Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (2): 370-378.doi: 10.3864/j.issn.0578-1752.2021.02.012

• HORTICULTURE • Previous Articles     Next Articles

Identification of F1 Hybrids in Blueberry (Vaccinium corymbosum L.) Based on Specific-Locus Amplified Fragment Sequencing (SLAF-seq)

LIU YouChun1,2(),LIU WeiSheng1,WANG XingDong1,SUN Bin1,LIU XiuLi1,YANG YanMin1,WEI Xin1,YANG YuChun1,ZHANG Duo1,LIU Cheng1(),LI TianZhong2()   

  1. 1Liaoning Institute of Pomology, Yingkou 115009, Liaoning
    2College of Horticulture, China Agricultural University, Beijing 100193
  • Received:2020-04-26 Accepted:2020-07-30 Online:2021-01-16 Published:2021-02-03
  • Contact: Cheng LIU,TianZhong LI;;


【Objective】 The aim of this investigation was to develop a method of identifying true F1 hybrids based on next-generation sequencing data, so as to provide an alternative strategy for fruit breeding, genetic analysis and genetic map construction.【Method】A total of 318 F1 population derived from reciprocal cross between two blueberry (Vaccinium corymbosum L.) accessions were sequenced by specific-locus amplified fragment sequencing (SLAF-seq). Based on the blueberry reference genome, the rare allelic variation, principal component analysis (PCA) and k-means clustering were used to identify the accidental hybrid in blueberry population. Furthermore, homozygous SNP were used to confirm the data.【Result】A total of 65.89 Gb sequence data was generated by Illumina sequencing, in which the GC content was 38.63% and Q30 value was 95.44%. The average sequencing depth of the two parents and F1 progenies were 12.86× and 5.41×, respectively. By aligning the sequencing reads onto the reference genome of Vaccinium corymbosum, a total of 73 543 and 114 851 SNPs were called in two reciprocal cross populations, respectively. According to these SNPs with minor allele frequency (MAF) more than 0.05, PCA, and k-means analysis, it was yielded four outlier individuals regarded as accidental hybrid progenies. Moreover, ten outliers were identified after total rare-alleles analysis and the private rare-alleles analysis by using SNP with MAF﹤0.05, including the above four individuals. By verifying the accidental hybrid progenies, homozygous SNP between the two parents was accounted for 34.56% and 38.95% of the total SNP, respectively, which were subjected to genotype the ten outliers and conflicted genotypes, and which were observed in individuals except H194-123 with the two parents, and the accuracy of our developed methods was validated.【Conclusion】For species with a reference genome, it was an effective strategy by using allele frequency (MAF) data set to identify the accidental hybrid progeny based on genetic relationship analysis and unique rare allele variation analysis.

Key words: blueberry, reciprocal population, SLAF-seq, SNP, unique rare allele variation, hybrid

Table 1

Summary statistics of sequencing results from Vaccinium samples"

Sample ID
Total reads
Total bases
Q30 percentage (%)
GC percentage (%)
Berkeley 10 350 711 2 067 990 444 95.12 39.14
N6 10 914 992 2 180 687 340 94.88 39.45
杂交后代 Offspring 971 057 193 850 201 95.04 39.72
水稻(对照样品) Rice (Control) 1 047 399 209 302 386 94.59 40.46
总和 Total 330 061 690 65 893 041 734 95.04 39.72

Table 2

SNP in different Vaccinium crossing populations"

Number of SNPs
(MAF > 0.05)
SNP标记数量(MAF < 0.05)
Number of SNPs
(MAF < 0.05)
MF185 70 243 3 200
FM133 111 527 3 324

Fig. 1

Principal component analysis in MF185 and FM133 populations in blueberry A-B:MF185 population; C-D:FM133 population. Cluster1, 2 and 3 corresponded to K-means clustering result at k=2 and 3"

Fig. 2

Boxplots analysis of total and private rare-allele in different populations ● represents each sample, * indicates outliers apart from population. The same as below"

