基于SSR分子标记的葡萄F1代杂交群体鉴定及灰霉病抗性遗传评价

doi:10.13560/j.cnki.biotech.bull.1985.2026-0737

摘要/Abstract

摘要：

目的利用SSR分子标记对‘赤霞珠’×‘北冰红’F₁代群体进行真实性鉴定，并开展灰霉病抗性遗传分析，为葡萄抗病育种提供理论依据和种质材料。方法以‘赤霞珠’为母本、‘北冰红’为父本，通过人工杂交获得106株F₁代群体。采用CTAB法提取亲本及其后代叶片DNA，利用SSR标记鉴定真实杂交后代，并对其离体叶片接种灰霉菌进行抗性鉴定和病斑统计。结果筛选获得引物Z3M12（ab×cd型）用于群体鉴定。在106株后代中，真实杂种91株，真实率85.85%。母本‘赤霞珠’病情指数40.0（感病），父本‘北冰红’病情指数20.0（抗病）。后代病斑面积连续分布，变异系数83.40%‒117.40%；抗性分离为高抗5株、抗病66株、感病17株、高感3株，抗病及以上占比78.02%。染色显示，抗性个体H₂O₂积累与菌丝生长呈负相关，高抗个体表现超亲抗性。3次重复病斑面积呈极显著正相关（r=0.731‒0.903，P<0.01）。子代平均病斑面积低于亲中值，组合传递率为57.67%‒66.38%，筛选出5株稳定高抗材料。结论 SSR分子标记可快速精准鉴定杂交后代，有效剔除假杂种。‘赤霞珠’×‘北冰红’杂交组合F₁代群体的灰霉病抗性呈连续分布，育种选择效率较高；5株高抗株系可为抗病新品种选育及基因定位提供核心种质。

关键词: 葡萄, SSR分子标记, F₁代杂交群体, 真实性鉴定, 灰霉病, 抗性遗传

Abstract:

Objective To authenticate the F₁ population derived from the cross between ‘Cabernet Sauvignon’ and ‘Beibinghong’ using SSR molecular markers, and to conduct genetic analysis of resistance to gray mold, thereby providing a theoretical basis and germplasm materials for grape disease-resistance breeding. Method Using ‘Cabernet Sauvignon’ as the female parent and ‘Beibinghong’ as the male parent, 106 F₁ individuals were obtained through artificial hybridization. DNA was extracted from leaves of the parents and their progeny using the CTAB method, true hybrids were identified using SSR markers, and detached leaves were inoculated with Botrytis cinerea for resistance identification and lesion statistics. Result Primer Z3M12 (ab × cd type) was screened for population identification. Among the 106 progeny, 91 were true hybrids, with a true hybrid rate of 85.85%. The disease index of the female parent ‘Cabernet Sauvignon’ was 40.0 (susceptible), and that of the male parent ‘Beibinghong’ was 20.0 (resistant). The lesion areas of the progeny showed a continuous distribution, with coefficients of variation ranging from 83.40% to 117.40%; resistance segregation was 5 highly resistant, 66 resistant, 17 susceptible, and 3 highly susceptible individuals, with resistant and highly resistant individuals accounting for 78.02%. Staining showed that H₂O₂ accumulation in resistant individuals was negatively correlated with hyphal growth, and highly resistant individuals exhibited transgressive resistance. The lesion areas of the three replicates were highly significantly and positively correlated (r=0.731–0.903, P<0.01). The mean lesion area of the progeny was lower than the mid-parent value, and the cross transmission rate was 57.67%–66.38%. Five stable highly resistant materials were screened. Conclusion SSR molecular markers can rapidly and accurately identify hybrid progeny and effectively eliminate false hybrids. The gray mold resistance of the F₁ population from the cross ‘Cabernet Sauvignon’ × ‘Beibinghong’ showed a continuous distribution, and the breeding selection efficiency was relatively high; the five highly resistant lines can provide core germplasm for breeding new disease-resistant cultivars and gene mapping.

Key words: grape, SSR molecular markers, F₁ hybrid population, authenticity identification, gray mold, resistance inheritance

马文玲, 刘国甜, 李承男, 陈明, 单守明, 尹晓. 基于SSR分子标记的葡萄F₁代杂交群体鉴定及灰霉病抗性遗传评价[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0737.

