基于极端混合池（BSA）全基因组重测序的羽衣甘蓝白色叶基因定位

doi:10.13560/j.cnki.biotech.bull.1985.2023-0275

摘要/Abstract

摘要：

筛选羽衣甘蓝白色叶形成相关的关键基因和位点以及心叶颜色性状的遗传规律，以羽衣甘蓝红叶亲本WR、白叶亲本WB及其构建的F₂分离群体为试验材料，从F₂群体中挑选红叶和白叶植株各20株，分别构建2个DNA混合池，对子代混合池和亲本分别开展50×和20×覆盖深度的全基因组重测序，定位白叶性状关联区间，并根据基因注释信息预测候选基因。结果表明，重测序获得3 987 718个单核酸多态性（SNP）标记，获得位于第2、3和9染色体的8个显著关联区间，根据基因注释和功能分析，筛选到8个候选基因（Bol030253、Bol029431、Bol012077、Bol007709、Bol030318、Bol030235、Bol030286和Bol005195）。将8个基因确定为羽衣甘蓝白叶的候选基因，可能在羽衣甘蓝叶片颜色形成过程中起着重要作用。

关键词: 羽衣甘蓝, 全基因组测序, BSA, 白叶, 基因定位

Abstract:

This study is aimed to screen for key genes and loci associated with white leaf formation in kale（Brassica oleracea L. var. acephala DC）and the genetic patterns of heart leaf color traits. The kale red-leaf parent WR, the white-leaf parent WB and their constructed F₂ segregating populations were used as test materials. Twenty red-leaf and white-leaf plants were selected from the F₂ population, and two DNA mixing pools were constructed. Whole genome sequencing to offspring mixing pool and parents at 50x and 20x cover depth was conducted respectively, and the association interval of white leaf traits was located and candidate genes were predicted according to the gene annotation information. The results showed that re-sequencing 3 987 718 single nucleic acid polymorphism（SNP）markers were obtained via re-sequencing, 8 significant association intervals on chromosomes 2,3, and 9 were obtained, and 8 candidate genes（Bol030253, Bol029431, Bol012077, Bol007709, Bol030318, Bol030235, Bol030286, and Bol005195）were obtained based on gene annotation and functional analysis. Identifying eight genes as candidate genes for white kale leaves may play an important role in the color formation of kale leaves.

Key words: kale, whole genome sequencing, BSA, white leaf, gene mapping

王腾辉, 葛雯冬, 罗雅方, 范震宇, 王玉书. 基于极端混合池（BSA）全基因组重测序的羽衣甘蓝白色叶基因定位[J]. 生物技术通报, 2023, 39(9): 176-182.

WANG Teng-hui, GE Wen-dong, LUO Ya-fang, FAN Zhen-yu, WANG Yu-shu. Gene Mapping of Kale White Leaves Based on Whole Genome Re-sequencing of Extreme Mixed Pool（BSA）[J]. Biotechnology Bulletin, 2023, 39(9): 176-182.

