欢迎访问作物学报，今天是
作物学报

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)

RichHTML

20

PDF

338

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
[1] 徐建飞, 金黎平. 马铃薯遗传育种研究: 现状与展望. 中国农业科学, 2017, 50: 990-1015.
Xu J F, Jin L P. Analysis for maturity of progenies from potato cultivar Zhongshu 3. Sci Agric Sin, 2017, 50: 990-1015. (in Chinese with English abstract)
[2] 孙慧生. 马铃薯育种学. 北京: 中国农业出版社, 2003. pp 30-59.
Sun H S. Potato Breeding. Beijing: China Agriculture Press, 2003. pp 30-59. (in Chinese)
[3] 徐建飞, 段绍光, 庞万福, 卞春松, 刘杰, 金黎平. 马铃薯品种中薯3号杂交后代熟性分析. 华北农学报, 2015, 30(增刊1): 92-96.
Xu J F, Duan S G, Pang W F, Bian C S, Liu J, Jin L P. Analysis for maturity of progenies from potato cultivar Zhongshu 3. Acta Agric Boreali-Sin, 2015, 30(S1): 92-96. (in Chinese with English abstract)
[4] Mackerron D K L, Davies H V. Markers for maturity and senescence in the potato crop. Potato Res, 1986, 29: 427-436.
doi: 10.1007/BF02357908
[5] Hvostova V V, Iacina I M. 马铃薯遗传学. 北京: 中国农业出版社, 1981. pp 280-283.
Hvostova V V, Iacina I M. Potato Genetics. Beijing: China Agriculture Press, 1981. pp 280-283. (in Chinese)
[6] Kloosterman B, Abelenda J A, Gomez M M C, Oortwijn M, De boer J M, Kowitwanich K, Horvath B M, Van Eck H J, Smaczniak C, Prat S, Visser R G F, Bachem C W B. Naturally occurring allele diversity allows potato cultivation in northern latitudes. Nature, 2013, 495: 246-250.
doi: 10.1038/nature11912
[7] Bonierbale M W, Plaisted R L, Tanksley S D. RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics, 1988, 120: 1095-1103.
doi: 10.1093/genetics/120.4.1095 pmid: 17246486
[8] Visker M H P W, Kerzer L C P, Van Eck H J, Jacobsen E, Colon L T, Struik P C. Can the QTL for late blight resistance on potato chromosome 5 be attributed to foliage maturity type? Theor Appl Genet, 2003, 106: 317-325.
pmid: 12582858
[9] Li X, Xu J, Duan S, Zhang J, Bian C, Hu J, Li G, Jin L. Mapping and QTL analysis of early-maturity traits in tetraploid potato (Solanum tuberosum L.). Int J Mol Sci, 2018, 19: 3065.
doi: 10.3390/ijms19103065
[10] Malosetti M, Visser R G F, Celis-Gamboa C, van Eeuwijk F A. QTL methodology for response curves on the basis of non-linear mixed models, with an illustration to senescence in potato. Theor Appl Genet, 2006, 113: 288-300.
pmid: 16791695
[11] Bradshaw J E, Hackett C A, Pande B, Waugh R, Bryan G J. QTL mapping of yield, agronomic and quality traits in tetraploid potato (Solanum tuberosum subsp. tuberosum). Theor Appl Genet, 2008, 116: 193-211.
doi: 10.1007/s00122-007-0659-1 pmid: 17938877
[12] Hackett C A, Bradshaw J E, Bryan G J. QTL mapping in autotetraploids using SNP dosage information. Theor Appl Genet, 2014, 127: 1885-1904.
doi: 10.1007/s00122-014-2347-2
