控制桃粘/离核PG基因的BAC克隆筛选与序列分析

doi:10.3864/j.issn.0578-1752.2021.20.013

摘要/Abstract

摘要：

【目的】控制桃果实粘/离核性状的多聚半乳糖醛酸酶（polygalacturonase,PG）基因存在串联重复和大片段的缺失。本研究对粘核桃所处的F-M基因座序列特征进行分析,为开发相关分子标记提供依据。【方法】本研究利用构建的粘核桃单株‘87-7-1’基因组的细菌人工染色体（bacterial artificial chromosome,BAC）文库,通过PCR筛选出含F-M基因座的阳性克隆,利用单分子纳米孔技术进行全长测序,并对BAC克隆中的插入片段进行基因注释、序列比对和生物信息学分析。【结果】利用已有桃品种的基因组重测序数据设计PCR引物,以BAC文库为模板,扩增F-M基因座上/下游稳定共有的序列,获得了扩增产物上/下游条带均为阳性的目标单克隆46-B-10。全长测序结果表明,BAC克隆的插入片段全长为111 612 bp,GC含量为37.03%。利用基因同源共线性方法对Prunus_persica_v2.0桃参考基因组和BAC克隆之间的同源信息进行比对分析,确定了同源区域。而与已知的桃参考基因组（测序品种为‘Lovell’,离核）序列进行比对,发现只有5个基因（Prupe.4G261700、Prupe.4G261800、Prupe.4G261900、Prupe.4G262000、Prupe.4G262500）序列能比对到该单克隆全长的区域,而‘87-7-1’在相应位置缺失了4个基因共34 kb,其中包括控制桃粘/离核的EndoPGF（Prupe.4G262200）。【结论】与桃参考基因组测序品种‘Lovell’相比,粘核桃单株‘87-7-1’的F-M基因座缺失了EndoPGF,只有EndoPGM,本研究明确了粘核桃F-M基因座的结构变异情况,为粘/离核性状分子标记的开发奠定了基础。

关键词: 桃, 粘/离核, PG基因, BAC文库

Abstract:

【Objective】Tandem repeats and large segment deletions of Polygalacturonase (PG) gene was related to the clingstone/freestone characteristic of peach (Prunus persica) fruit. In this study, the sequence character of F-M locus of clingstone peach was analyzed to provide a basis for the development of related molecular markers. 【Method】With a Bacterial Artificial Chromosome library of clingstone peach 87-7-1 constructed, the positive BAC clone contained F-M locus was screened from the BAC library by PCR analysis. The screened BAC clone was sequenced by single-molecule nanopore technology. Gene annotation and sequence alignment were performed by bioinformatics. 【Result】PCR primers were designed based on re-sequencing data of existing peach varieties, and PCR reactions were performed with all the BAC library clones. Amplification products of the sequences in upstream/downstream of F-M loci were corrected, and then the target clone 46-B-10 was obtained. Full-length sequencing showed that the fragment with the length of 111612 bp and GC content of 37.03% was inserted between upstream and downstream primers. The homologous region of 46-B-10 was determined by sequence alignment with the reference genome Prunus_persica_v2.0. Five genes (Prupe.4G261700, Prupe.4G261800, Prupe.4G261900, Prupe.4G262000, and Prupe.4G262500) in F-M locus were found in the BAC clone 46-B-10. In comparison, 87-7-1 with 34 kb sequence including four genes was discarded, and one of them was EndoPGF, which controlled peach freestone and was reported previously. 【Conclusion】Compared with the reference genome freestone variety ‘Lovell’, there was only EndoPGM (Prupe.4G261900) in F-M locus of clingstone peach individual 87-7-1, while EndoPGF (Prupe.4G262200) was discarded. In this study, the structural variation of F-M locus in clingstone peach was determined, which has laid an important foundation for the development of molecular markers for clingstone/freestone trait in peach.

Key words: peach, clingstone/freestone, PG gene, BAC library

孟君仁,牛良,邓丽,潘磊,鲁振华,崔国朝,王志强,曾文芳. 控制桃粘/离核PG基因的BAC克隆筛选与序列分析[J]. 中国农业科学, 2021, 54(20): 4396-4404.

MENG JunRen,NIU Liang,DENG Li,PAN Lei,LU ZhenHua,CUI GuoChao,WANG ZhiQiang,ZENG WenFang. Screening and Sequence Analysis of BAC Clone Contained PG Gene Controlling Clingstone/Freestone Characteristic of Peach[J]. Scientia Agricultura Sinica, 2021, 54(20): 4396-4404.

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位置 Location	引物名称 Primer code	引物序列 Primer sequence	片段大小 Size ( bp)	扩增序列 Amplification sequence
F-M基因座上游 Upstream of F-M	8771-PG F1	CACTAATGCTATGCGATTGTGA	178	CACTAATGCTATGCGATTGTGATCTTAACATGTAAAAATTTGCGATTAATACCTATTACAGAGAAGACTCTGCTAGATTATTGATAGATCCCTCCTTGTTTGTCCAAGTAGGGATTAAGGATAGTTTAAGTAGTGGGCTCACTTCTCTCGAGTTTATCCAAACGGAAATCTGAGAGTG
F-M基因座上游 Upstream of F-M	8771-PG R1	CACTCTCAGATTTCCGTTTGG	178
F-M基因座下游 Downstream of F-M	8771-PG F2	TCCAGCTTAAGGCATCCACT	207	TCCAGCTTAAGGCATCCACTTGTTTCGGTTATGCAAGTGGATTAAGAATTGGATTGATTGTTGATGATGATATCACAACAAAAAAATTAGGCATGTACCATTATTCCAAGACTTCAGTTGGCCAAAGTCTCTTCCATGTGGAAATCTCTTCCTTGGTTCTAAGCTTCCTGATGCCCCCGTAAAACCCATCTGCCTCATTTCCCTCCT
F-M基因座下游 Downstream of F-M	8771-PG R2	AGGAGGGAAATGAGGCAGAT	207

基因ID Gene ID	参考基因组基因序列长度 Gene length of reference sequence (bp)	BAC克隆基因序列长度 Gene length of BAC clone (bp)	基因序列相似度 Similarity of gene sequences (%)	编码氨基酸序列相似度 Similarity of coding amino acid sequences (%)
Prupe.4G261700	1519	1545	94.24	99.49
Prupe.4G261800	1771	1751	98.52	98.98
Prupe.4G261900	3262	3225	96.36	99.75
Prupe.4G262000	1516	1552	83.30	79.22
Prupe.4G262100	2380	/	/	/
Prupe.4G262200	2660	/	/	/
Prupe.4G262300	2544	/	/	/
Prupe.4G262400	3648	/	/	/
Prupe.4G262500	4553	4547	99.08	99.75