Fig. 3

The false-hybrids verification based on homozygous dominant SNP of parental genotype"

[1] 朱骏驰, 郭印山, 刘镇东, 李坤, 杨晓旭, 石广丽, 牛早柱, 李成祥, 郭修武. 利用SSR分子标记鉴定葡萄F1代杂种. 沈阳农业大学学报, 2016,47(2):148-152.
ZHU J C, GUO Y S, LIU Z D, LI K, YANG X X, SHI G L, NIU Z Z, LI C X, GUO X W. Identification of the F1 hybrids of grape using SSR molecular markers. Journal of Shenyang Agricultural University, 2016,47(2):148-152. (in Chinese)
[2] 颜廷进, 谭振新. 农作物种子纯度鉴定技术研究进展. 种子科技, 2004,22(3):153-155.
YAN T J, TAN Z X. Purity vertification research prospect of crops seed. Seed Science & Technology, 2004,22(3):153-155. (in Chinese)
[3] 曾明, 杨柏云. 同工酶技术在柑橘研究中的应用. 江西科学, 2006,24(1):100-104.
ZENG M, YANG B Y. The application of isoenzyme technology in citrus studies. Jiangxi Science, 2006,24(1):100-104. (in Chinese)
[4] 鹿金颖, 毛永民, 申莲英, 彭士琪, 刘敏. 用AFLP分子标记鉴定冬枣自然授粉实生后代杂种的研究. 园艺学报, 2005,32(4):680-683.
LU J Y, MAO Y M, SHEN L Y, PENG S Q, LIU M. Application of AFLP markers for identification of hybrids from open pollinated Dongzao (Zizyphus jujuba Mill.) progenies. Acta Horticulturae Sinica, 2005,32(4):680-683. (in Chinese)
[5] 马鸿翔, 陈佩度, 余桂红, 任丽娟. 东北草莓×凤梨草莓种间杂种一代的细胞遗传学观察与RAPD分析. 园艺学报, 2007,34(3):597-604.
MA H X, CHEN P D, YU G H, REN L J. Cytogenetics and RAPD analysis of interspecific hybrids from the cross of Fragaria mandschurica Staudt and F. ananassa Duch. Acta Horticulturae Sinica, 2007,34(3):597-604. (in Chinese)
[6] 乔燕春, 林顺权, 何小龙, 杨向晖. 普通枇杷种内和种间杂种苗的RAPD鉴定. 果树学报, 2010,27(3):385-390.
QIAO C Y, LIN S Q, HE X L, YANG X H. Identification of intraspecific and interspecific hybridizati0ns in Loquat (Eriobotrya) using RAPD molecular markers. Journal of Fruit Science, 2010,27(3):385-390. (in Chinese)
[7] 郭修武, 张鹏翔, 郭印山, 刘镇东, 李坤, 李成祥. 应用SRAP分子标记技术鉴定葡萄种间杂交后代. 分子植物育种, 2011. doi: 10.5376/
GUO X W, ZHANG P X, GUO X S, LIU Z D, LI K, LI C X. Authenticity of identification of progenies from interspecific cross Red Globe (Vitis vinifera) × Shuangyou (Vitisamurensis) by SRAP markers. Molecular Plant Breeding, 2011. doi: 10.5376/ 09.0052. (in Chinese)
[8] 樊秀彩, 张颖, 姜建福, 孙海生, 焦建, 刘崇怀. SSR分子标记鉴定山葡萄和河岸葡萄种间杂种. 西北植物学报, 2012,32(11):2195-2200.
FAN X C, ZHANG Y, JIANG J F, SUN H S, JIAO J, LIU C H. Identification of interspecific hybrids derived from Vitis tiparia × Vitis amurensis by SSR marker. Acta Botanica Boreali-Occidentalia Sinica, 2012,32(11):2195-2200. (in Chinese)
[9] 苏聪聪, 金燕, 徐丰, 白描, 石雪晖, 杨国顺, 钟晓红, 刘昆玉, 陈陈恒, 李含晰. 利用SSR分子标记鉴定刺葡萄F1代杂种. 江苏农业科学, 2018,46(17):35-38.
SU C C, JIN Y, XU F, BAI M, SHI X H, YANG G S, ZHONG X H, LIU K Y, CHEN C H, LI H X. Identification of the F1 hybrids of thorn grape using SSR molecular markers. Jiangsu Agricultural Sciences, 2018,46(17):35-38. (in Chinese)
[10] 韩燕, 马登超, 刘译阳, 崔凤, 孙秀芹, 李荣冲, 万书波, 李国卫. 利用特异性SNP位点鉴定花生杂交F1代真假杂种. 山东农业科学, 2016,48(4):14-17.
