甜瓜果柄离区细胞学观察及成熟脱落基因AL3的初步定位

doi:10.16420/j.issn.0513-353x.2020-1041

摘要/Abstract

摘要：

以甜瓜果实成熟不脱蒂材料M2-10为母本,易脱蒂材料ZT091为父本,配制杂交组合,获得F₂、F₃、F₄及回交群体,利用6世代群体分析脱蒂性状的遗传规律,显示由两对基因控制,不脱蒂与脱蒂分离比符合两对互补基因遗传规律。对亲本果实成熟期果柄与果肉连接处离区组织石蜡切片及电镜扫描观察结果表明,亲本间离区细胞存在差异,ZT091果柄离区细胞体积增大、排列松散,细胞间隙较大。利用SLAF-BSA测序,对83-F₃家系单基因分离群体开展控制脱蒂性状的AL3基因初步定位研究,将其定位在第8号染色体10 686 821 ~ 11 042 074 bp区间,覆盖0.355 Mb。通过亲本重测序数据挖掘SNP位点,设计CAPs标记,利用F₄群体将AL3最后定位在8号染色体约64.7 kb区间内（10 774 778 ~ 10 839 486 bp）,包含10个候选基因。

关键词: 甜瓜, 脱落, 细胞学观察, 基因定位

Abstract:

The F₂,F₃,F₄and backcross populations derived from a cross between M2-10 （none-mature fruit abscission,NMFA）and ZT091（mature fruit abscission,MFA）was used to study the cytological observation of abscission zone and gene mapping of one of mature fruit abscission（MFA）gene（AL3）.The analysis of genetic inheritance indicated that the MFA was controlled by two genes. Paraffin section and scanning electron microscopy observation of the parent fruit at the maturity stage showed that the cells in the abscission zone of peduncle of ZT091 were larger in size,loosen arrangement and larger spaces between cells,there were significant difference between parental peduncle abscission zone. AL3 was primary mapped into a 0.355 Mb region（from 10 686 821 to 11 042 074 bp）on chromosome 8 with the strategy of SLAF-BSA using 83-F₃ family only segregated with AL3. F₄family and CAPs markers were used for narrowing down the mapping region. Finally, AL3 was fined mapped into a 64.7 kb（from 10 774 778 to 10 839 486 bp）region with 10 annotated candidate genes.

Key words: melon, fruit abscission, cyotological observation, gene mapping

中图分类号:

S652

盛云燕, 杨丽敏, 戴冬洋, 张佳欣, 王岭, 王迪, 才羿, 田丽美. 甜瓜果柄离区细胞学观察及成熟脱落基因AL3的初步定位[J]. 园艺学报, 2022, 49(2): 341-351.

SHENG Yunyan, YANG Limin, DAI Dongyang, ZHANG Jiaxin, WANG Ling, WANG Di, CAI Yi, TIAN Limei. Cytological Observation of Fruit Peduncle Abscission Zone and Preliminary Mapping of Mature Fruit Abscission AL3 gene in Melon[J]. Acta Horticulturae Sinica, 2022, 49(2): 341-351.

图/表 7

图1 甜瓜亲本及用于脱落基因定位的83-F3群体

Fig. 1 Materials of parent lines and 83-F3 generations for gene mapping

表1 引物信息

Table 1 Primer sequence information

引物编号 Marker ID	染色体 Chr.	基因组位点 Position	正向引物序列（5′-3′） Forward sequence	反向引物序列（5′-3′） Reverse sequence	酶 Enzyme	酶切位点 Recognition site
SSR20531	8	9 968 964	GCAGACCAAAAATGGGAAAA	GGAATGGATGCAGTGATGC
SNP269	8	10 698 869	GCCAAACGGGTTATGAGGTA	TATTCCATGATTGGACGCCT	AluⅠ	AGCT
SNP361	8	10 761 364	TACCACCGGTTCCATGTTTT	ATGGTGGTTCCAATGACGTT	AluⅠ	AGCT
SNP392	8	10 774 778	TGCATAATACTCATTCAACGCA	TTGATGCTTACTTTGGTGATGC	AluⅠ	AGCT
SNP485	8	10 839 486	TGGAAAAATGGCTTCAAATCT	TGCAAACGATTCGTCATTTC	AluⅠ	AGCT
SNP523	8	10 857 497	CATGGCGCTATGAGGTTTTT	TGCAAAATTTTCTTCCAATCG	MseⅠ	TTAA
SNP700	8	10 934 492	TGGAATGCAAACAAATGTGAA	GGACATCCGGTGTTGAACTT	AluⅠ	AGCT

表2 甜瓜果实成熟脱落性状分离情况

Table 2 Segregation of fruit mature abscission in melon

群体 Generation	植株数 Plant number			分离比 Segregation ratio	卡方 Chi-test	P 值 P value
群体 Generation	总株数 Total	不脱蒂 None-abscission	脱蒂 Abscission	分离比 Segregation ratio	卡方 Chi-test	P 值 P value
P₁（M2-10）	30	30	0
P₂（ZT091）	30	0	30
F₁	30	30	0
F₂	189	110	79	9:7	0.173	0.678
BC₁P₁	153	153	0
BC₁P₂	146	115	31	3:1	0.481	0.817

图2 甜瓜M2-10和ZT091果蒂与果肉连接处显微观察 C：形成层;AZ：离层区域;M：髓。

Fig. 2 Microscopic observation of the junction between peduncle and fruit of M2-10 and ZT091 C：Cambium;AZ：Abscission zone;M：Medullary.

图3 甜瓜M2-10和ZT091果柄离区电镜扫描图 C：形成层;AZ：离层区域;M：髓;MR：髓射线;PX：初生木质部;PP：初生韧皮部。

Fig. 3 Electron microscope scanning of parental peduncle abscission zone of M2-10 and ZT091 C：Cambium;AZ：Abscission zone;M：Medullary;MR：Medullary ray;PX：Primary xylem;PP：Primary phloem.

表3 利用两种分析方法2种计算方式获得的连锁区间

Table 3 The linkage region using by two calculation with 2 analysis ways

分析方法 Analysis way	ED		Index		合集 Combination
分析方法 Analysis way	开始位点 Start	终止位点 Ending	开始位点 Start	终止位点 Ending	开始位点 Start	终止位点 Ending
SNP	10 685 509	11 693 628	10 686 821	11 693 628	10 686 821	11 693 628
InDel	10 428 865	11 108 980	10 459 389	11 042 074	10 459 389	11 042 074
合并区域 Combination region	10 686 821	11 042 074

图4 甜瓜脱蒂基因AL3初步定位 A：利用SLAF-BSA测序技术对F3群体基因池初步定位的结果;B：利用F4家系开展精细定位的结果;C：候选区域包含10候选基因。

Fig. 4 Preliminary mapping of mature fruit abscission AL3 gene A：Preliminary mapping by SLAF-BSA using F3 bulks;B：Mapping of candidate region using F4 families;C：Candidate region contains 10 candidate genes.

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