甜瓜果肉硬度分子标记的开发与利用

doi:10.7668/hbnxb.20194125

摘要/Abstract

摘要：

为提高不同果肉硬度类型甜瓜材料的选择效率,加速良种培育,以脆肉甜瓜材料19A21、19A75和软肉甜瓜材料N84、20S66为亲本,分别构建F₂与BC₁F₁群体,采用感官测试统计,脆肉材料与软肉材料符合3∶1与1∶1理论值,说明甜瓜果肉硬度性状为单基因遗传,且脆肉相对于软肉为显性;以4份甜瓜亲本为供试材料,分析不同硬度类型果实的ACO活性变化及CmACO1的表达模式,结果显示,软肉甜瓜果实发育中ACO活性出现峰值,而脆肉甜瓜未出现,软肉甜瓜CmACO1表达量显著高于脆肉材料,说明该基因可能参与调控甜瓜果肉硬度。根据CmACO1在不同材料中的插入缺失位点(Insert/Deletion)差异,开发InDel-Pf标记,利用该InDel标记对32份甜瓜材料进行基因型检测,其中脆肉甜瓜表现为缺失带型,软肉甜瓜表现为非缺失带型,标记多态性与果肉硬度共分离。利用2个F₂群体对InDel-Pf标记进行验证,分子标记鉴定与表型鉴定符合率达到95.3%,98.1%。研究结果表明,InDel-Pf标记对甜瓜果肉硬度的实际鉴定有较高准确性,能够有效提高育种选择效率,缩短良种培育周期。

关键词: 甜瓜, 果肉硬度, CmACO1, InDel标记, 辅助选择育种

Abstract:

In order to improve the selection efficiency of melon materials with different pulp firmness types and accelerate developing varieties.The F₂ and BC₁F₁ populations were constructed with crisp melon materials 19A21,19A75 and soft melon materials N84,20S66 as parents.According to sensory testing statistics,the theoretical values of 3∶1 and 1∶1 for crispy and soft materials were consistent,indicating that the melon pulp firmness was controlled by a single gene,and crisp was dominant to soft.The changes of ACO activity and expression pattern of CmACO1 with different firmness types were analyzed with 4 materials.The results showed that the ACO activity of soft melon appeared at a peak during fruit development,but the crispy melon did not appear.The expression of CmACO1 in soft melon was significantly higher than that in crispy melon.This gene may be involved in regulating pulp firmness.According to the difference of insertion and deletion sites of CmACO1 in different materials,the InDel-Pf marker was developed.The InDel marker was used to detect the genotypes of 32 melon materials.Among them,crisp melon showed deletion band type,soft melon showed non-deletion band type,the marker polymorphism and pulp firmness were co-separated.Two F₂ populations were used to validate the InDel-Pf,the genotype and phenotype coincidence rates were 95.3% and 98.1%.The results showed that InDel-Pf marker had high accuracy in the actual identification of melon pulp firmness,which could effectively improve the efficiency of breeding selection and shorten the breeding cycle of improved varieties.

Key words: Melon, Pulp firmness, CmACO1, InDel marker, Assisted selection breeding

李肯, 张伟, 武云鹏, 潘静怡, 彭冬秀, 张若纬. 甜瓜果肉硬度分子标记的开发与利用[J]. 华北农学报, 2023, 38(5): 94-101. doi: 10.7668/hbnxb.20194125.

