中国古老月季‘月月粉’等位基因不平衡分析与分子标记开发利用

doi:10.16420/j.issn.0513-353x.2023-0970

摘要/Abstract

摘要：

利用已报道的中国古老月季‘月月粉’（Rosa chinensis‘Old Blush’）的两套单倍型基因组——RcOB_hap1与RcOB_hap2，对其基因组进行全面的比较分析。同时利用生物信息方法鉴定‘月月粉’中的不平衡等位基因，开发相应的分子标记。结果表明，两套单倍型基因组共线性较好，但RcOB_hap2组装的完整性明显优于RcOB_hap1。‘月月粉’杂合度为3.48%，含有大量的单碱基变异、小片段插入或缺失以及大片段染色体变异。全基因组范围可能存在1 157对蛋白功能差异的等位基因，720对表达显著差异的等位基因。GO注释以及KEGG富集结果表明，不平衡等位基因主要参与了代谢合成调控、生长发育以及环境适应等过程。围绕‘月月粉’的花器官发育、挥发物质合成与抗病免疫反应，分别挑选了不平衡等位基因AP2、MBY4、DSC1进行分子标记开发。在183份月季材料中的分子标记结果显示，蛋白功能不完整的Hap2型AP2在现代栽培月季中被强选择，使月季具有重瓣性状；表达量低的Hap2型MBY4被多数现代月季选择，可能导致栽培月季品种酚类物质合成减少；针对DSC1，现代月季品种广泛携带了表达量低且蛋白功能受影响的Hap1型，可能使现代月季抗病性降低。

关键词: 月季, 等位基因不平衡, 等位基因蛋白功能差异, 等位基因差异表达, 分子标记

Abstract:

In this study，a comprehensive comparative analysis of the two haplotype genomes of Rosa chinensis‘Old Blush’were conducted using RcOB_hap1 and RcOB_hap2. The allele-specific imbalance analysis was conducted by bioinformatics methods，and the corresponding molecular markers were developed，providing a basis for the breeding of the cultivated rose. The results showed that two haplotype genomes had a good collinearity relationship，but the assembly integrity of RcOB_hap2 is better than RcOB_hap1. The heterozygosity of‘Old Blush’rose was 3.48%，containing a large number of single nucleotide polymorphisms，fragment insertions or deletions and chromosomal variations. A total of 1 157 allele-specific protein function genes and 720 allele-specific expression genes were identified in the whole genome. GO annotation and KEGG enrichment analyses of all imbalanced alleles showed that they were mainly involved in the processes of metabolism and synthesis，plant growth and development，and environmental adaptation. Imbalanced alleles AP2，MBY4 and DSC1 were selected for developing molecular markers for floral organ development，aroma synthesis and immune response，respectively. Based on the results of the molecular markers within 183 materials found that the Hap2 AP2 allele had incomplete protein function and was strongly selected in cultivated roses with double flower；For the MBY4，its Hap2 allele showed lower expression and was adopted by most cultivated roses，that might lead cultivated rose emitting less benzenoid volatile；DSC1’s Hap1 allele had lower expression and possible defect protein function，but it was selected in cultivated varieties，that might result in a reduction of the disease resistance in cultivated roses.

Key words: rose, allele-specific imbalance, allelic protein function differences, allele-specific expression, molecular marker

阎旭, 李月, 傅小鹏, 宁国贵, 梁梅. 中国古老月季‘月月粉’等位基因不平衡分析与分子标记开发利用[J]. 园艺学报, 2025, 52(2): 365-379.

YAN Xu, LI Yue, FU Xiaopeng, NING Guogui, LIANG Mei. Allele-specific Imbalance Analysis and Related Molecular Marker Development in Rosa chinensis‘Old Blush’[J]. Acta Horticulturae Sinica, 2025, 52(2): 365-379.

图/表 9

表1 分子标记引物序列

Table 1 Primers sequence of molecular marker

引物 Primer	正引物序列（5′-3′） Forward primer	反引物序列（5′-3′） Reverse primer	片段大小/bp Fragment size	基因ID Gene ID
AP2	TCATCTATGAAAGAATTTGGAG	GAAGGAATGCGACTGTTAGG	Hap1：962 Hap2：807	Rc3g0243000 Rc3g0468481
MYB4	TAGGACTCAAGT TACCACAA	TCGTGAGGTACATTATCATCTA	Hap1：240 Hap2：430	Rc7g0018900 Rc7g0178681
DSC1	CGTGAGGTACAT TATCATCTA	AAGATGACTGGACCAGAGCAA	Hap1：599 Hap2：1051	Rc3g0384800 Rc3g0451671

图1 ‘月月粉’不同单倍型基因组比较与杂合度分析 A：RcOB_hap1和RcOB_hap2大片段比对结果；B：两个单倍型基因组特有染色体区域上的基因与转座子占比；C：RcOB_hap1和RcOB_hap2共线性基因点阵图；D：‘月月粉’重测序数据的读段频率分布图（K = 17）

Fig. 1 Comparative analysis of the RcOB_hap1 and RcOB_hap2 genomes in Rosa chinensis‘Old Blush’ A：Large segment alignment of RcOB_hap1 and RcOB_hap2；B：Distribution percentage of the gene and transposon on the specific chromosome regions for two genomes；C：Dot-plots of the whole-genomic gene alignment between RcOB_hap1 and RcOB_hap2；D：Distribution survey of the K-mer for‘Old Blush’re-sequence genome（K = 17）

