板栗总苞和坚果主要性状与SSR标记的关联分析

doi:10.3864/j.issn.0578-1752.2022.13.011

摘要/Abstract

摘要：

【目的】通过测定113份板栗品种（系）的数量、质量和假质量性状,分析其遗传变异,比较不同组群之间的性状差异,将SSR标记与性状进行关联,获得更多与SSR标记显著关联的性状,挖掘优异的等位变异位点,为开展板栗分子辅助育种的研究提供参考。【方法】测定并分析板栗总苞和坚果的38个数量、质量和假质量性状,利用SPSS和Graphpad软件对数量性状进行差异显著性和相关性分析,基于SSR标记进行遗传多样性分析,最后用TASSEL 2.1软件通过一般线性模型（general linear model,GLM）和混合线性模型（mixed linear model,MLM）分别对性状和标记进行关联分析。【结果】在遗传多样性分析中,21对SSR引物的有效等位基因数（N_e）、Shannon指数（I）、多态性信息含量（PIC>0.5）、观察杂合度（H_o）和期望杂合度（H_e）的平均值分别为3.164、1.269、0.589、0.593和0.635。根据群体结构分析分为2个主要组群,为了更好地比较性状差异,在聚类分析时将中间类型的品种单独划分为一组。在质量和假质量性状分析中,多样性指数变化范围为0.139—1.567,遗传多样性最高的性状是坚果光泽,最低的是底座接线;坚果形状、光泽、颜色等性状组群间频率分布差异明显。在数量性状分析中,变异系数的范围在3.96%—36.31%,苞重、总苞重和单粒重的变异系数均在30%以上,遗传变异程度高;果形指数和含水量变异系数均在10%以下,具有稳定的遗传特性;总苞和坚果的外观性状之间有强相关性,相关系数均在0.6以上;组1-组2和组2-组3在果实形状和重量中存在显著差异（P<0.05）。在关联分析中,GLM模型中有13个标记位点与18个表型性状极显著关联,表型变异解释率范围为15.12%—54.99%;MLM模型中有6个标记位点与7个表型性状极显著关联,表型变异解释率范围在8.66%—26.93%。【结论】本研究将SSR标记与表型性状进行关联分析,共发现13个标记位点与坚果单粒重等20个表型性状极显著关联,为开展板栗分子辅助育种的研究奠定了基础。

关键词: 板栗, 性状, SSR, 遗传多样性, 关联分析

Abstract:

【Objective】In the experiment, the quantitative, qualitative and pseudo-qualitative characteristics of 113 chestnut varieties (lines) were measured, their genetic variation were analyzed, characteristics differences among different groups were compared, SSR markers were associated with characteristics, more characteristics significantly associated with SSR markers were obtained, and the excellent allelic variation sites were excavated, so as to provide the references for the research on molecular assisted breeding of Chinese chestnut.【Method】Firstly, 38 quantitative, qualitative and pseudo-qualitative characteristics of bur and nut were determined and analyzed. Secondly, the significance and correlation analysis of quantitative characteristics were performed by SPSS and Graphpad software, and the genetic diversity of SSR markers was analyzed. Finally, the association analysis of characteristics and markers was carried out by general linear model (GLM) and mixed linear model (MLM) of TASSEL 2.1.【Result】In genetic diversity analysis, the average number of effective number of alleles (N_e), Shannon index (I), polymorphism information content (PIC>0.5), observed heterozygosity (H_o), and expected heterozygosity (H_e) of 21 pairs of SSR markers were 3.164, 1.269, 0.589, 0.593, and 0.635, respectively. They were divided into two main groups according to the analysis of population structure. On the other hand, the intermediate type of varieties was defined as one group in the cluster analysis, so that the characteristics differences could be compared precisely. In the analysis of qualitative and pseudo-qualitative characteristics, the diversity index varied from 0.139 to 1.567, and the characteristic with the highest genetic diversity was nut glossiness, while the lowest was the hilum margin. There were obvious differences in the frequency distribution of nut shape, glossiness, color and other characteristics in different groups. In the analysis of quantitative characteristics, the range of coefficient of variation was between 3.96% and 36.31%, the coefficient of variation in bur weight, total bur weight and single nut weight were all above 30% with a high degree of genetic variation; the coefficient of variation of nut shape index and water content were all below 10%, which had stable genetic characteristics. The correlation coefficients were all above 0.6, indicating the appearance characteristics of bur and nut were highly correlated. There were significant differences (P<0.05) in fruit shape and weight between Group 1 vs. Group 2 and Group 2 vs. Group 3. In the association analysis, 13 SSR markers were extremely significantly associated with 18 phenotypic characteristics in the GLM model and the interpretation rate of phenotypic variation ranged from 15.12% to 54.99%, while there were 6 SSR markers in the MLM model that were extremely significantly associated with 7 phenotypic characteristics and the interpretation rate of phenotypic variation ranged from 8.66% to 26.93%. 【Conclusion】In this study, SSR markers were associated with phenotypic characteristics, thirteen SSR markers were extremely significantly correlated with 20 phenotypic characteristics, such as nut single weight, which laid a foundation for molecular assisted breeding of Chinese chestnut.

