马铃薯红色薯肉调控基因的精细定位与候选基因分析

doi:10.3864/j.issn.0578-1752.2019.15.011

摘要/Abstract

摘要：

【目的】薯肉颜色是马铃薯重要的农艺性状,它直接影响马铃薯的营养和商品价值,一直是马铃薯遗传研究和育种改良的重要目标。本研究通过对二倍体红色薯肉分离群体的混池分析、基因精细定位和候选基因表达分析,确定调控红色薯肉的候选基因,为下一步基因功能、遗传调控研究及彩色马铃薯的分子育种奠定基础。【方法】本研究通过向二倍体红色薯肉亲本导入自交不亲和抑制基因Sli获得BC₁S₁群体,从300个单株中挑选18株红色薯肉和21株黄色薯肉个体提取基因组DNA,分别测序进行混池分析。通过集团分离分析法（bulked segregation analysis,BSA）对基因进行初步定位;在定位区间内开发分子标记,对796份BC₁S₁植株进行基因型分析,筛选交换单株,并结合表型对基因进行精细定位;借助参考基因组注释信息和qRT-PCR表达量分析确定候选基因。【结果】本研究通过构建薯肉颜色分离的二倍体BC₁S₁群体,利用BSA-seq分析把调控薯肉花青素合成的主效位点定位在第10号染色体48.70—52.20 Mb。最终,利用分子标记将该基因定位于51.47—51.85 Mb的377 kb区间内。基于参考基因组注释信息,此区间包括5个基因,其中2个基因注释为MYB类转录因子,结合表达量数据推测这2个基因为候选基因,编号分别为PGSC0003DMG400013966、PGSC0003DMG400013965。【结论】本研究将调控马铃薯薯肉花青素积累的一个主效位点定位于第10号染色体51.47—51.85 Mb之间,推测PGSC0003DMG400013966和PGSC0003DMG400013965为候选基因。

关键词: 红色薯肉, 花青素代谢, BSA-seq, 精细定位, 候选基因

Abstract:

【Objective】 The color of tuber flesh is an important agronomic trait, which directly affects the nutritional and commercial value of potatoes. Accordingly, it is an important goal for potato genetic research and breeding improvement. In this study, we mapped the locus controlling red flesh color by bulked segregant analysis (BSA) and fine mapping, and predicted the candidate genes aided by expression analysis, which provides a foundation for gene function characterization, regulation analysis as well as the molecular breeding of colored potatoes. 【Method】 We crossed a red flesh diploid clone with the Sli gene donor to produce F₁, and the F₁ was backcrossed with the red flesh clone, and finally generated a BC₁S₁ diploid population. From the BC₁S₁ segregation population with 300 individuals, the genomic DNA of 18 red-colored flesh and 21 yellow-colored flesh individuals were extracted respectively and used for sequencing, and the locus controlling red flesh was preliminarily mapped by BSA-seq. The locus was fined mapped through genotyping and phenotyping of the recombination plants from 796 BC₁S₁ individuals. Based on reference genomic annotation and qRT-PCR expression analysis, candidate genes were predicted. 【Result】 In this study, BC₁S₁segregating population with red and yellow-colored tuber flesh was constructed. The major locus which regulates related anthocyanin synthesis in potato flesh was mapped on chromosome 10 within a physical distance between 48.70 Mb and 52.20 Mb. Finally, molecular markers were used to locate the gene into a 377 kb genomic interval from 51.47 Mb to 51.85 Mb. There are five annotated genes in this genomic region based on reference genome annotation information, two of them were MYB transcription factors. We predicted that the two genes, PGSC0003DMG400013966 and PGSC0003DMG400013965, were candidate genes controlling red tuber flesh. 【Conclusion】 In this research, the major locus which regulates accumulation of anthocyanin in potato tuber flesh finally was mapped to chromosome 10 with an interval from 51.47 Mb to 51.85 Mb and predicted the candidate genes, PGSC0003DMG400013966 and PGSC0003DMG400013965.

Key words: red-colored flesh, anthocyanin metabolism, BSA-seq, fine mapping, candidate genes

许芸梅, 李玉梅, 贾玉鑫, 张春芝, 李灿辉, 黄三文, 祝光涛. 马铃薯红色薯肉调控基因的精细定位与候选基因分析[J]. 中国农业科学, 2019, 52(15): 2678-2685.

XU YunMei, LI YuMei, JIA YuXin, ZHANG ChunZhi, LI CanHui, HUANG SanWen, ZHU GuangTao. Fine Mapping and Candidate Genes Analysis for Regulatory Gene of Anthocyanin Synthesis in Red-Colored Tuber Flesh[J]. Scientia Agricultura Sinica, 2019, 52(15): 2678-2685.

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引物名称 Primer name	Indel位置 Indel position	正向引物 Forward (5′-3′)	反向引物 Reverse (5′-3′)
P1	48710951	GAAGAATGGTAGCTGGAGTG	CCGAATATGCATGTATTAGC
P2	49255127	TGTGTCAACGCTAACAACAT	TACCCCAGACAGAATGAGTC
P3	49962534	CCATGGCCTCTAACTTTAGTC	GTAGTTGCAAATGGGTGATT
P4	51413788	AACTCAATAACGGACCAGTG	GATCGGAGAACAAGTTGAAA
P5	52271249	TGGAGGGACTAAAAGTGCTA	TACGTACGTTTCCCTATGCT
P6	51427985	TCCCAGAGACTCCATTAAAA	GTTTGATGAAATGTGCAGTG
P7	51460414	ATTAGCGGTGAAAGAAGACA	AGTAAATGAGAGAGTAGTGCGA
P8	51469924	GTTGTTGAATTATGTTCCTCATACAACA	GGAAAATTGGAAAGAATTTGAGGAA
P9	51473988	AGGAAGAGAGTTCCTTCACCACG	GCTTCTAGCTACTTCTATAATGACGCAG
P10	51723735	GGATCATCATTAGTGGATCG	GAAAGTGTCACGACTTGGAT
P11	51725907	TGTGAAGAACTATTGGAACACTCACTT	CCGAGGTTGAGGTCTTATTA
P12	51732426	GCGCCGAAAGGTTTATATT	CTATGTAAGTCGATTTGGGC
P13	51744513	GGATTATGACGAACCTAATAACCCAA	TTCACCATTCTTGAAGCTAAATGTAA
P14	51845484	TGTTGTTGAACATGCACTCT	AGAAAATAATATAAGTTTAATCCGTTCACT
P15	51850970	ATTTTTCCTACCGTAATCCC	TCACTCTTCAAGATCAATGC

引物名称 Primer name	正向引物 Forward (5′-3′)	反向引物 Reverse (5′-3′)
Actin	GGGATGGAGAAGTTTGGTGGTGG	CTTCGACCAAGGGATGGTGTAG
MYB	GGCTAAATTATCTAAGGCCACATATCAAGAGA	CGAGGAGGAGGAGGAGGAGCAA
R2R3-MYB	GGAGTAATAAGGAAAGGTTCATGGACTGAAG	AGCTTCCTTAGAAGGTTAGTGTTCCAATAGTTT