图2 ‘月月粉’单倍型基因组RcOB_hap1和RcOB_hap2之间DNA序列变异 SNP：单核苷酸多态性；InDel：小片段插入/缺失；FeS：移码突变；StL：起始密码子丢失突变；SSA：剪接受体位点突变；SSD：剪接供体位点突变；SpG：终止密码子获得/提前终止突变；SpL：终止密码子丢失突变

Fig. 2 The DNA sequence variation between RcOB_hap1 and RcOB_hap2 genomes in Rosa chinensis‘Old Blush’ SNP：Single nucleotide polymorphism；InDel：Insertion or deletion；FeS：Frame shift；SpL：Stop codon lost；SSA：Splice site acceptor；SSD：Splice site donor；SpG：Stop codon gained or premature stop；StL：Start codon lost

表3 示例基因在花瓣组织中的ASE分析结果

Table 3 ASE analysis result of gene examples in petal

基因 Gene	SNP编号 SNP ID	Hap1型读长数 Hap1-type read number	Hap2型读长数 Hap2-type read number	变化倍数的log₂值 log₂fold change	ASE基因（是/否） ASE gene (yes/no)	P值 P-value	基因注释 Annotation
Rc1g0350201	Chr01_43344427	0	131	-10.17	是 Yes	8.28e^-108	未知蛋白 Unknown protein
	Chr01_43344491	0	149
	Chr01_43344521	0	136
	Chr01_43344550	0	158
	小计Subtotal	0	574
Rc2g0129651	Chr02_45318393	31	53	-0.83	否 No	1.15e^-4	未知蛋白 Unknown protein
	Chr02_45318518	30	53
	Chr02_45318530	30	56
	Chr02_45318549	30	53
	小计Subtotal	121	215
Rc3g0468121	Chr03_14202662	86	13	2.83	是 Yes	1.70e^-47	磷酸酶 Protein phosphatase
	Chr03_14202709	81	13
	Chr03_14202963	69	10
	Chr03_14203072	75	8
	Chr03_14204073	84	11
	小计Subtotal	395	55
Rc5g0077891	Chr05_83740197	173	133	0.35	否 No	2.74e^-4	未知蛋白 Unknown protein
Rc5g0077891	Chr05_83740290	132	107	0.35	否 No	2.74e^-4	未知蛋白 Unknown protein
	小计Subtotal	305	240

图3 ‘月月粉’不同组织中的ASE基因数（A）、七维韦恩图（B）和三维韦恩图（C）

Fig. 3 ASE genes in different tissues of Rosa chinensis‘Old Blush’（A），seven-dimensional Venn diagram（B）and three-dimensional Venn diagram（C）

图4 Rc3g0480151、Rc7g0179391与Rc4g0437551的基因对应的总读长数

Fig. 4 The gene read count of the Rc3g0480151，Rc7g0179391 and Rc4g0437551

图5 4 024个ASI基因GO功能分类（A）与KEGG富集分析（B）

Fig. 5 GO function classification（A）and KEGG enrichment analysis（B）of a total of 4 024 ASI genes

表4 AP2、MYB4与DSC1的两个等位拷贝比较与在183份材料中的频率

Table 4 Comparison of two alleles of AP2，MYB4 and DSC1 and their frequency in 183 materials

基因 Gene	等位基因比较 Allele comparison		基因型频率 Frequency of different genotypes
基因 Gene	基因表达（有/无差异） Gene expression（DIF./No DIF.）	蛋白功能（有/无差异） Protein function（DIF./No DIF.）	36份野生种 36 wild species	147份栽培种 147 cultivated varieties
AP2	有差异（茎和刺：Hap1 < Hap2） DIF.（Hap1 < Hap2 in stem & prickle）	有差异（Hap2型：提前终止） DIF.（Hap2-type：Premature）	Hap2: 8 Hap1&2: 6 Hap2&3: 6 Hap3: 9 No band: 7	Hap2: 45 Hap1&2: 18 Hap2&3: 70 Hap3: 5 Hap1&3: 6 No band: 3
MYB4	有差异（茎和雌蕊：Hap1 < Hap2） DIF.（Hap1 < Hap2 in stem & pistil）	无差异 No DIF.	Hap1: 17 Hap2: 1 Hap1&2: 11 No band: 7	Hap1: 94 Hap2: 5 Hap1&2: 47 No band: 1
DSC1	有差异（所有组织：Hap1 < Hap2） DIF.（Hap1 < Hap2 in all tissues）	有差异（Hap1型：移码）（Hap1-type：Frame shift）	Hap1: 3 Hap2: 3 Hap1&2: 6 No band: 24	Hap1: 83 Hap2: 5 Hap1&2: 30 No band: 29

图6 分子标记在月季及近缘野生种（泳道1 ~ 24）与栽培月季（泳道25 ~ 48）中的扩增图谱泳道1为‘月月粉’月季；泳道46为单瓣‘甜美人’月季

Fig. 6 Amplification results of molecular markers among roses and related wild species（line 1 to 24） and cultivated varieties（line 25 to 48） Line 1 is Rosa chinensis‘Old Blush’；Line 46 is Rosa hybrida var.‘Sweet Pretty’with simple flower

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