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作物学报 ›› 2022, Vol. 48 ›› Issue (9): 2274-2284.doi: 10.3724/SP.J.1006.2022.14138

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于2b-RAD测序的四倍体马铃薯熟性相关的分子标记开发

惠志明(), 徐建飞, 简银巧, 卞春松, 段绍光, 胡军, 李广存, 金黎平*()   

  1. 中国农业科学院蔬菜花卉研究所 / 农业农村部薯类作物生物学和遗传育种重点实验室, 北京 100081
  • 收稿日期:2021-08-05 接受日期:2021-11-29 出版日期:2022-09-12 网络出版日期:2021-12-16
  • 通讯作者: 金黎平
  • 作者简介:E-mail: huizhiming@aliyun.com
  • 基金资助:
    宁夏回族自治区农业育种专项马铃薯新品种选育项目(2019NYYZ01-1)

2b-RAD based maturity associated molecular marker identification in tetraploid potato (Solanum tuberosum L.)

HUI Zhi-Ming(), XU Jian-Fei, JIAN Yin-Qiao, BIAN Chun-Song, DUAN Shao-Guang, HU Jun, LI Guang-Cun, JIN Li-Ping*()   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences / Key Laboratory of Biology and Genetic Improvement of Tuber and Root Crop, Ministry of Agriculture and Rural Affairs, Beijing 100081, China
  • Received:2021-08-05 Accepted:2021-11-29 Published:2022-09-12 Published online:2021-12-16
  • Contact: JIN Li-Ping
  • Supported by:
    New Potato Varieties Breeding of Agricultural Breeding Special Project of the Ningxia Hui Autonomous Region of China(2019NYYZ01-1)

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摘要：

马铃薯熟性是由多基因控制的数量性状, 是我国不同栽培区划选择适宜品种的重要指标之一。本研究以晚熟品种中薯18号和早熟品种中薯5号及其F1分离群体为材料, 2018—2019年连续2年对“中薯18号(母本) × 中薯5号(父本)”杂交分离群体进行熟性评价, 从中筛选出极端晚熟和极端早熟的基因型各30个, 并分别构建极端晚熟和极端早熟基因组DNA混池。利用简化基因组2b-RAD (2b-restriction site-associated DNA)技术测序, 寻找差异标签开发出3个与熟性连锁的分子标记SCARA2-2、SCARA4-21和SCARA5-16, 3个分子标记联合使用对熟性分离群体子代进行验证, 晚、早熟表型符合率分别到达了87.5%和93.0%, 这些分子标记的开发和联合使用对辅助马铃薯熟性选择具有重要的参考价值。

关键词: 马铃薯, 熟性, 2b-RAD, 分子标记

Abstract:

Potato maturity is a quantitative trait controlled by multiple genes and it is one of the major agronomic characteristics for selecting suitable varieties in different agro-ecological zones. In this study, the maturity of the segregating population derived from Zhongshu 18 (♀) × Zhongshu 5 (♂) was evaluated in 2018 and 2019. Respectively, 30 offsprings with extremely late or early maturity were selected and used to construct early and late maturing genomic pool. Three molecular markers (SCARA2-2, SCARA4-21, and SCARA5-16) linked to maturity were identified by simplified genome 2b-RAD (2b-restriction site-associated DNA) sequencing. The maturity phenotypic association rate of the three-marker-combination for the late and early maturity genotype verification reached 87.5% and 93.0%, respectively. Thus, these molecular markers are valuable for markers assisted selection in potato maturity breeding.

Key words: potato, maturity, 2b-RAD, molecular markers

表1

至今已经发表的马铃薯熟性分子标记"

分子标记
Molecular marker		 染色体(连锁群)
Chromosome
(linkage group)		 组合
Cross		 倍性
Ploidy		 群体
Population		 参考文献
Reference
GP179, GP76 Chr.V (Major QTL), Chr.VI (Minor QTL) G87D2.4.1 × I88.55.6
G87D2.4.1 × I86.102.1		 Diploid 109
29		 Oberhagemann et al. [21]
STM1100-STM1056, STM0028-STM3009, TM1057-STM0024 Chr.V, Chr.VI, Chr.VII, Chr.VIII G87D2.4.1 × I88.55.6 Diploid 113 Collins et al. [22]
GP21 Chr.V USW5337.3 × 77.2102.37 Diploid 67 Visker et al. [8]
STM3179 Chr.V 12601ab1 × Stirling Tetraploid 227 Bradshaw et al. [23]
分子标记
Molecular marker		 染色体(连锁群)
Chromosome
(linkage group)		 组合
Cross		 倍性
Ploidy		 群体
Population		 参考文献
Reference
GP21 Chr.III (Minor QTL), Chr.V (Major QTL) SH82-44-111 × CE51
DH84-19-1659 × dI88.55.6		 Diploid 227
300		 Visker et al. [24]
C51, C52, C53, C61, C62 Chr.V, Chr.VI S. phureja × S. tuberosum Diploid 205 Mal.osetti et al. [10]
GP186, BA47f2t7, CP113 Chr.V DG 83-1520 ×DG 84-195 Diploid 156 Sliwka et al. [25]
STM3179 Chr.V 12601ab1 × Stirling Tetraploid 227 Bradshaw et al. [11]
solcap_snp_c2_47609 Chr.V 12601ab1 × Stirling Tetraploid 190 Hackett et al. [12]
solcap_snp_c2_22986 Chr.V Jacqueline Lee × MSG227-2 Tetraploid 156 Massa et al. [13]
solcap_snp_c2_11605 Chr.V Rio Grande Russet ×
Premier Russet		 Tetraploid 162 Massa et al. [14]
SSR5-85-1, SCAR5-8 Chr.V Zhongshu 19 × Zhongshu 3 Tetraploid 221 Li et al. [9]
PM_Z_05 Chr.II, IV, V, VII, XI Longshu 8 × Zaodabai Tetraploid 192 Li et al. [26]

