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作物学报 ›› 2018, Vol. 44 ›› Issue (7): 1095-1102.doi: 10.3724/SP.J.1006.2018.01095

• 研究简报 • 上一篇    

四倍体马铃薯块茎蛋白含量分子标记的开发与验证

单洪波,史佳文,石瑛()   

  1. 东北农业大学农学院, 黑龙江哈尔滨 150030
  • 收稿日期:2018-01-16 接受日期:2018-03-26 出版日期:2018-07-10 网络出版日期:2018-04-27
  • 通讯作者: 石瑛
  • 基金资助:
    本研究由国家重点研发计划项目(2017YFD0101906)资助

Development and Validation of Molecular Marker for Protein Content in Tetraploid Potato Tuber

Hong-Bo SHAN,Jia-Wen SHI,Ying SHI()   

  1. College of Agronomy, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
  • Received:2018-01-16 Accepted:2018-03-26 Published:2018-07-10 Published online:2018-04-27
  • Contact: Ying SHI
  • Supported by:
    This study was supported by the National Key Research and Development Programs (2017YFD0101906).

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摘要：

块茎蛋白含量是马铃薯的重要品质性状之一, 相关分子标记的开发研究较少。本研究以马铃薯高蛋白品种大西洋、低蛋白品种定薯1号及包含173份子代的蛋白含量分离群体为材料, 通过高通量简化基因组测序技术与集群分离分析(BSA)相结合, 开发了SCAR8-107标记, 并以此标记对分离群体的74份高蛋白、42份低蛋白子代及54个四倍体马铃薯品种验证表明, SCAR8-107标记在后代分离群体和四倍体马铃薯品种中的检测结果与表型蛋白含量的对应度分别达到了90.51%和72.97%, 经过Pearson’s双侧相关性分析, 相关性均达到了极显著水平。SCAR8-107的开发对于四倍体马铃薯块茎蛋白含量标记辅助选择与加速马铃薯品质育种具有重要意义。

关键词: 马铃薯蛋白, 标记开发, BSA混池测序, 分子标记辅助育种

Abstract:

Protein content is one of the most important quality traits in potato. In this study, SCAR8-107, a protein content linked molecular marker, was developed using Atlantic, Dingshu 1, and their F1 segregation population consisting of 173 individuals by reduced-representation sequencing and bulked segregant analysis. A further testing of marker SCAR8-107 was performed using 74 high protein content individuals, 42 low protein content individuals and some tetraploid varieties, showing the selection accuracy of 90.51% and 72.97% between marker test and phenotyping in segregation population and tetraploid varieties respectively. Pearson’s correlation analysis also showed that the marker SCAR8-107 was significantly correlated to protein content in potato. The SCAR8-107 marker could be used in marker-assisted selection.

Key words: potato protein, marker development, BSA mixing pool sequencing, marker-assisted selection

表1

马铃薯品种信息"

序号
Code		 品种
Variety		 蛋白含量
Protein content (%)		 序号
Code		 品种
Variety		 蛋白含量
Protein content (%)
1 -37 1.18 28 Shlin-6 1.54
2 东农305 Dongnong 305 1.39 29 坝薯10号 Bashu 10 1.50
3 东农308 Dongnong 308 1.45 30 BC 1.45
4 东农310 Dongnong 310 2.21 31 大200305-32 Da 200305-32 1.50
5 东农311 Dongnong 311 1.17 32 大200410-19 Da 200410-19 2.15
6 东农312 Dongnong 312 1.68 33 大地3号 Dadi 3 1.53
7 东农313 Dongnong 313 1.26 34 定薯3号 Dingshu 3 2.01
8 东农314 Dongnong 314 1.68 35 东农03-1 Dongnong 03-1 1.44
9 东农316 Dongnong 316 1.44 36 费乌瑞它 Favourite 1.69
10 东农317 Dongnong 317 1.63 37 H9900-1 1.58
11 Allora 1.56 38 H99-9 2.12
12 B190 1.59 39 克新12 Kexin 12 2.06
13 B192 1.41 40 克新22 Kexin 22 1.98
14 B200 1.64 41 克新27 Kexin 27 1.68
15 B201 1.71 42 希森3号 Xisen 3 1.95
16 Carlar 2.30 43 夏坡蒂 Shepody 2.55
17 CIP39048 1.33 44 鑫科1号 Xinke 1 1.51
18 D412 1.45 45 兴佳2号 Xingjia 2 1.47
19 DESIREE 1.22 46 延薯3号 Yanshu 3 1.79
20 FW5 1.75 47 延薯4号 Yanshu 4 2.20
21 Gala 1.53 48 延薯6号 Yanshu 6 1.47
22 K200410-137 2.32 49 延薯7号 Yanshu 7 1.51
23 NY128 1.02 50 延薯8号 Yanshu 8 0.94
24 Q12 1.59 51 延薯97-8 Yanshu 97-8 1.71
25 早大白 Zaodabai 1.37 52 云薯201 Yunshu 201 1.18
26 中薯3号 Zhongshu 3 1.90 53 云薯301 Yunshu 301 2.09
27 中薯5号 Zhongshu 5 0.94 54 早50 Zao 50 1.59