HAN Y, MA D C, LIU Y Y, CUI F, SUN X Q, LI R C, WAN S B, LI G W. Identification of true F1 peanut hybrid with specific SNP locus. Shandong Agricultural Sciences, 2016,48(4):14-17. (in Chinese)
[11] ZHANG J, YUAN H, LI M, LI Y J, WANG Y, MA X J, ZHANG Y, TAN F, WU R L. A high-density genetic map of tetraploid Salix matsudana using specific length amplified fragment sequencing (SLAF-seq). PLoS ONE, 2016,11(6):e0157777.
[12] NIU D, DU Y, WANG Z, WANG Z, XIE S, NGUYEN H, DONG Z G, SHEN H D, LI J L. Construction of the first high-density genetic linkage map and analysis of quantitative trait loci for growth-related traits in Sinonovacula constricta. Marine Biotechnology, 2017,19(5):488-496.
pmid: 28725940
[13] DOYLE J J T, DOYLE J L. Isolation of plant DNA from fresh tissue. Focus, 1990,12:13-15.
[14] POLASHOCK J, ZELZION E, FAJARDO D, ZALAPA J, GEORGI L, BHATTACHARYA D, VORSA N. The American cranberry: First insights into the whole genome of a species adapted to bog habitat. BMC Plant Biology, 2014,14(1):165.
[15] COLLE M, LEISNER C P, WAI C M, QU S J, BRID K A, WANG J, WISECAVER J H, YOCCA A E, ALGER E I, TANG H B, XIONG Z Y, CALLOW P, BEN-ZVI B, BRODT A, BARUCH K, SWALE T, SHIUE L, SONG G Q, CHUIDS K L, SCHILMILLER A, CORSA N, BUELL C R, VANBUREN R, JIANG N, EDGER P P. Haplotype- phased genome and evolution of phytonutrient pathways of tetraploid blueberry. GigaScience, 2019, 8: giz012.
[16] VAN DER AUWERA G A, CARNEIRO M O, HARTL C, POPLIN R, DEL ANGEL G, LEVY-MOONSHINE A, JORDAN T, SHAKIR K, ROAZEN D, THIBAULT J, BANKS E, GARIMELLA K V, ALSHULER D, GABRIEL S, DEPRISTO M A. From FastQ data to high-confidence variant calls: The genome analysis toolkit best practices pipeline. Current Protocols in Bioinformatics, 2013, 43(1110): 11.10.1-11.10.33.
[17] MEIRMANS P G, VAN TIENDEREN P H. Genotype and genodive: Two programs for the analysis of genetic diversity of asexual organisms. Molecular ecology notes, 2004,4(4):792-794.
[18] EXCOFFIER L, SMOUSE P E, QUATTRO J M. Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics, 1992,131(2):479-491.
pmid: 1644282
[19] MICHALAKIS Y, EXCOFFIER L. A generic estimation of population subdivision using distances between alleles with special reference for microsatellite loci. Genetics, 1996,142(3):1061-1064.
[20] KIRKPATRICK S, GELATT C D, VECCHI M P. Optimization by simulated annealing. Science, 1983,220(4598):671-680.
doi: 10.1126/science.220.4598.671 pmid: 17813860
[21] LIGGES U, MACHIER M. Scatterplot3d-an R package for visualizing multivariate data (No. 2002, 22). Technical Report, 2002.
pmid: 24545891
[22] WICKHAM H. ggplot2: Elegant Graphics for Data Analysis. Springer, 2016.
[23] SU J S, ZHANG F, CHONG X R, SONG A P, GUAN Z Y, FANG W M, CHEN F D. Genome-wide association study identifies favorable SNP alleles and candidate genes for waterlogging tolerance in chrysanthemums. Horticulture Research, 2019,6(1):1-13.
