‘鲁丽’×‘红1#’苹果杂交群体全基因组KASP标记开发及验证

doi:10.3864/j.issn.0578-1752.2023.05.010

摘要/Abstract

摘要：

【目的】利用‘鲁丽’×‘红1#’亲本及杂交后代，开发苹果叶片性状竞争性等位基因特异PCR（kompetitive allele-specific PCR，KASP）标记，为苹果光能高效利用育种提供参考。【方法】以‘鲁丽’和I型红肉苹果‘红1#’及其134个杂交后代的幼嫩叶片为材料，调查叶片性状表型（包括叶片长度、宽度、叶绿素含量、花青苷含量、光合参数和叶绿素荧光参数等）数据。采用Illumina HiSeq 2500测序平台进行全基因组重测序，通过短序列比对软件将测序序列（reads）回贴到苹果参考基因组，利用GATK软件工具包检测单核苷酸多态性（single nucleotide polymorphisms, SNP）标记。【结果】‘鲁丽’和‘红1#’及其134个杂交后代重测序得到有效reads数分别为164 776 660、149 482 876和3 927 370 200。与苹果参考基因组比对率分别为98.77%、98.89%和97.79%。共检测到6 445 766个SNP。经过滤后获得94 208个特异性强，准确性高的SNP位点。对每个SNP进行KASP引物设计，最终开发了5 802个苹果全基因KASP标记。这些KASP标记的多态性信息含量（polymorphism information content，PIC）平均值为0.31，PIC大于0.35的占30.13%。Simpson遗传多样性指数在0.01—0.67，平均每个位点遗传多样性指数为0.53。观测杂合度平均值为0.32。KASP标记所在基因的KEGG功能聚类分析显示，“叶酸介导的一碳代谢途径”是差异基因富集最显著的途径。通过对‘鲁丽’×‘红1#’杂交群体24个叶片相关性状进行表型调查，结果显示亲本‘鲁丽’和‘红1#’在叶片净光合速率、气孔导度（Gs）等性状的表型值均存在显著差异。杂交后代表型变异幅度较大，各性状分离广泛。随后对杂交群体叶片性状表型值与KASP标记基因型进行相关性分析，结合KASP标记注释，筛选得到21个与叶片大小、Gs和色素含量性状显著相关的KASP标记。【结论】利用‘鲁丽’和‘红1#’及其134个杂交后代全基因组重测序数据开发了5 802个苹果全基因组KASP标记，获得21个与叶片性状显著相关的KASP标记，为开展标记辅助苹果光能高效利用育种提供了理论参考。

关键词: 苹果, 全基因组重测序, 叶片性状, KASP标记

Abstract:

【Objective】 A series of kompetitive allele-specific PCR (KASP) markers for traits related to apple leaves were developed to provide a reference to effectively utilize light in apple breeding using a Luli ×Red No. 1 hybrid population as materials. 【Method】This study investigated the leaf trait phenotypic data (including leaf length, width, contents of chlorophyll and anthocyanin, photosynthetic rate and chlorophyll fluorescence parameters) of Luli, a type I red-fleshed apple, Red No. 1, and 134 hybrids. Whole-genome resequencing was conducted on an Illumina HiSeq 2500 platform. The clean sequencing reads were mapped to the apple reference genome using BWA software. GATK software was used to identify single nucleotide polymorphisms (SNPs). 【Result】A total of 164 776 660, 149 482 876, and 3 927 370 200 clean reads were obtained by whole-genome resequencing from Luli, Red No. 1 and 134 hybrid individuals, respectively. The data were aligned to the reference genome sequences with mapping rates of 98.77%, 98.89%, and 97.79%, respectively. A total of 6 445 766 SNPs were identified. The SNPs were then filtered, and 94 208 accurate and high-quality SNPs were selected for subsequent KASP primer design. Finally, 5 802 KASP markers were developed based on a high-throughput whole-genome resequencing approach. The average polymorphism information content (PIC) of these KASP markers was 0.31, and there were 30.13% PICs>0.35. The Simpson genetic diversity index ranged from 0.01 to 0.67, with an average value of 0.53. The mean observed heterozygosity was 0.32. A Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the genes for KASP marker revealed that “one carbon pool by folate” was the most significant pathway. An analysis of the phenotypic data of leaf-related traits in both the parents and individuals from the Luli ×Red No. 1 cross showed that there were significant differences in the phenotypic values of leaf net photosynthetic rate, stomatal conductance (Gs) and other traits between the parents Luli and Red No. 1. The F1 generation exhibited a wide phenotypic variation, and the most traits segregated extensively. Α detailed study of the phenotypic data of leaf traits of the hybrid population with the genotype and annotations of the KASP markers finally resulted in the development of 21 KASP markers, which was correlated with leaf-related traits, including chlorophyll content, Gs and leaf length and width.【Conclusion】Use of the whole-genome resequencing data of Luli, Red No. 1 and 134 hybrid individuals resulted in the development of 5 802 KASP. A total of 21 of these KASP markers significantly correlated with leaf traits, which provided a theoretical reference for the high utilization of light energy in apple breeding.

