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 E-mail:liuyouchun911@126.com;stevecliu@hotmail.com;litianzhong1535@163.com

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Abstract:

【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"

样品名
Sample ID		 总测序条数
Total reads		 总碱基数量
Total bases		 高质量reads百分比
Q30 percentage (%)		 GC含量百分比
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"

越橘杂交
群体
Population		 SNP标记数量（MAF>0.05）
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"

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