Key words: Chinese chestnut, characteristic, SSR, genetic diversity, association analysis

陈旭,郝雅琼,聂兴华,杨海莹,刘松,王雪峰,曹庆芹,秦岭,邢宇. 板栗总苞和坚果主要性状与SSR标记的关联分析[J]. 中国农业科学, 2022, 55(13): 2613-2628.

CHEN Xu,HAO YaQiong,NIE XingHua,YANG HaiYing,LIU Song,WANG XueFeng,CAO QingQin,QIN Ling,XING Yu. Association Analysis of Main Characteristics of Bur and Nut with SSR Markers in Chinese Chestnut[J]. Scientia Agricultura Sinica, 2022, 55(13): 2613-2628.

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https://www.chinaagrisci.com/CN/Y2022/V55/I13/2613

图/表 10

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板栗品种（系）的质量性状频率分布差异"

性状 Character	组别 Groups	频率分布 Frequency distribution (%)
性状 Character	组别 Groups	1	2	3	4	5
开裂方式 Bur crack	1	47.06	47.06	5.88
	2	29.33	68.00	2.67
	3	33.33	61.90	4.76
刺束分支角 Prickle angle	1	5.88	58.82	35.29
	2	5.33	65.33	29.33
	3		71.43	28.57
刺束密度 Prickle density	1	5.88	23.53	70.59
	2	5.33	32.00	62.67
	3	4.76	23.81	71.43
刺束颜色 Prickle color	1		11.76	88.24
	2		10.67	89.33
	3		14.29	85.71
坚果颜色均匀度 Nut color evenness	1	70.59	23.5	5.88
	2	76.00	17.33	6.67
	3	57.14	38.10	4.76
坚果光泽 Nut glossiness	1	29.41	41.18	11.76	11.76	5.88
	2	20.00	26.67	10.67	22.67	20.00
	3	19.05	33.33	23.81	19.05	4.76
果顶果肩 Apex	1	17.65	29.41	52.94
	2	28.00	36.00	33.33	2.67
	3	28.57	38.10	28.57	4.76
茸毛分布 Fuzz distribution	1	88.24	5.88	5.88
	2	48.00	25.33	26.67
	3	61.90	23.81	14.29
茸毛颜色 Fuzz color	1	23.53	76.47
	2	26.67	73.33
	3	23.81	76.19
茸毛密度 Fuzz density	1	35.29	41.18	11.76	11.76
	2	29.33	18.67	32.00	20.00
	3	33.33	38.10	28.57
筋线明显程度 Obvious of stripes	1	76.47	23.53
	2	78.67	16.00	5.33
	3	66.67	33.33
底座大小 Hilum size	1	23.53	58.82	17.65
	2	6.67	41.33	52.00
	3	9.52	76.19	14.29
性状 Character	组别 Groups	频率分布 Frequency distribution (%)
性状 Character	组别 Groups	1	2	3	4	5
底座光滑度 Hilum smoothing	1	29.41	70.59
	2	48.00	52.00
	3	42.86	57.14
涩皮剥离难易程度 Difficulty of peeling inner skin	1	23.53	76.47
	2	9.33	90.67
	3	14.29	85.71
刺苞形状 Bur shape	1	17.65	82.35
	2	24.00	74.67	1.33
	3	33.33	66.67
坚果颜色 Nut color	1		17.65		70.59	11.76
	2	2.67	42.67	18.67	33.33	2.67
	3	4.76	28.57	9.52	47.62	9.52
边果形状 Nut shape	1	47.06	52.94
	2	85.33	14.67
	3	76.19	23.81
底座接线 Hilum margin	1	88.24		11.76
	2	98.67	1.33
	3	100.00
种仁颜色 Kernel color	1	17.65	82.35
	2	4.00	92.00	4.00
	3		76.19	23.81