[24] MCKENZIE K S, ADAYA V C, JODARI F, SAMONTE P B, OSTER J J, ADAYA C B. Rice breeding at the california rice experiment station. SABRAO Journal of Breeding & Genetics, 2015,47(1):1-13.
[25] KUJURA A, BAJAJ D, UPADHYAYA H D, DAS S, RANJAN R, SHREE T, GOWDA C L L. A genome-wide SNP scan accelerates trait-regulatory genomic loci identification in chickpea. Scientific reports, 2015,5:11166. doi: 10.1038/srep11166.
[26] FILZMOSER P, HRON K, REIMANN C. Principal component analysis for compositional data with outliers. Environmetrics: The Official Journal of the International Environmetrics Society, 2009,20(6):621-632.
[27] GODSTEIN M, UCHIDA S. A comparative evaluation of unsupervised anomaly detection algorithms for multivariate data. PLoS ONE, 2016,11(4):e0152173.
pmid: 27093601
[28] LENZ M, MüLLER F J, ZENKE M, SCHUPPERT A. Principal components analysis and the reported low intrinsic dimensionality of gene expression microarray data. Scientific Reports, 2016,6(1):1-11.
pmid: 28442746
[29] CHAWLA S, GIONIS A. K-means: A unified approach to clustering and outlier detection//Proceedings of the 2013 SIAM International Conference on Data Mining. Society for Industrial and Applied Mathematics, 2013: 189-197.
[30] BENEVENUTO J, FERRãO L F V, AMADEU R R, MUNOZ P. How can a high-quality genome assembly help plant breeders? GigaScience, 2019, 8(6): giz068.
[31] KREBS S L, HANCOCK J F. Early-acting inbreeding depression and reproductive success in the highbush blueberry, Vaccinium corymbosum L. Theoretical and Applied Genetics, 1990,79:825-832.
[32] 王哲. 植物杂交后代中基因偏分离的产生原因及其进化意义. 遗传, 2016,38(9):801-810.
doi: 10.16288/j.yczz.16-084 pmid: 27644741
WANG Z. Distorted segregation in plant hybrids and its implication for evolution. Hereditas, 2016,38(9):801-810. (in Chinese)
doi: 10.16288/j.yczz.16-084 pmid: 27644741
[1] LIN Ping, WANG KaiLiang, YAO XiaoHua, REN HuaDong. Development of DNA Molecular ID in Camellia oleifera Germplasm Based on Transcriptome-Wide SNPs [J]. Scientia Agricultura Sinica, 2023, 56(2): 217-235.
[2] LI ZhouShuai,DONG Yuan,LI Ting,FENG ZhiQian,DUAN YingXin,YANG MingXian,XU ShuTu,ZHANG XingHua,XUE JiQuan. Genome-Wide Association Analysis of Yield and Combining Ability Based on Maize Hybrid Population [J]. Scientia Agricultura Sinica, 2022, 55(9): 1695-1709.
[3] TANG HuaPing,CHEN HuangXin,LI Cong,GOU LuLu,TAN Cui,MU Yang,TANG LiWei,LAN XiuJin,WEI YuMing,MA Jian. Unconditional and Conditional QTL Analysis of Wheat Spike Length in Common Wheat Based on 55K SNP Array [J]. Scientia Agricultura Sinica, 2022, 55(8): 1492-1502.
[4] LIAO Ping,MENG Yi,WENG WenAn,HUANG Shan,ZENG YongJun,ZHANG HongCheng. Effects of Hybrid Rice on Grain Yield and Nitrogen Use Efficiency: A Meta-Analysis [J]. Scientia Agricultura Sinica, 2022, 55(8): 1546-1556.