表2

马铃薯群体熟性分级"

熟性等级
Maturity class		 生育期
Growth period (d)
1 ≤60
2 61-70
3 71-80
4 81-90
5 91-100
6 ≥101

图1

早熟性分子标记SCAR5-8 (A)和基因StCDF1.2 (B)特异引物在早熟品种中薯5号和晚熟品种中薯18号中条带检测 M1: DL2000; M2: marker III; P1: 中薯5号; P2: 中薯18号。"

图2

2018-2019年分离群体熟性鉴定平均结果"

表3

测序数据分析"

材料
Material		 原始reads
Clean reads		 高质量Reads
Enzyme reads		 百分数
Percentage (%)		 特异标签数
No. of uniq tags		 测序深度
Depth
中薯18号Zhongshu 18 58,046,070 51,631,527 88.95 121,964 162.5 ×
中薯5号Zhongshu 5 58,046,070 52,205,695 89.94 122,940 162.1 ×
极端晚熟混池Extremely late pool 58,046,070 52,383,011 90.24 128,108 158.2 ×
极端早熟混池Extremely early pool 58,046,070 53,881,077 92.82 127,564 161.1 ×
平均Mean 58,046,070 52,525,328 90.49 125,144 161.0 ×

图3

标签在染色体上分布密度(A)及早晚熟混池差异标签密度图(B) 0~80 M: 染色体物理距离(单位: M); Chr.1~Chr.12分别为马铃薯的12条染色体。图A左侧标注EPool表示早熟混池, LPool表示晚熟混池; 右侧柱状图, 不同颜色表示不同的标签数量, 数字表示标签数量。图B中, 每条染色体中上边柱形图表示早熟池特异标签, 中间柱形图为晚熟池特异标签, 下边柱形图为两池之间差异标签绝对值; 右侧柱状图, 不同颜色表示不同的标签数量, 数字表示标签数量。"

图4

SCARA5-16标记在部分极早熟(A)与极晚熟(B)子代中的扩增 M: DL2000; P1: 中薯5号; P2: 中薯18号。A图中数字27~51表示极端早熟子代材料编号; B图中数字298~172表示极端晚熟子代材料编号。"

表4

极端早熟和极端晚熟材料的标记验证"

极端基因型
Extreme genotype		 样本总数(个)
Total number of samples		 标记名称
Marker name		 单标记表型符合率
Single coincidence rate (%)		 染色体
Chr.		 引物序列
Primer sequences(5°-3°)		 标记鉴定
Marker
identification		 标记联合使用表型符合率
Comprehensive coincidence rate (%)
极端晚熟
Extremely
late maturity		 30 SCARA2-2 56.7 2 F: ACAGCTCGGCGAGAAAACAG
R: TCAAGCAATTAGGGCGGTG		 阳性
Positive		 90.0
SCARA4-21 60.0 4 F: TCACTTTGGCGACCACACTT
R: CAGACCCGCTTACGCTAAGAT		 阳性
Positive
SCARA5-16 60.0 5 F: TTTTTGTGATCAGGGGCGG
R: TGCATTGCATCCTCCCAAC		 阳性
Positive
极端早熟
Extremely early maturity		 30 SCARA2-2 56.7 2 F: ACAGCTCGGCGAGAAAACAG
R:TCAAGCAATTAGGGCGGTG		 阴性
Negative		 96.7
SCARA4-21 60.0 4 F: TCACTTTGGCGACCACACTT
R: CAGACCCGCTTACGCTAAGAT		 阴性
Negative
SCARA5-16 76.7 5 F: TTTTTGTGATCAGGGGCGG
R: TGCATTGCATCCTCCCAAC		 阴性
Negative

表5

熟性表型鉴定与标记检测统计结果"

表型
Phenotype		 样本总数(个)
Total number of
samples		 标记名称
Marker name		 单标记表型符合率
Single coincidence rate (%)		 染色体
Chr.		 引物序列
Primer sequences (5°-3°)		 标记鉴定
Marker
identification		 标记联合使用表型符合率Comprehensive coincidence rate (%)
晚熟
Late maturity		 48 SCARA2-2 62.5 2 F: ACAGCTCGGCGAGAAAACAG
R: TCAAGCAATTAGGGCGGTG		 阳性
Positive		 87.5
SCARA4-21 58.3 4 F: TCACTTTGGCGACCACACTT
R: CAGACCCGCTTACGCTAAGAT		 阳性
Positive
SCARA5-16 60.4 5 F: TTTTTGTGATCAGGGGCGG
R: TGCATTGCATCCTCCCAAC		 阳性
Positive
早熟
Early maturity		 71 SCARA2-2 54.9 2 F: ACAGCTCGGCGAGAAAACAG
R: TCAAGCAATTAGGGCGGTG		 阴性
Negative		 93.0
SCARA4-21 66.2 4 F: TCACTTTGGCGACCACACTT
R: CAGACCCGCTTACGCTAAGAT		 阴性
Negative
SCARA5-16 76.1 5 F: TTTTTGTGATCAGGGGCGG’
R:TGCATTGCATCCTCCCAAC		 阴性
Negative
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