[13] Massa A N, Manriquew-Carpintero N C, Coombs J J, Zarka D G, Boone A E, Wirk W W, Hackett C A, Bryan G J, Douches D S. Genetic linkage mapping of economically important traits in cultivated tetraploid potato (Solanum tuberosum L.). Genes Genom Genet, 2015, 5: 2357-2364.
[14] Massa A N, Manrique-Carpintero N C, Coombs J, Haynes K G, Bethke P C, Brandt T L, Gupta S K, Yencho G C, Novy R G, Douches D S. Linkage analysis and QTL mapping in a tetraploid russet mapping population of potato. BMC Genet, 2018, 19: 87.
doi: 10.1186/s12863-018-0672-1
[15] Jia L, Zhang B, Liu K, Zheng D, Wang X. Analysis of population genetic diversity of Cynoglossus cynoglossus based on 2b-RAD simplified genome sequencing. Fisher Res, 2017, 4: 125-133.
[16] Blanco-Bercial L, Buchlin A. New view of population genetics of zooplankton: RAD-seq analysis reveals population structure of the North Atlantic planktonic copepod Centropages typicus. Mol Ecol, 2016, 25: 1566-1580.
doi: 10.1111/mec.13581 pmid: 26857348
[17] Gao W, Qu J, Zhang J, Sonnenberg A, Chen Q, Zhang Y, Huang C. A genetic linkage map of Pleurotus tuoliensis integrated with physical mapping of the de novo sequenced genome and the mating type loci. BMC Genomics, 2018, 19: 18.
doi: 10.1186/s12864-017-4421-z
[18] Seetharam A S, Stuart G W. Whole genome phylogeny for 21 Drosophila species using predicted 2b-RAD fragments. PeerJ, 2013, 1: e226.
doi: 10.7717/peerj.226
[19] Dou J, Li X, Fu Q, Jiao W, Li Y, Li T, Wang Y, Hu X, Wang S, Bao Z. Evaluation of the 2b-RAD method for genomic selection in scallop breeding. Sci Rep, 2016, 6: 19244.
[20] 李兴翠, 李广存, 徐建飞, 段绍光, 卞春松, 庞万福, 刘杰, 金黎平. 四倍体马铃薯熟性连锁SCAR标记的开发与验证. 作物学报, 2017, 43: 821-828.
doi: 10.3724/SP.J.1006.2017.00821
Li X C, Li G C, Xu J F, Duan S G, Bian C S, Pang W F, Liu J, Jin L P. Development and verification of SCAR marker linked to maturity in tetraploid potato. Acta Agron Sin, 2017, 43: 821-828. (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2017.00821
[21] Oberhagemann P, Chatot-Balandras C, Schäfer-Pregl R, Wegener D, Palomino C, Salamini F, Bonnel E, GebhardT C. A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection. Mol Breed, 1999, 5: 399-415.
doi: 10.1023/A:1009623212180
[22] Collins A, Milbourne D, RamsaY L, Meyer R, Chatot-Balandras C, Oberhagemann P, De Jong W, Gebhardt C, Bonnel E, Waugh R. QTL for field resistance to late blight in potato are strongly correlated with maturity and vigour. Mol Breed, 1999, 5: 387-398.
doi: 10.1023/A:1009601427062
[23] Bradshaw J E, Pande B, Bryan G J, Hackett C A, McLean K, Stewart H E, Waugh R. Interval mapping of quantitative trait loci for resistance to late blight [Phytophthora infestans (Mont.) de Bary], height and maturity in a tetraploid population of potato (Solanum tuberosum subsp. tuberosum). Genetics, 2004, 168: 983-995.
pmid: 15514069