图/表 7

参考文献 28

[1]	孙日飞, 张淑江, 章时蕃, 等. 紫红色大白菜种质的创新研究[J]. 园艺学报, 2006, 33(5): 1032.
	Sun RF, Zhang SJ, Zhang SF, et al. Research on creation of purple Chinese cabbage germplasm[J]. Acta Hortic Sin, 2006, 33(5): 1032.
[2]	Li HB, Zhu LX, Yuan GG, et al. Fine mapping and candidate gene analysis of an anthocyanin-rich gene, BnaA.PL1, conferring purple leaves in Brassica napus L.[J]. Mol Genet Genomics, 2016, 291(4): 1523-1534. doi: 10.1007/s00438-016-1199-7 URL
[3]	Guo N, Wu J, Zheng SN, et al. Anthocyanin profile characterization and quantitative trait locus mapping in zicaitai(Brassica rapa L. ssp. chinensis var. purpurea)[J]. Mol Breeding, 2015, 35(5): 113. doi: 10.1007/s11032-015-0237-1 URL
[4]	Zhu PF, Cheng MM, Feng X, et al. Mapping of Pi, a gene conferring pink leaf in ornamental kale(Brassica oleracea L. var. acephala DC)[J]. Euphytica, 2016, 207(2): 377-385. doi: 10.1007/s10681-015-1555-4 URL
[5]	He Q, Lu QQ, He YT, et al. Dynamic changes of the anthocyanin biosynthesis mechanism during the development of heading Chinese cabbage(Brassica rapa L.) and Arabidopsis under the control of BrMYB2[J]. Front Plant Sci, 2020, 11: 593766. doi: 10.3389/fpls.2020.593766 URL
[6]	叶沈华, 马晓伟, 杨杰, 等. 甘蓝型油菜叶片白化分子机理研究[J]. 植物遗传资源学报, 2023, 1: 1-18
	Ye SH, Ma XW, Yang J, et al. Molecular mechanisms of albino leaves in Brassica napus[J]. J Plant Gen Res, 2023, 1: 1-18.
[7]	Jiang XF, Zhao H, Guo F, et al. Transcriptomic analysis reveals mechanism of light-sensitive albinism in tea plant Camellia sinensis ‘Huangjinju ’[J]. BMC Plant Biol, 2020, 20(1): 216. doi: 10.1186/s12870-020-02425-0
[8]	王容, 李博, 刘刚, 等. 大麦阶段性低温诱导白化突变体的转录组分析[J]. 麦类作物学报, 2023, 43(1): 64-74.
	Wang R, Li B, Liu G, et al. Transcription analysis of stage specific low temperature induced albinism in barley[J]. J Triticeae Crops, 2023, 43(1): 64-74.
[9]	Lu HF, Lin T, Klein J, et al. QTL-seq identifies an early flowering QTL located near flowering locus T in cucumber[J]. Theor Appl Genet, 2014, 127(7): 1491-1499. doi: 10.1007/s00122-014-2313-z URL
[10]	Illa-Berenguer E, Van Houten J, Huang ZJ, et al. Rapid and reliable identification of tomato fruit weight and locule number loci by QTL-seq[J]. Theor Appl Genet, 2015, 128(7): 1329-1342. doi: 10.1007/s00122-015-2509-x pmid: 25893466
[11]	张尧锋, 张冬青, 余华胜, 等. 基于极端混合池(BSA)全基因组重测序的甘蓝型油菜有限花序基因定位[J]. 中国农业科学, 2018, 51(16): 3029-3039. doi: 10.3864/j.issn.0578-1752.2018.16.001
	Zhang YF, Zhang DQ, Yu HS, et al. Location and mapping of the determinate growth habit of Brassica napus by bulked segregant analysis(BSA)using whole genome re-sequencing[J]. Sci Agric Sin, 2018, 51(16): 3029-3039.
[12]	乔军, 刘婧, 李素文, 等. 基于极端混合池全基因组重测序的茄子萼下果色基因预测[J]. 园艺学报, 2022, 49(3): 613-621. doi: 10.16420/j.issn.0513-353x.2021-0018
	Qiao J, Liu J, Li SW, et al. Prediction of fruit color genes under the Calyx of eggplant based on genome-wide resequencing in an extreme mixing pool[J]. Acta Hortic Sin, 2022, 49(3): 613-621.
[13]	严昕, 项超, 刘荣, 等. 基于BSA-seq技术对豌豆花色基因的精细定位[J]. 作物学报, 2023, 49(4): 1006-1015. doi: 10.3724/SP.J.1006.2023.24055
	Yan X, Xiang C, Liu R, et al. Fine mapping of pea flower color genes based on BSA-seq technology[J]. Acta Agron Sin, 2023, 49(4): 1006-1015.
[14]	Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30(15): 2114-2120. doi: 10.1093/bioinformatics/btu170 pmid: 24695404
[15]	Li H, Handsaker B, Wysoker A, et al. The sequence alignment/map format and SAMtools[J]. Bioinformatics, 2009, 25(16): 2078-2079. doi: 10.1093/bioinformatics/btp352 pmid: 19505943
[16]	Mckenna A, Hanna M, Banks E, et al. The genome analysis toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data[J]. Genome Res, 2010, 20(9): 1297-1303. doi: 10.1101/gr.107524.110 pmid: 20644199
[17]	Wang K, Li MY, Hakonarson H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data[J]. Nucleic Acids Research, 2010, 38(16): e164. doi: 10.1093/nar/gkq603 URL
[18]	Takagi H, Abe A, Yoshida K, et al. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations[J]. Plant J, 2013, 74(1): 174-183. doi: 10.1111/tpj.2013.74.issue-1 URL
[19]	Song B, Stöcklin J, Armbruster WS, et al. Reversible colour change in leaves enhances pollinator attraction and reproductive success in Saururus chinensis(Saururaceae)[J]. Ann bot, 2018, 121(4): 641-650. doi: 10.1093/aob/mcx195 URL
[20]	Sun JF, Gong YB, Renner SS, et al. Multifunctional bracts in the dove tree Davidia involucrata(Nyssaceae: Cornales): rain protection and pollinator attraction[J]. Am Nat, 2008, 171(1): 119-124. doi: 10.1086/523953 URL
[21]	Maiwald D, Dietzmann A, Jahns P, et al. Knock-out of the genes coding for the Rieske protein and the ATP-synthase delta-subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression[J]. Plant Physiol, 2003, 133(1): 191-202. doi: 10.1104/pp.103.024190 URL
[22]	Colcombet J, Lopez-Obando M, Heurtevin L, et al. Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles[J]. RNA Biol, 2013, 10(9): 1557-1575. doi: 10.4161/rna.26128 pmid: 24037373
[23]	Liu XY, Jiang RC, Wang Y, et al. ZmPPR26, a DYW-type pentatricopeptide repeat protein, is required for C-to-U RNA editing at atpA-1148 in maize chloroplasts[J]. J Exp Bot, 2021, 72(13): 4809-4821. doi: 10.1093/jxb/erab185 URL
[24]	刘胜坤, 孙世磊, 董鲁朋, 等. 玉米黄叶基因ZmNPPR5的克隆[J]. 植物遗传资源学报, 2023, 24(2): 388-395. doi: 10.13430/j.cnki.jpgr.20220918002
	Liu SK, Sun SL, Dong LP, et al. Cloning of a new yellow leaf gene ZmNPPR5 in maize[J]. J Plant Genet Resour, 2023, 24(2): 388-395.
[25]	Wagner R, von Sydow L, Aigner H, et al. Deletion of FtsH11 protease has impact on chloroplast structure and function in Arabidopsis thaliana when grown under continuous light[J]. Plant Cell Environ, 2016, 39(11): 2530-2544. doi: 10.1111/pce.v39.11 URL
[26]	Xia H, Zhou Y, Lin Z, et al. Characterization and functional validation of β-carotene hydroxylase AcBCH genes in Actinidia chinensis[J]. Hortic Res, 2022, 9: uhac063. doi: 10.1093/hr/uhac063 URL
[27]	Diretto G, Wwlsch R, Tavazza R et al. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers[J]. BMC Plant Biol, 2007, 7(1): 11. doi: 10.1186/1471-2229-7-11
[28]	宋江华, 张立新. 植物跨膜蛋白研究进展[J]. 生物学杂志, 2009, 26(6): 62-64.
	Song JH, Zhang LX. Progress on the transmembrane protein in plants[J]. J Biol, 2009, 26(6): 62-64.