图1

群体蛋白含量分布"

表2

群体蛋白含量的测定结果"

蛋白含量
Protein content (%)		 总数
Total number		 极端材料数量
Number of extreme individuals
极高蛋白 Extremely high protein >2.5 18 25 (>2.4)
高蛋白 High protein 2.0-2.5 56
中蛋白 Medium protein 1.5-2.0 57
中低蛋白 Medium-low protein 1.0-1.5 39
极低蛋白 Extremely low protein <1.0 3 25 (<1.3)

表3

引物信息"

引物名称
Primer name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 退火温度
Anneal temp. (°C)		 产物大小
Product size (bp)
chr8-100 CTGTTGGTCCGTGGTCTGG CGAGAGAGTGAATGGGCTAAGG 58 798
chr8-101 TCGCACAAGAACAACCACAATC ATACATTCAGCCATTCAGACTTCG 59 474
chr8-102 CAACAAAGTCTGAATGGCTGAATG AGGCTATGCTGCTAAGGATGC 59 488
chr8-103 TGAGAGGTCATCGTCTGGTTG TGGCTGTGGTTTAAGAAGTTGG 59 717
chr8-104 GGATGGCTCGTGATTGTCTTG TTTACTTCATTGGGTCGTTCGTC 59 699
chr8-105 TTCAGCGAGGACATAGCACAG CTCACTACACTCTTGGCACATTG 58 892
chr8-106 ACTCATAAGCATCCTCATTCTCAG CTAACCTCCTTCACTCTTCTATCC 60 569
chr8-107 GATGTCTAATACTCCGTCAAGGTG TCAAAGCATTACAACAACAAAGGG 58 724
chr8-108 CGCCAAGCCTCCAACTACTG ACTTTGAATCCTCTTTCCAACTCC 58 651
chr8-109 TTACCGTCACATTACATTTCACAG CTCCCATACTCTCTTCTCTAAACC 58 544
chr8-110 ATGGGATGTGTCAAGGGTCTC CCTCTCACTTCACTCTTCTCAAC 59 772
chr8-111 TCAATCCACGGACACAGACC GCCTAAACCCTCAACTCAAACG 58 515
chr8-112 CAAAAGTGATGGACAAAGTGACG CCTATCTCCCTCTGCTGTTCC 58 807
chr8-113 CACCTGAATGGACGGATATAAGC TGGGAGTGATAAGTCTGGTATGG 58 686
chr8-114 ATCAAGAAGAGAACGAGCAAGAC CAGACCCACCACAACATCAATC 58 671
chr8-115 GCTATCCTGCTATTCTCCGAAG TCTGACCATCACGACATCTCC 59 461
chr8-116 AACATCTGACTGTCTATGAAGCC GAGCACCGATATGAGGGAGAG 61 733
chr8-117 CTTACCGCTTCAGGCTTTCTC TTGATAGTCCTCAGGCATAAACG 58 752
chr8-118 CTGGCTTATGAACTCGGTGTC ATTCCTTGCTGTGTCCTTTGC 58 575
chr8-119 GGTTCAATTAAAGGTGTGCTGTG ATGAGGTGAGGACAATGCTAGG 58 745
chr8-120 TCAACAAAGACCCACATCACAAG TCACCTTCACGAGCCACTTC 58 686
chr8-121 TGCCGCATTATTGTTGTTATTCG GCCGCTCTCAGTTCTCTCC 59 484
chr8-89 TCAGATCACGCATAAATCGGTTC TGGACATAAACACGGGACTTAGG 59 406
chr8-90 TTGCTTCCGTGAAAGTGTTATGG TCATCAGTGGCTTGGCTTACC 60 748
chr8-91 CCTCCAGAGCCACCACTTAC TTGAACTTCCAATGCTACCATCC 58 626
chr8-92 GAGGGTGGAAAGTCGTGTTATG AATGAATGTGGTGGTGGATGC 58 831
chr8-93 GAGGGTGGAAAGTCGTGTTATG TGGATGCTCTTAATTGGCTATGG 58 817
chr8-94 AGGTGTAAGTGTAGGTGTAGGTG GCGAGCATCTTGGAGTTAGC 58 464
chr8-95 ACAAGGTGAATTGGAAGATGCTC TGGAAGGACAATCTGCTACGAC 58 578
chr8-96 GCGATGCTGCTATATGGTTCAC CTCCTCACGAATCTCCTCTGC 58 453
chr8-97 CATCCTCCGCCACCCAAAG CAGAATCAGCAGCAGCATCC 58 835
chr8-98 GGAGGAATTTGCGTGGGAAAG GCCGTATGTTGTGAAGGAAGC 58 705
chr8-99 CTCATCAGCATCACACTTCACTC AAGGCAAGGGTAAAGGCAAATC 58 571