表3

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引物 Marker	观察等位基因数 N_a	有效等位基因数 N_e	Shannon指数 I	观测杂合度 H_o	期望杂合度 H_e	多态性信息含量 PIC
CmSI0396	4	2.553	1.069	0.708	0.608	0.542
CmSI0509	5	2.217	1.040	0.504	0.549	0.503
CmSI0561	8	1.988	1.132	0.460	0.497	0.477
CmSI0617	8	4.414	1.637	0.814	0.773	0.738
CmSI0658	6	4.454	1.589	0.796	0.775	0.740
CmSI0702	5	2.887	1.177	0.637	0.654	0.589
CmSI0735	6	2.753	1.199	0.646	0.637	0.568
CmSI0742	6	3.149	1.280	0.558	0.682	0.624
CmSI0800	13	5.240	1.959	0.708	0.809	0.786
CmSI0809	4	3.015	1.193	0.625	0.668	0.604
CmSI0853	6	3.835	1.518	0.681	0.739	0.699
CmSI0871	5	1.895	0.768	0.531	0.472	0.384
CmSI0883	6	3.776	1.531	0.681	0.735	0.702
CmSI0933	3	1.822	0.799	0.469	0.451	0.408
CmSI0938	4	1.774	0.740	0.204	0.436	0.368
CmSI0922	8	4.884	1.766	0.664	0.795	0.769
CmSI0930	11	4.617	1.838	0.779	0.783	0.761
CmSI0881	6	3.278	1.442	0.478	0.695	0.658
CmSI0614	6	4.464	1.590	0.805	0.776	0.740
CmSI0404	3	1.399	0.510	0.212	0.285	0.253
CmSI0747	3	2.042	0.880	0.496	0.510	0.456
均值 Average	6	3.164	1.269	0.593	0.635	0.589

性状 Character	频率分布 Frequency distribution (%)					多样性指数H' Diversity index
性状 Character	1	2	3	4	5	多样性指数H' Diversity index
开裂方式Bur crack	32.74	63.72	3.54			0.771
刺束分支角Prickle angle	4.42	65.49	30.09			0.777
刺束密度Prickle density	5.31	29.20	65.49			0.793
刺束颜色Prickle color		11.50	88.50			0.357
坚果颜色均匀度Nut color evenness	71.68	22.12	6.19			0.745
坚果光泽Nut glossiness	21.24	30.09	13.27	20.35	15.04	1.567
果顶果肩Apex	26.55	35.40	35.40	2.65		1.184
茸毛分布Fuzz distribution	56.64	22.12	21.24			0.985
茸毛颜色Fuzz color	25.66	74.34				0.570
茸毛密度Fuzz density	30.97	25.66	28.32	15.04		1.354
筋线明显程度Obvious of stripes	76.11	20.35	3.54			0.650
底座大小Hilum size	9.73	50.44	39.82			0.939
底座光滑度Hilum smoothing	44.25	55.75				0.687
涩皮剥离难易程度Difficulty of peeling inner skin	12.39	87.61				0.375
刺苞形状Bur shape	24.78	74.34	0.88			0.608
坚果颜色Nut color	2.65	36.28	14.16	41.59	5.31	1.262
边果形状Nut shape	77.88	22.12				0.528
底座接线Hilum margin	97.35	0.88	1.77			0.139
种仁颜色Kernel color	5.31	87.61	7.08			0.459

指标 Character	极小值 Minimum	极大值 Maximum	平均值 Average	标准差 S	变异系数 CV (%)
总苞重 Total bur weight (g)	11.93	120.83	65.91	22.02	33.41
苞重 Bur weight (g)	7.76	79.95	38.66	14.04	36.31
刺苞宽Bur width (mm)	50.80	105.31	80.54	10.77	13.38
刺苞厚Bur thickness (mm)	42.58	88.06	66.45	8.14	12.25
刺苞高Bur height (mm)	39.55	80.09	61.99	7.87	12.69
苞肉厚Bur skin thickness (mm)	1.52	5.48	3.63	0.974	26.80
刺束长Prickle length (mm)	6.80	18.66	13.77	2.21	16.08
刺苞的坚果数Nuts No./Bur	1.38	3.10	2.50	0.305	12.21
单粒重Single nut weight (g)	2.67	20.68	10.99	3.81	34.69
坚果厚Nut thickness (mm)	13.86	32.05	20.60	3.25	15.76
坚果宽Nut width (mm)	19.42	39.53	31.08	3.67	11.79
坚果高Nut height (mm)	17.45	32.63	26.60	2.71	10.19
果形指数Nut shape index	0.788	0.953	0.858	0.034	3.96
出实率Kernel yield (%)	22.23	56.84	41.27	5.80	14.05
含水量Water content (%)	44.46	60.69	52.03	3.57	6.85
可溶性糖含量Soluble sugar (%, DW)	8.63	19.75	13.21	2.08	15.76
直链淀粉含量Amylose (%, DW)	4.44	18.53	11.86	3.20	26.94
支链淀粉含量Amylopectin (%, DW)	28.52	75.64	53.15	10.15	19.09
总淀粉含量Total starch (%, DW)	40.22	85.18	65.01	9.39	14.45