群体 Group	总株数 Total plants	红叶株数 No. of red-leaf plants	白叶株数 No. of white-leaf plants	红叶∶白叶 Red leaf∶White leaf	卡方值 χ²	检验值 χ²_0.05
WR	20	20	0	-	-	-
WB	20	0	20	-	-	-
F₁	20	20	0	-	-	-
F₂	1 300	982	318	3.09∶1	0.20	3.84

群体 Group	总株数 Total plants	红叶株数 No. of red-leaf plants	白叶株数 No. of white-leaf plants	红叶∶白叶 Red leaf∶White leaf	卡方值 χ²	检验值 χ²_0.05
WR	20	20	0	-	-	-
WB	20	0	20	-	-	-
F₁	20	20	0	-	-	-
F₂	1 300	982	318	3.09∶1	0.20	3.84

样品 Sample	Clean reads数 Number of clean reads	比对reads数 Mapped reads	参考基因组比对 Map ratio/%	平均覆盖深度 Average cover depth	基因组覆盖度 Coverage/%	碱基数质量值大于Q20/%
W12	122 570 282	117 542 515	95.90	29.03	88.05	98.35
W06	156 059 918	148 403 662	95.09	35.68	88.38	98.17
L-bulk	370 780 056	352 633 985	95.11	81.94	91.05	98.19
B-bulk	324 884 700	307 268 196	94.58	73.98	91.12	97.81

样品 Sample	Clean reads数 Number of clean reads	比对reads数 Mapped reads	参考基因组比对 Map ratio/%	平均覆盖深度 Average cover depth	基因组覆盖度 Coverage/%	碱基数质量值大于Q20/%
W12	122 570 282	117 542 515	95.90	29.03	88.05	98.35
W06	156 059 918	148 403 662	95.09	35.68	88.38	98.17
L-bulk	370 780 056	352 633 985	95.11	81.94	91.05	98.19
B-bulk	324 884 700	307 268 196	94.58	73.98	91.12	97.81

变异位点信息 Variation sites information	WR	WB	R-bulk	B-bulk
基因区间 Intergenic region	525 758	529 072	555 294	555 413
非同义突变 Non-synonymous	266 962	270 929	283 962	283 910
同义突变 Synonymous	150 618	152 757	159 396	159 535
起始密码子丢失 Start lost	611	628	672	668
终止密码子丢失 Stop_lost	6 645	6 703	7 028	7 075
终止密码子获得 Stop_Gained	13 829	13 994	14 600	14 596
5'非翻译区 UTR_5_Prime	489	484	511	512
SNP总数 SNP total	964 912	979 644	1 021 463	1 021 699