LI Ken, ZHANG Wei, WU Yunpeng, PAN Jingyi, PENG Dongxiu, ZHANG Ruowei. Development and Utilization of Molecular Markers for Identification of Pulp Firmness in Melon[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 94-101. doi: 10.7668/hbnxb.20194125.

http://www.hbnxb.net/CN/Y2023/V38/I5/94

图/表 7

参考文献 22

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序号 Number	材料 Accession	TPA	穿刺 Puncture		类型 Type	果肉硬度 Pulp firmness
序号 Number	材料 Accession	硬度/g Hardness	平均硬度/N Average hardness	脆性/(N/S) Brittleness	类型 Type	果肉硬度 Pulp firmness
1	20S66	375.27±68.25c	0.72±0.02b	1.31±0.02b	厚皮甜瓜	软肉
2	19A21	2 532.25±348.78a	1.75±0.10a	4.13±0.46a	厚皮甜瓜	脆肉
3	N84	193.83±29.10cd	0.43±0.04b	0.88±0.07b	薄皮甜瓜	面肉
4	19A75	2 213.51±377.99a	1.58±0.12a	4.17±0.32a	薄皮甜瓜	脆肉
5	R-13	329.28±51.17c	0.69±0.07b	1.26±0.09b	厚皮甜瓜	软肉
6	R-14	306.95±80.22c	0.66±0.07b	1.24±0.92b	厚皮甜瓜	软肉
7	R-20	339.14±87.26c	0.70±0.06b	1.29±0.19b	厚皮甜瓜	软肉
8	S-19	347.20±87.25c	0.71±0.09b	1.29±0.40b	厚皮甜瓜	软肉
9	S-28	391.27±45.39c	0.74±0.11b	1.33±0.31b	厚皮甜瓜	软肉
10	Sa-85	374.50±77.21c	0.72±0.00b	1.31±0.42b	厚皮甜瓜	软肉
11	Sa-86	367.98±42.87c	0.70±0.04b	1.28±0.33b	厚皮甜瓜	软肉
12	Sa-93	335.85±71.59c	0.68±0.06b	1.26±0.20b	厚皮甜瓜	软肉
13	R-12	2 487.16±611.05a	1.72±0.11a	4.03±0.78a	厚皮甜瓜	脆肉
14	R-15	2 584.13±145.39a	1.79±0.17a	3.92±0.90a	厚皮甜瓜	脆肉
15	R-62	2 479.60±639.51a	1.70±0.06a	3.88±0.39a	厚皮甜瓜	脆肉
16	R-65	2 529.74±611.05a	1.75±0.10a	4.22±0.53a	厚皮甜瓜	脆肉
17	R-71	2 394.25±495.60a	1.69±0.03a	3.96±0.56a	厚皮甜瓜	脆肉
18	Ra-18	1 976.75±635.97ab	1.42±0.09a	2.85±0.16ab	厚皮甜瓜	酥脆肉
19	Ra-26	1 942.36±395.96ab	1.38±0.12a	2.49±0.23ab	厚皮甜瓜	酥脆肉
20	Ra-72	1 890.80±356.01ab	1.34±0.08a	2.46±0.79ab	厚皮甜瓜	酥脆肉
21	N85	184.65±33.10cd	0.40±0.02b	0.80±0.09b	薄皮甜瓜	面肉
22	N86	213.25±36.87cd	0.46±0.06b	0.93±0.05b	薄皮甜瓜	面肉
23	N87	199.06±21.15cd	0.42±0.03b	0.84±0.06b	薄皮甜瓜	面肉
24	N88	215.60±35.27cd	0.46±0.03b	0.85±0.16b	薄皮甜瓜	面肉
25	N89	247.88±41.88c	0.51±0.10b	0.89±0.13b	薄皮甜瓜	面肉
26	R-155	523.25±53.59c	0.68±0.12b	1.12±0.79b	薄皮甜瓜	软肉
27	R-156	478.71±83.58c	0.62±0.09b	0.90±0.02b	薄皮甜瓜	软肉
28	R-157	516.20±77.21c	0.66±0.04b	1.04±0.16b	薄皮甜瓜	软肉
29	R-162	493.34±69.53c	0.64±0.03b	0.93±0.08b	薄皮甜瓜	软肉
30	R-163	488.29±53.59c	0.63±0.08b	0.95±0.22b	薄皮甜瓜	软肉
31	R-170	502.36±68.46c	0.64±0.02b	0.99±0.18b	薄皮甜瓜	软肉
32	R-101	2 185.23±196.41ab	1.52±0.16a	4.09±1.01a	薄皮甜瓜	脆肉
33	R-102	2 009.46±495.42ab	1.48±0.09a	3.86±0.22a	薄皮甜瓜	脆肉
34	R-103	2 089.30±457.92ab	1.51±0.12a	3.95±0.19a	薄皮甜瓜	脆肉
35	R-133	1 288.91±185.36c	1.12±0.02ab	2.48±0.71ab	薄皮甜瓜	酥脆肉
36	R-136	1 308.27±99.65c	1.20±0.03ab	2.51±0.23ab	薄皮甜瓜	酥脆肉