[24] Visker M H P W, Heilersig H J B, Kodde L P, Van de Weg W E, Voorrips R E, Struik P C, Colon L T. Genetic linkage of QTLs for late blight resistance and foliage maturity type in six related potato progenies. Euphytica, 2005, 143: 189-199.
doi: 10.1007/s10681-005-3444-8
[25] Sliwka J, Jakuczun H, Lebecka R, Marczewski W, Gebhardt C, Zimnoch-Guzowska E. Tagging QTLs for late blight resistance and plant maturity from diploid wild relatives in a cultivated potato (Solanum tuberosum L.) background. Theor Appl Genet, 2007, 115: 101-112.
pmid: 17468842
[26] Li J, Wang Y, Wen G, Li G, Li Z, Zhang R, Ma S, Zhou J, Xie C. Mapping QTL underlying tuber starch content and plant maturity in tetraploid potato. Crop J, 2019, 7: 261-272.
doi: 10.1016/j.cj.2018.12.003
[27] 罗育, 黄春喜, 吴耀生, 蔡丹昭, 郭宏伟, 朱丹. 3种DNA分子标记法联合鉴别草珊瑚及其混伪品. 中草药, 2020, 51: 733-740.
Luo Y, Huang C X, Wu Y S, Cai D Z, Guo H W, Zhu D. Molecular authentication of Sarcandra glabra and its adulterants using three DNA molecular markers. Chin Trad Herbal Drugs, 2020, 51: 733-740. (in Chinese with English abstract)
[28] 黄艺宁. RAPD、ISSR分子标记联合鉴定毛木耳菌株的研究. 河南农业科学, 2020, 49(7): 118-125.
Huang Y N. RAPD and ISSR molecular identification of Auricularia cornea Ehrenb. J Henan Agric Sci, 2020, 49(7): 118-125. (in Chinese with English abstract)
[1] 陈国欢, 张锐, 李艳迪, 赵佳琪, 任湧涛, 张天赐, 郭华春, 李俊, 杨芳. 外源硒叶面喷施对浅紫色马铃薯块茎花青素合成的影响[J]. 作物学报, 2026, 52(6): 1876-1890.
[2] 翟胜男, 曹新有, 李豪圣, 李吉虎, 李法计, 刘金栋, 夏先春, 吕莹莹, 马瑞峰, 王颖, 耿洪伟, 刘建军. 小麦Pod-A1、Pod-D1和Pod-2D位点等位变异对籽粒过氧化物酶活性的遗传效应分析[J]. 作物学报, 2026, 52(6): 1593-1603.
[3] 彭佳泺, 李颖, 李丹丹, 杨军宁, 郭学峰, 张文姣, 俞晓雪, 周亚荣, 王振玉, 王彩香, 马雄风, 宿俊吉. 陆地棉I类LBD家族成员鉴定及GhLBD6调控开花期的功能和单倍型分析[J]. 作物学报, 2026, 52(6): 1682-1697.
[4] 王文辕, 燕雪嘉, 刘玉霖, 孙晓彤, 李亚楠, 唐鑫华, 石瑛. 耐弱光马铃薯品种筛选及转录因子编码基因StPIF3的克隆与功能分析[J]. 作物学报, 2026, 52(6): 1631-1645.
[5] 张献丰, 郭利建, 李康春, 孔斌雪, 刘玉芳, 车卓, 杨德龙. 小麦ABHD6基因家族鉴定与粒重功能标记开发[J]. 作物学报, 2026, 52(6): 1711-1727.
[6] 田立涛, 丁宁, 王树林, 齐恩芳, 张荣, 王蕊蕊, 马利雯, 李建武, 杨江伟. 马铃薯Argonaute基因家族鉴定及晚疫病诱导表达分析[J]. 作物学报, 2026, 52(4): 1116-1126.
[7] 汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[8] 王楚锐, 李开祥, 赵志, 肖麓, 唐国永, 赵志刚, 徐亮, 杜德志, 柳海东. 甘蓝型春油菜早花基因BnCRY2功能位点KASP标记的开发及应用[J]. 作物学报, 2026, 52(3): 708-721.
[9] 张宇, 刘芳, 蔡诚诚, 杨小华, 吉阿么石扎, 杨元军, 王西瑶. 溴乙烷与赤霉素协同处理破除马铃薯块茎休眠的机理初探[J]. 作物学报, 2026, 52(3): 825-838.
[10] 徐强, 谢奎忠, 胡新元, 岳云, 董博, 罗爱花. 连作对马铃薯根际土壤线虫群落结构与功能的影响[J]. 作物学报, 2026, 52(2): 527-538.
[11] 杨飚, 杜帅康, 张继旺, 石瑛, 张丽莉. 马铃薯III类POD基因家族的全基因组鉴定及其表达谱分析[J]. 作物学报, 2026, 52(2): 405-420.
[12] 王粤生, 葛冬冬, 程兰斐, 陈春环, 王长有, 刘新伦, 李停栋, 邓平川, 吉万全, 赵继新. 小麦-华山新麦草二体异代换系16DH25-7的分子细胞遗传学及抗病性鉴定[J]. 作物学报, 2026, 52(2): 433-445.
[13] 田甲春, 葛霞, 李守强, 李梅, 田世龙, 张亚倩, 程建新, 李玉梅. 低O2高CO2贮藏环境延缓马铃薯块茎衰老的作用机制[J]. 作物学报, 2026, 52(1): 262-278.
[14] 王雅致, 杨飚, 季香林, 石瑛, 张丽莉. 二倍体马铃薯抗旱资源鉴定及抗旱基因初步筛选[J]. 作物学报, 2026, 52(1): 72-84.
[15] 姬炫彤, 卞春松, 金黎平, 李森, 秦军红, 李广存. 不同耐旱型马铃薯根际微生物对干旱的响应[J]. 作物学报, 2026, 52(1): 165-177.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2