189份桃种质肉质性状形成相关位点基因型鉴定及组合分析

doi:10.3864/j.issn.0578-1752.2023.12.011

摘要/Abstract

摘要：

【目的】通过分子标记与生物信息学手段，鉴定189份桃种质与肉质形成相关的F-M基因座、YUC11启动子区转座子插入、NAC72编码区9 bp插入的基因型及3个位点组合情况，为深入研究桃果实肉质形成机制及育种亲本选配提供理论基础。【方法】通过PCR扩增、竞争性等位基因特异PCR（kompetitive allele specific PCR，KASP）、高分辨率溶解曲线（high resolution melting analysis，HRM）技术，对F-M基因座倍型、YUC11转座子插入、NAC72 9 bp插入进行检测；同时结合重测序数据利用生物信息学方法进一步验证，从而准确鉴定189份桃种质肉质性状形成相关位点基因型及组合情况。【结果】F-M基因座编码多聚半乳糖醛酸酶（endo-PG）的PGM与PGF是调控桃果实肉质形成的两个重要基因。189份桃种质中，PGM与PGF分别有159份（84%）、99份（52%）。F-M基因座共发现H0、H1、H2、H3 4种单倍型，占比分别为33%、39%、4%、24%。在F-M基因座，不溶质粘核以H3H3、H2H3组合为主，溶质粘核以H0H0、H0H1组合为主，溶质离核以H1H1、H0H1组合为主。发现18份含有硬质基因型的种质，在YUC11启动子区含有转座子纯合插入。HRM结果表明，NAC72编码区9 bp插入纯合（早熟）有45份种质、插入杂合（中熟）有71份种质、无插入（晚熟）有73份种质，中熟与晚熟种质占比77%；其中6份种质基因型与表型差别较大。不溶质粘核、溶质粘核、溶质离核3种常见肉质类型，在3个位点最多的基因型组合分别是mmffHdHdI、MMffHdHdI、MMFFHdHdL。【结论】通过分子试验与生物信息学相结合进一步证实了F-M基因座存在4种单倍型；发现18份含有硬质基因型的种质；开发了一种用于鉴定桃成熟期的分子标记；鉴定出不同种质在肉质相关位点的基因型组合形式。

关键词: 桃, 肉质, 基因型鉴定, 基因型组合

Abstract:

【Objective】 The molecular markers and bioinformatics were used to identify the genotypes and combinations of the F-M locus, transposon insertion in YUC11 promoter region, and 9 bp insert in NAC72 coding region by 189 peach germplasms, in order to provide the theoretical foundation for the mechanism of peach flesh formation and the selection of breeding parents.【Method】PCR amplification, KASP, and HRM were used to identify genotypes. Results of loci associated with the formation of flesh trait were further validated using bioinformatics by resequencing data in 189 peach germplasms.【Result】Two genes encoding endopolygalacturonase in F-M locus, designated PGM and PGF, are associated with peach texture. Through primer amplification, 159 (84%) PGM and 99 (52%) PGF were detected in 189 peach germplasms. Four haplotypes (H0, H1, H2, and H3) were found in F-M locus, while H0 and H1 were the major genotypes. The haplotype combination of non-melting flesh, melting flesh, free-stone melting flesh were H3H3 and H2H3, H0H0 and H0H1, H1H1 and H0H1, respectively. In addition, it was found that 18 germplasms contain homozygous transposon insertion. HRM results demonstrated that 45 germplasms were homozygous insertion (early ripening), 71 germplasms were heterozygous insertion (middle ripening), and 73 germplasms were no insertion (late ripening). But six germplasm genotypes were inconsistent with the phenotypes. The most frequent genotypic combinations at the three loci of non-melting flesh, melting flesh, and free-stone melting flesh were mmffHdHdI, MMffHdHdI, MMFFHdHdL, respectively.【Conclusion】The existence of four haplotypes at the F-M locus further was confirmed, 18 germplasms containing stony hard genotype were identified, a molecular marker for the identification of maturity date was developed, and genotype combinations of different germplasms at flesh-related loci were identified.

Key words: peach, flesh texture, genotype identification, genotype combinations

王朝晖, 李勇, 曹珂, 朱更瑞, 方伟超, 陈昌文, 王新卫, 吴金龙, 王力荣. 189份桃种质肉质性状形成相关位点基因型鉴定及组合分析[J]. 中国农业科学, 2023, 56(12): 2367-2379.

WANG ZhaoHui, LI Yong, CAO Ke, ZHU GengRui, FANG WeiChao, CHEN ChangWen, WANG XinWei, WU JinLong, WANG LiRong. Genotype Identification and Combination Analysis of Loci Related to the Peach Flesh Texture Trait via 189 Peach Accessions[J]. Scientia Agricultura Sinica, 2023, 56(12): 2367-2379.

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名称 Code	引物序列 Primer sequence	退火温度 Annealing temperature (℃)	片段长度 Size (bp)
PGM-F	ACTAACCCAACCAATTACTCAAACC	56	3066
PGM-R	TGTCCTTAGTACTGGAGGGCAAAC
PGF-F	AGGAGAAATTGGAGACGCCG	58	3353
PGF-R	CGTCTTCCGGACATAATCTTACA
YUC-F	TAAAGCCGCCCAAAAATAAA	58	600
YUC-R1	TGGGAAGGAAGAAAATAGTCACA
YUC-R2	ATTTTCAACTTTCCCGAGCA	58	448/3015
PGH0-F	TACGACTACAAGCCAAGTCGG	56	591
PGH0-R	ACTCCAGTTGCGCTGAAAGA
PGH2-F	CATATTGAAGAACCAGCCA	56	1364
PGH2-R	TGGTATATTGCATCGACGTG
PGH3-F	AAAGATAGGAAGGGGGAAGG	56	524
PGH3-R	GTTTGCACTGGGGGTTTTAGA

表型 Phenotype				基因型出现个数 Number of genotype occurrences
溶质粘核		溶质离核	不溶质粘核	1	2	3	4
MMffHdHdI	MMFFHdHdL	MMFFHdHdL	mmffHdHdL	12	2	22	11
MMFFHdHdE	MmffHdhdI	MMFfHdhdI	mmffHdHdE	9	1	8	5
MMFfHdHdI	MmffhdhdE	MMFfHdHI	mmffHdHdI	9	1	7	5
MMffHdHdL	MMFFHdhdE	MMFfHdHdL	MmffHdHdE	8	1	5	3
MMFfHdHdE	MMFFHdhdL	MmFfHdHdL	MMffHdHdI	4	1	5	3
MmFfHdhdE	MMFfHdhdI	MMFFhdhdI	MMffHdHdL	4	1	4	2
MmffHdHdL	MmFfHdHdI	MMffHdHdL	mmffhdhdE	4	1	3	2
MmffHdHdL	MmFfHdHdL	MmFfHdhdE	MmffHdHdI	4	1	2	1
MMffhdhdI	MMffHdHdE	MmFfHdhdI	MmffHdHdL	3	1	2	1
MmffHdHdI	MMffHdhdI	MMFFHdHdI	MmffHdhdI	3	1	2	1
MmFfHdHdE	MMffHdhdI	MMFfhdhdE	MMffHdhdI	3	1	1	1
MMFFHdHdI	MMffhdhdL	MmFfHdHdE	MmFfHdHdI	2	1	1	1
MMffHdhdL		MMFFhdhdL	MMFfHdHdI	2		1	1
MMffhdhdE		MMFFhdhdE		2		1
MmffHdHdE		MmFfhdhdE		2		1
mmffHdHdE		MMFfHdhdE		2		1