[5] WANG YaLiang,ZHU DeFeng,CHEN RuoXia,FANG WenYing,WANG JingQing,XIANG Jing,CHEN HuiZhe,ZHANG YuPing,CHEN JiangHua. Beneficial Effects of Precision Drill Sowing with Low Seeding Rates in Machine Transplanting for Hybrid Rice to Improve Population Uniformity and Yield [J]. Scientia Agricultura Sinica, 2022, 55(4): 666-679.
[6] LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon [J]. Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.
[7] TU YunJie,JI GaiGe,ZHANG Ming,LIU YiFan,JU XiaoJun,SHAN YanJu,ZOU JianMin,LI Hua,CHEN ZhiWu,SHU JingTing. Screening of Wnt3a SNPs and Its Association Analysis with Skin Feather Follicle Density Traits in Chicken [J]. Scientia Agricultura Sinica, 2022, 55(23): 4769-4780.
[8] JU Ming, MIAO HongMei, HUANG YingYing, MA Qin, WANG HuiLi, WANG CuiYing, DUAN YingHui, HAN XiuHua, ZHANG HaiYang. Analysis of Cross Compatibility Variation Among Diverse Sesamum Species and Biological Characteristics of the Interspecific Hybrid Progenies [J]. Scientia Agricultura Sinica, 2022, 55(20): 3897-3909.
[9] HU Xin, ZHANG ZhiLiang, ZHANG Fei, DENG Bo, FANG WeiMin. Comprehensive Evaluation and Selection of Hybrid Offsprings of Large-Flowered Tea Chrysanthemum [J]. Scientia Agricultura Sinica, 2022, 55(20): 4036-4051.
[10] XiaoChuan LI,ChaoHai WANG,Ping ZHOU,Wei MA,Rui WU,ZhiHao SONG,Yan MEI. Deciphering of the Genetic Diversity After Field Late Blight Resistance Evaluation of Potato Breeds [J]. Scientia Agricultura Sinica, 2022, 55(18): 3484-3500.
[11] CHEN XueSen,WANG Nan,ZHANG ZongYing,MAO ZhiQuan,YIN ChengMiao. Understanding and Thinking About Some Problems of Fruit Tree Germplasm Resources and Genetic Breeding [J]. Scientia Agricultura Sinica, 2022, 55(17): 3395-3410.
[12] LI Ting,DONG Yuan,ZHANG Jun,FENG ZhiQian,WANG YaPeng,HAO YinChuan,ZHANG XingHua,XUE JiQuan,XU ShuTu. Genome-Wide Association Study of Ear Related Traits in Maize Hybrids [J]. Scientia Agricultura Sinica, 2022, 55(13): 2485-2499.
[13] XU ZhiYing,WANG BaiCui,MA XiaoLan,JIA ZiMiao,YE XingGuo,LIN ZhiShan,HU HanQiao. Polymorphism Analysis Among Chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and Wheat 6A and 6D Based on Wheat SNP Chip [J]. Scientia Agricultura Sinica, 2021, 54(8): 1579-1589.
[14] FAN XiaoJing, YU WenTao, CAI ChunPing, LIN Yi, WANG ZeHan, FANG WanPing, ZHANG JianMing, YE NaiXing. Construction of Molecular ID for Tea Cultivars by Using of Single- nucleotide Polymorphism (SNP) Markers [J]. Scientia Agricultura Sinica, 2021, 54(8): 1751-1760.
[15] LÜ TengFei,SHEN Jie,MA Peng,DAI Zou,YANG ZhiYuan,XU Hui,ZHENG ChuanGang,MA Jun. Effects of Combined Application of Slow Release Nitrogen Fertilizer and Urea on the Nitrogen Utilization Characteristics in Machine- Transplanted Hybrid Rice [J]. Scientia Agricultura Sinica, 2021, 54(7): 1410-1423.
Full text



No Suggested Reading articles found!