序号 Number	材料 Accession	TPA	穿刺 Puncture		类型 Type	果肉硬度 Pulp firmness
序号 Number	材料 Accession	硬度/g Hardness	平均硬度/N Average hardness	脆性/(N/S) Brittleness	类型 Type	果肉硬度 Pulp firmness
1	20S66	375.27±68.25c	0.72±0.02b	1.31±0.02b	厚皮甜瓜	软肉
2	19A21	2 532.25±348.78a	1.75±0.10a	4.13±0.46a	厚皮甜瓜	脆肉
3	N84	193.83±29.10cd	0.43±0.04b	0.88±0.07b	薄皮甜瓜	面肉
4	19A75	2 213.51±377.99a	1.58±0.12a	4.17±0.32a	薄皮甜瓜	脆肉
5	R-13	329.28±51.17c	0.69±0.07b	1.26±0.09b	厚皮甜瓜	软肉
6	R-14	306.95±80.22c	0.66±0.07b	1.24±0.92b	厚皮甜瓜	软肉
7	R-20	339.14±87.26c	0.70±0.06b	1.29±0.19b	厚皮甜瓜	软肉
8	S-19	347.20±87.25c	0.71±0.09b	1.29±0.40b	厚皮甜瓜	软肉
9	S-28	391.27±45.39c	0.74±0.11b	1.33±0.31b	厚皮甜瓜	软肉
10	Sa-85	374.50±77.21c	0.72±0.00b	1.31±0.42b	厚皮甜瓜	软肉
11	Sa-86	367.98±42.87c	0.70±0.04b	1.28±0.33b	厚皮甜瓜	软肉
12	Sa-93	335.85±71.59c	0.68±0.06b	1.26±0.20b	厚皮甜瓜	软肉
13	R-12	2 487.16±611.05a	1.72±0.11a	4.03±0.78a	厚皮甜瓜	脆肉
14	R-15	2 584.13±145.39a	1.79±0.17a	3.92±0.90a	厚皮甜瓜	脆肉
15	R-62	2 479.60±639.51a	1.70±0.06a	3.88±0.39a	厚皮甜瓜	脆肉
16	R-65	2 529.74±611.05a	1.75±0.10a	4.22±0.53a	厚皮甜瓜	脆肉
17	R-71	2 394.25±495.60a	1.69±0.03a	3.96±0.56a	厚皮甜瓜	脆肉
18	Ra-18	1 976.75±635.97ab	1.42±0.09a	2.85±0.16ab	厚皮甜瓜	酥脆肉
19	Ra-26	1 942.36±395.96ab	1.38±0.12a	2.49±0.23ab	厚皮甜瓜	酥脆肉
20	Ra-72	1 890.80±356.01ab	1.34±0.08a	2.46±0.79ab	厚皮甜瓜	酥脆肉
21	N85	184.65±33.10cd	0.40±0.02b	0.80±0.09b	薄皮甜瓜	面肉
22	N86	213.25±36.87cd	0.46±0.06b	0.93±0.05b	薄皮甜瓜	面肉
23	N87	199.06±21.15cd	0.42±0.03b	0.84±0.06b	薄皮甜瓜	面肉
24	N88	215.60±35.27cd	0.46±0.03b	0.85±0.16b	薄皮甜瓜	面肉
25	N89	247.88±41.88c	0.51±0.10b	0.89±0.13b	薄皮甜瓜	面肉
26	R-155	523.25±53.59c	0.68±0.12b	1.12±0.79b	薄皮甜瓜	软肉
27	R-156	478.71±83.58c	0.62±0.09b	0.90±0.02b	薄皮甜瓜	软肉
28	R-157	516.20±77.21c	0.66±0.04b	1.04±0.16b	薄皮甜瓜	软肉
29	R-162	493.34±69.53c	0.64±0.03b	0.93±0.08b	薄皮甜瓜	软肉
30	R-163	488.29±53.59c	0.63±0.08b	0.95±0.22b	薄皮甜瓜	软肉
31	R-170	502.36±68.46c	0.64±0.02b	0.99±0.18b	薄皮甜瓜	软肉
32	R-101	2 185.23±196.41ab	1.52±0.16a	4.09±1.01a	薄皮甜瓜	脆肉
33	R-102	2 009.46±495.42ab	1.48±0.09a	3.86±0.22a	薄皮甜瓜	脆肉
34	R-103	2 089.30±457.92ab	1.51±0.12a	3.95±0.19a	薄皮甜瓜	脆肉
35	R-133	1 288.91±185.36c	1.12±0.02ab	2.48±0.71ab	薄皮甜瓜	酥脆肉
36	R-136	1 308.27±99.65c	1.20±0.03ab	2.51±0.23ab	薄皮甜瓜	酥脆肉