图2

部分引物在亲本定薯1号和大西洋的扩增产物电泳图引物编号: 8-106、8-107、8-108、8-109、8-110; 每对引物的第1泳道和第2泳道分别为亲本定薯1号和大西洋。 "

图3

引物8-107标记在部分极端高蛋白与极端低蛋白分离后代中的PCR扩增结果P1: 低蛋白含量亲本定薯1号; P2: 高蛋白含量亲本大西洋; 编号39、46、49、50、51为极端高蛋白后代材料; 编号32、43、45、53、54为极端低蛋白后代材料。"

图4

SCAR8-107标记在部分高蛋白(A)与低蛋白(B)分离后代中的PCR扩增结果P1: 低蛋白含量亲本定薯1号; P2: 高蛋白含量亲本大西洋; 数字依次为低蛋白含量与高蛋白含量的后代群体材料编号。"

表4

F1群体部分样品的蛋白含量表型鉴定与标记检测结果统计"

标记检测
Marker test		 样本总数
Total number of samples		 表型
Phenotype		 样本数
Number of samples		 百分比
Percentage (%)
阳性
Positive		 70 高蛋白含量 High protein content 67 95.71
低蛋白含量 Low protein content 3
阴性
Negative		 46 高蛋白含量 High protein content 8 82.61
低蛋白含量 Low protein content 38

表5

部分品种蛋白含量表型鉴定与标记检测结果统计"

标记检测
Marker test		 样本总数
Total number of samples		 表型
Phenotype		 样本数
Number of samples		 百分比
Percentage (%)
阳性
Positive		 14 高蛋白含量 High protein content 10 71.42
低蛋白含量 Low protein content 4
阴性
Negative		 23 高蛋白含量 High protein content 6 73.91
低蛋白含量 Low protein content 17

图5

SCAR8-107标记在部分高蛋白(A)与低蛋白(B)品种中的PCR扩增结果P1: 低蛋白含量亲本定薯1号; P2: 高蛋白含量亲本大西洋; 1~12: 夏坡蒂、K200410-137、Carlora、东农310、延薯4号、大200410-19、呼H99-9、云薯301、克新12、定薯3号、克新22、希森3号; 13~24: 东农316、B192、东农305、早大白、CIP39048、东农313、DESIREE、云薯201、东农311、NY128、中薯5号、延薯8号。"

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