Key words: apple, whole-genome resequencing, leaf-related traits, KASP marker

郑文燕, 常源升, 何平, 何晓文, 王森, 高文胜, 李林光, 王海波. ‘鲁丽’×‘红1#’苹果杂交群体全基因组KASP标记开发及验证[J]. 中国农业科学, 2023, 56(5): 935-950.

ZHENG WenYan, CHANG YuanSheng, HE Ping, HE XiaoWen, WANG Sen, GAO WenSheng, LI LinGuang, WANG HaiBo. Development and Validation of KASP Markers Based on a Whole- Genome Resequencing Approach in a Hybrid Population of Luli × Red No. 1[J]. Scientia Agricultura Sinica, 2023, 56(5): 935-950.

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样品 Sample	Reads数 Clean reads	碱基数 Bases (bp)	Q30百分比Q30 (%)	GC含量 GC (%)
‘鲁丽’ Luli	164776660	24684144730	93.59	38.64
‘红1#’ Red No. 1	149482876	22391415716	94.24	38.72
杂交后代 Offsprings	3927370200	588153323320	92.87	38.61
总计 Total	4241629736	635228883766	92.88	38.61

染色体Chromosome	染色体长度 Length of chromosome (Mb)	SNP数 SNP number	SNP密度（个/Mb） SNP density (No./Mb)	过滤后SNP数 SNP number after filtration	过滤后SNP密度（个/Mb） SNP density after filtration (No./Mb)
Chr01	32.625	278600	8539	3216	99
Chr02	37.578	404259	10758	5965	159
Chr03	37.524	372841	9936	5747	153
Chr04	32.302	331145	10252	5380	167
Chr05	47.952	486261	10141	6655	139
Chr06	37.137	291836	7858	3291	89
Chr07	36.691	352192	9599	5113	139
Chr08	31.609	302356	9566	4445	141
Chr09	37.605	373414	9930	5601	149
Chr10	41.762	442804	10603	6965	167
Chr11	43.060	406159	9432	5570	129
Chr12	33.050	366784	11098	6256	189
Chr13	44.340	417525	9416	6019	136
Chr14	32.513	329508	10135	4574	141
Chr15	54.945	525072	9556	7431	135
Chr16	41.389	406794	9829	5892	142
Chr17	34.749	358216	10309	6088	175