指标 Character	组1 Group1	组2 Group2	组3 Group3
总苞重Total bur weight (g)	56.29±17.47a	72.26±21.37b	51.03±16.40a
苞重Bur weight (g)	32.80±10.55a	42.68±13.88b	29.05±9.71a
刺苞宽Bur width (mm)	75.29±10.62a	83.62±9.81b	73.78±9.07a
刺苞厚Bur thickness (mm)	62.15±7.98a	68.94±7.12b	61.05±7.40a
刺苞高Bur height (mm)	57.34±7.09a	64.58±6.58b	56.54±7.96a
苞肉厚Bur skin thickness (mm)	3.30±0.82a	3.84±0.96b	3.18±0.89a
刺束长Prickle length (mm)	12.97±2.02a	14.18±2.10a	12.95±2.31a
刺苞的坚果数Nuts No./Bur	2.53±0.28a	2.49±0.30a	2.52±0.32a
单粒重Single nut weight (g)	9.24±3.05a	12.03±3.74b	8.67±2.87a
坚果厚Nut thickness (mm)	19.22±2.52a	21.42±3.12b	18.75±2.99a
坚果宽Nut width (mm)	29.78±3.47a	32.06±3.34b	28.63±3.35a
坚果高Nut height (mm)	25.63±2.71a	27.32±2.38b	24.82±2.71a
果形指数Nut shape index	0.86±0.04a	0.85±0.04a	0.87±0.02a
出实率Kernel yield (%)	41.69±4.50a	40.71±6.31a	42.95±4.10a
含水量Water content (%)	51.58±3.17a	52.13±3.56a	52.04±3.77a
可溶性糖含量Soluble sugar (%, DW)	12.62±2.11a	13.19±1.98a	13.83±2.19a
直链淀粉含量Amylose (%, DW)	12.94±2.93a	11.77±3.26a	11.21±2.81a
支链淀粉含量Amylopectin (%, DW)	52.51±11.56a	52.82±9.68a	55.09±9.96a
总淀粉含量Total starch (%, DW)	65.45±10.82a	64.58±9.02a	66.29±8.96a

性状 Character	位点 Loci	GLM		MLM
性状 Character	位点 Loci	P<0.01	R² (%)	P<0.01	R² (%)
总苞重Total bur weight	CmSI0883	0.0017	36.84
总苞重Total bur weight	CmSI0930	0.0021	46.90
苞重Bur weight	CmSI0883	0.0023	36.43
苞重Bur weight	CmSI0930	0.001	49.07	0.0095	16.31
刺苞宽Bur width	CmSI0883	0.0072	33.37
刺苞厚Bur thickness	CmSI0883	0.0045	33.90
刺苞高Bur height	CmSI0883	7.96E-04	37.47
刺束长Prickle length	CmSI0702	0.0018	28.87
刺束长Prickle length	CmSI0883	0.0084	34.38
刺苞的坚果数Nuts No./Bur	CmSI0800	0.0074	53.33	0.0027	19.99
单粒重Single nut weight	CmSI0853	0.0021	38.84
单粒重Single nut weight	CmSI0930	0.0092	42.86
坚果宽Nut width	CmSI0930	0.0065	45.21
坚果高Nut height	CmSI0883	0.0035	36.59
含水量Water content	CmSI0735	6.71E-04	36.42
可溶性糖含量Soluble sugar	CmSI0800			0.0085	26.93
刺束密度Prickle density	CmSI0735	0.0064	26.79	0.0065	8.66
刺束密度Prickle density	CmSI0742	0.0053	29.04
坚果颜色均匀度Nut color evenness	CmSI0871	8.14E-05	30.05
筋线明显程度Obvious of stripes	CmSI0800			0.0037	20.21
涩皮剥离难易程度Difficulty of peeling inner skin	CmSI0561	7.94E-05	42.52
刺苞形状Bur shape	CmSI0404	0.0061	15.12
坚果颜色Nut color	CmSI0881	0.0076	36.49
底座接线Hilum margin	CmSI0509	1.73E-06	45.52	1.20E-03	14.41
底座接线Hilum margin	CmSI0881	5.96E-07	54.99	3.60E-03	16.29
种仁颜色Kernel color	CmSI0617	0.0037	47.72	0.0041	17.93
种仁颜色Kernel color	CmSI0881	0.0033	37.95	0.0013	15.01