MA Wen-ling, LIU Guo-tian, LI Cheng-nan, CHEN Ming, SHAN Shou-ming, YIN Xiao. Identification of F₁ Hybrid Population in Grape Using SSR Molecular Markers and Genetic Evaluation of Resistance to Gray Mold[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0737.

图/表 9

表1 F1代杂交群体的遗传多样性分析

Table 1 Genetic diversity analysis of the F1 hybrid population

赤霞珠

Cabernet Sauvignon

北冰红

Beibinghong

F₁群体

F₁hybrid population

表2 ‘赤霞珠’×‘北冰红’及F1代抗灰霉病遗传分析

Table 2 Genetic analysis of gray mold resistance in 'Cabernet Sauvignon' × 'Beibinghong' and F1 progeny

次数

Run

母本病斑面积Maternal lesion area （cm²）

父本病斑面积Paternal lesion area （cm²）

亲中值Mid-parent value （cm²）

子代病斑面积平均值 Mean lesion area of the progeny （cm²）

变异系数Coefficient of variation（%）

超低亲率

Ultra-low parent rate （%）

超高亲率

Ultra-high parent rate （%）

组合传递率

Combinationtransmission rate （%）

遗传力

Heritability（%）

偏度

Skewness

图1 SSR多态性引物的凝胶电泳筛选M：2 000 bp marker，1‒48：引物编号

Fig. 1 Screening of SSR polymorphic primers by gel electrophoresisM: 2 000 bp marker; 1-48: primer number

图2 部分SSR多态性引物的毛细管筛选A：引物Z3M12的扩增结果；B：引物VVMD7的扩增结果；X轴代表DNA片段大小，即等位基因；Y轴代表荧光强度（RFU）

Fig. 2 Capillary screening of some polymorphic SSR primersA: Amplification results of primer Z3M12. B: Amplification results of primer VVMD7; the X-axis represents DNA fragment size, i.e., alleles; the Y-axis represents fluorescence intensity (RFU)

图3 引物Z3M12对部分杂种后代的鉴定结果

Fig. 3 Identification results of some hybrid progeny using primer Z3M12

图4 亲本和部分杂交后代的表型♂：父本‘北冰红’；♀：母本‘赤霞珠’；真杂交后代：2、14、53；假杂交后代：94、101；比例尺=1 cm

Fig. 4 Phenotypes of parents and some hybrid progeny♂: Male parent Beibinghong. ♀: Female parent Cabernet Sauvignon. True hybrid progeny: 2, 14, 53. False hybrid progeny: 94, 101. Scale bar = 1 cm

图5 亲本间灰霉病的抗性鉴定A：亲本间灰霉病的抗性表型，比例尺=1 cm；B：亲本的病情严重度；**：在P<0.01水平差异极显著。下同

Fig. 5 Identification of resistance to gray mold between the parentsA: Resistance phenotypes to gray mold between the parents, scale bar = 1 cm. B: Disease severity of the parents. ** indicates extremely significant difference at the P < 0.01 level. The same below

图6 ‘赤霞珠’×‘北冰红’部分杂交后代对灰霉病的抗性鉴定结果（A）及后代群体抗性频率分布（B）比例尺=1 cm；HS：高感病；S：感病；R：抗病；HR：高抗病；1‒64：品种编号

Fig. 6 Identification results of resistance to gray mold in some hybrid progeny of 'Cabernet Sauvignon' × 'Beibinghong' (A) and frequency distribution of resistance in the progeny population (B)Scale bar = 1 cm. HS： Highly susceptible. S： Susceptible. R： Resistant. HR： Highly resistant. 1-64： Variety numbers

图7 亲本和F1代接种灰霉菌4 d后的微观表现A：台盼蓝染色；B：DAB染色

Fig. 7 Microscopic phenotypes of parents and F1 generation at 4 d after inoculation with Botrytis cinereaA： Trypan blue staining； B： DAB staining

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基于SSR分子标记的葡萄F₁代杂交群体鉴定及灰霉病抗性遗传评价

Identification of F₁ Hybrid Population in Grape Using SSR Molecular Markers and Genetic Evaluation of Resistance to Gray Mold

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