGGTGGCAT R: GAACTTTAGCAGTAGTTCAGCTTCATGAT
VIT_14_29590769	C/T	F: GAAGGTGACCAAGTTCATGCTTTCCTCCAAGTCTCCATGTGCG F: GAAGGTCGGAGTCAACGGATTGTTCCTCCAAGTCTCCATGTGCA R: CAAAGCCCTTTGACAGACAACCTTCAA
VIT_15_15145042	A/C	F: GAAGGTGACCAAGTTCATGCTGTATATTTGTTCATCAGACATACTAACACT F: GAAGGTCGGAGTCAACGGATTTATATTTGTTCATCAGACATACTAACACG R: GCAGAGAGAAGCTCAGACATCTCATTA
VIT_15_18031506	A/C	F: GAAGGTGACCAAGTTCATGCTTAACTGATACATAAAACTGAAAAATCGGGT F: GAAGGTCGGAGTCAACGGATTACTGATACATAAAACTGAAAAATCGGGG R: GGGAGTAGAACCTGAAAGCAAGCATA
VIT_15_18127737	T/G	F: GAAGGTGACCAAGTTCATGCTGGATGTGATGTAGGGGTCCTTGT F: GAAGGTCGGAGTCAACGGATTGATGTGATGTAGGGGTCCTTGG R: CATATCACCTTTTCTAGGACTAAAACCCAA
VIT_15_18567587	T/G	F: GAAGGTGACCAAGTTCATGCTCTGTATTAAAGCTCTTGGGTGAAAAACT F: GAAGGTCGGAGTCAACGGATTTGTATTAAAGCTCTTGGGTGAAAAACG R: ATTGTTGGGCACAAATACGCTCAAGATT
VIT_16_13454358	A/G	F: GAAGGTGACCAAGTTCATGCTGCTTTTGACGTGAGAGAGCAAGAATT F: GAAGGTCGGAGTCAACGGATTCTTTTGACGTGAGAGAGCAAGAATC R: CTGGTGCATATAAGGTTGCAGTTGTTAA
VIT_16_16198599	A/G	F: GAAGGTGACCAAGTTCATGCTGCACAACAATTTCTCCAGCTTCGTT F: GAAGGTCGGAGTCAACGGATTCACAACAATTTCTCCAGCTTCGTC R: AGAAATGGACTCACGTAGGTGTTAAGTT
VIT_16_17950801	A/G	F: GAAGGTGACCAAGTTCATGCTTCTGCTTGAGGGGGAGTGACAA F: GAAGGTCGGAGTCAACGGATTCTGCTTGAGGGGGAGTGACAG R: TAGTGGAAACATCTCCCACTTGATTCATA
VIT_16_21202286	A/G	F: GAAGGTGACCAAGTTCATGCTACCCAATGGAATCGATCCAATGTCA F: GAAGGTCGGAGTCAACGGATTCCAATGGAATCGATCCAATGTCG R: CTGGTTAATTTCAGACGAAGCCAAGTATA
VIT_17_126505	A/G	F: GAAGGTGACCAAGTTCATGCTTCAGTATTATGAGGAATGCTATTTATGAGT F: GAAGGTCGGAGTCAACGGATTCAGTATTATGAGGAATGCTATTTATGAGC R: GGTCCTGCAAGATATCCCAGAACATAT
VIT_17_6000914	C/T	F: GAAGGTGACCAAGTTCATGCTAACTGTTCCCAGCCGGATTGAG F: GAAGGTCGGAGTCAACGGATTGAACTGTTCCCAGCCGGATTGAA R: GCCGACCCCATCCTGTCACTAT
VIT_17_12206201	A/G	F: GAAGGTGACCAAGTTCATGCTCGCCATCTTATGACAATTGATGAACTA F: GAAGGTCGGAGTCAACGGATTGCCATCTTATGACAATTGATGAACTG R: CCTTTTAGTGACAGGTGGAATAAGAGAAAT
VIT_18_3829207	A/C	F: GAAGGTGACCAAGTTCATGCTTGCAAACTGGGAAGCAAATGTCTCT F: GAAGGTCGGAGTCAACGGATTCAAACTGGGAAGCAAATGTCTCG R: CAAGGATATTGTTGAGCTCCGAAATCAA
VIT_18_6487636	A/C	F: GAAGGTGACCAAGTTCATGCTAGATTGAATATCTCCATACCTTCGTCA F: GAAGGTCGGAGTCAACGGATTGATTGAATATCTCCATACCTTCGTCC R: GGGATAAAACATTCAAAGCCCATAAATTAAATTT
VIT_18_11138668	A/G	F: GAAGGTGACCAAGTTCATGCTGACATGTTTCCATACTGATCCTCCTA F: GAAGGTCGGAGTCAACGGATTACATGTTTCCATACTGATCCTCCTG R: CTGGACATTTTCAACCATGATGATGATGAT
VIT_19_7217380	A/T	F: GAAGGTGACCAAGTTCATGCTGGTCTTCATCCAAGATATGTATCTCATT F: GAAGGTCGGAGTCAACGGATTGGTCTTCATCCAAGATATGTATCTCATA R: CATACGATTAAATGGATGCGGGCAGTT
VIT_19_17751334	G/T	F: GAAGGTGACCAAGTTCATGCTCATGAGGCGTTTTTCATCACCAAC F: GAAGGTCGGAGTCAACGGATTGCATGAGGCGTTTTTCATCACCAAA R: GTTTGGCTCCAGACTTGCCTGAATTT

附表1

图1

图2

图3

图4

图5

图6

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