世代 Generations	总数 Total	脆肉 Crisp	软(面)肉 Soft	分离比 Segregation ratio	χ²
P₁(19A21)	20	20	0
P₂(20S66)	20	0	20
F₁(P₁×P₂)	39	39	0
F₁(P₂×P₁)	36	36	0
BC₁(F₁×19A21)	84	84	0
BC₁(F₁×20S66)	80	42	38	1.11∶1	1.19
F₂(P₁×P₂®)	423	313	110	2.85∶1	1.32
P₃(19A75)	20	20	0
P₄(N84)	20	0	20
F₁(P₃×P₄)	40	40	0
F₁(P₄×P₃)	36	36	0
BC₁(F₁×19A75)	79	79	0
BC₁(F₁×N84)	80	44	36	1.22∶1	1.25
F₂(P₃×P₄®)	415	309	106	2.92∶1	1.07

世代 Generations	总数 Total	脆肉 Crisp	软(面)肉 Soft	分离比 Segregation ratio	χ²
P₁(19A21)	20	20	0
P₂(20S66)	20	0	20
F₁(P₁×P₂)	39	39	0
F₁(P₂×P₁)	36	36	0
BC₁(F₁×19A21)	84	84	0
BC₁(F₁×20S66)	80	42	38	1.11∶1	1.19
F₂(P₁×P₂®)	423	313	110	2.85∶1	1.32
P₃(19A75)	20	20	0
P₄(N84)	20	0	20
F₁(P₃×P₄)	40	40	0
F₁(P₄×P₃)	36	36	0
BC₁(F₁×19A75)	79	79	0
BC₁(F₁×N84)	80	44	36	1.22∶1	1.25
F₂(P₃×P₄®)	415	309	106	2.92∶1	1.07

杂交组合 Hybrid combination	类型 Type	F₂群体植株数量 Number of F₂ population
杂交组合 Hybrid combination	类型 Type	分子标记鉴定 Molecular marker	表型鉴定 Phenotypic	误差 Error	符合率/% Matching rate
19A21×20S66	脆肉	323	313	20	95.3
	软肉	100	110
	总计	423	423
19A75×N84	脆肉	313	309	8	98.1
	面肉	102	106
	总计	415	415

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