性状 Traits	亲本 Parents		杂交后代 Hybrids
性状 Traits	鲁丽 Luli	红1# Red No. 1	范围 Range	平均值±标准差 Mean±SD	变异系数 CV (%)	正态性检验 Shapiro-Wilk test
Ci	206a	212a	105-245	184±30	16.47	Normal
Pn	20.42a	14.68b	8.20-24.50	17.40±3.37	19.22	Non
Gs	178a	145b	77-226	147±36	24.64	Normal
Tr	4.68a	3.95b	2.00-5.10	3.60±0.70	18.47	Normal
VPD	2.96a	3.06a	2.00-3.30	2.70±0.20	7.57	Non
WUE	4.38a	3.72b	2.90-8.40	4.90±1.20	24.17	Non
Vc	0.10a	0.07a	0.04-0.19	0.10±0.03	33.90	Normal
Chla	0.27a	0.20b	0.11-0.27	0.18±0.03	17.83	Normal
Chlb	0.11a	0.06b	0.03-0.11	0.06±0.01	22.35	Normal
Chl(a+b)	0.38a	0.26b	0.14-0.38	0.24±0.05	18.74	Normal
Anthocyanin	0.09b	0.31a	0.00-0.55	0.20±0.13	64.17	Non
Area	31.25a	29.49a	11.20-44.13	29.81±6.03	20.10	Normal
Length	11.95a	11.15b	6.25-14.94	11.12±1.41	12.57	Normal
Width	4.61a	4.85a	3.01-5.79	4.76±0.49	10.17	Non
Perimeter	30.99a	27.87a	17.44-39.09	30.23±3.47	11.43	Normal
Fv/Fm	0.81a	0.82a	0.30-0.84	0.73±0.09	11.77	Non
ABS/RC	1.09b	1.17a	1.05-4.75	1.81±0.67	36.78	Non
DIo/RC	0.20a	0.22a	0.17-3.19	0.58±0.49	84.19	Non
TRo/RC	0.89b	0.96a	0.88-2.08	1.25±0.24	18.78	Normal
ETo/RC	0.55a	0.51b	0.44-0.94	0.63±0.08	12.92	Non
ABS/CSo	6146a	6202a	5921-10932	7746±1090	14.01	Non
DIo/CSo	1143a	1137a	973-6117	2212±1051	7.35	Non
TRo/CSo	5003b	5065a	4251-6912	5575±343	6.12	Non
ETo/CSo	3097a	2700b	1669-3586	2929±347	11.81	Normal

KASP名称 KASP name	变异 Variation type	性状 Traits	亲本基因型 Parental genotype		杂交后代基因型 Hybrid genotype		卡方检验 χ ²
KASP名称 KASP name	变异 Variation type	性状 Traits	鲁丽 Luli	红1# Red No. 1	20个高表型极端个体 20 extremely high-phynotype individuals	20个低表型极端个体 20 extremely low-phynotype individuals	卡方检验 χ ²
KASP3343	C/A	Anthocyanin	CA	CA	CC:CA:AA=12:2:4	CC:CA:AA=3:2:13	10.17**
KASP5095	A/C	Chlb	AC	AA	AA:AC=8:6	AA:AC=15:1	5.59*
KASP5105	G/A	Chla	GA	GG	GA:GG=8:6	GA:GG=15:1	5.59*
KASP3508	C/T	Chlb	CT	CT	CC:CT:TT=3:9:2	CC:CT:TT=10:1:6	11.99**
KASP2936	T/C	Gs	TT	TC	TT:TC=8:9	TT:TC=14:3	4.64*
KASP3022	A/G	Gs	AG	GG	AG:GG=0:15	AG:GG=6:7	8.81**
KASP2608	A/T	Length	TT	AT	AT:TT=4:11	AT:TT=9:4	5.07*
KASP2718	T/A	Length	TT	TA	TT:TA=7:11	TT:TA=11:4	3.92*
KASP2744	G/A	Length	GG	GA	GG:GA=7:11	GG:GA=11:4	3.92*
KASP4678	A/T	Length	TT	AT	AT:TT=6:7	AT:TT=1:13	5.34*
KASP3350	A/G	Width	AG	AA	AA:AG=10:2	AA:AG=5:7	4.44*
KASP4591	G/T	Width	GT	GG	GG:GT=14:2	GG:GT=7:7	5.00*
KASP1587	T/C	Width	TC	TC	TT:TC:CC=9:6:3	TT:TC:CC=6:2:10	6.37*
KASP1588	C/T	Width	CT	CT	CC:CT:TT=12:2:5	CC:CT:TT=4:6:7	6.24*
KASP1584	G/T	ETo/CSo	GT	GG	GG:GT=6:5	GG:GT=17:1	6.62*
KASP1586	T/C	ETo/CSo	TC	TT	TT:TC=6:9	TT:TC=14:2	7.63**
KASP1589	C/T	ETo/CSo	CT	CC	CC:CT=7:9	CC:CT=13:3	4.80*
KASP1587	T/C	ETo/CSo	TC	TC	TT:TC:CC=4:4:9	TT:TC:CC=12:2:2	9.10**
KASP1588	C/T	ETo/CSo	CT	CT	CC:CT:TT=4:5:10	CC:CT:TT=11:4:1	10.56**
KASP1592	G/T	ETo/CSo	GT	TT	GT:TT=2:17	GT:TT=8:6	8.29**
KASP4533	G/A	ETo/CSo	GA	AA	GA:AA=2:14	GA:AA=6:6	4.73*