芸薹属同源异源六倍体(BcBcCcCcCoCo)染色体片段缺失的鉴定及其在色泽形成基因定位中的应用

doi:10.3724/SP.J.1006.2023.24287

摘要/Abstract

摘要：

多倍化导致物种的基因组趋于复杂, 基因拷贝数增多, 为性状的遗传解析及基因定位、功能分析带来挑战。染色体片段缺失往往导致相应的表型变化, 而控制这些表型的基因就位于缺失片段内。根据这个原理可以利用染色体缺失品系迅速、准确地将相关基因定位于特定染色体上/染色体区段内。本研究以紫株、黄花埃塞俄比亚芥(Brassica carinata, B^cB^cC^cC^c, 2n=34)为母本与绿株、白花芥蓝(Brassica oleracea, C^oC^o, 2n=18)杂交合成同源异源六倍体(B^cB^cC^cC^cC^oC^o)。在杂种幼胚培养、加倍过程中, 同一个幼胚愈伤组织中分化出2种表型不同的六倍体植株: 紫株、淡黄花与绿株、白花。细胞学观察、荧光原位杂交、基因组重测序分析表明与紫株相比, 绿株基因组B04染色体存在大片段缺失。转录组分析表明, 缺失区段内的DFR基因以及位于参考基因组B03染色体上的MYB90基因可能为控制紫株六倍体紫色形成的关键基因; 而位于缺失区段内的Z-ISO以及CRTISO2则是控制黄色花瓣形成的关键基因。

关键词: 芸薹属, 六倍体, 染色体缺失, 花青素, 类胡萝卜素

Abstract:

Polyploidy leads to the complexity of individual genomes and an increase of gene copies, which brings challenges to the genomic and genetic analysis. The deletion of a chromosome or chromosome fragment often leads to corresponding phenotypic changes, and the genes controlling these traits are located within the deleted fragments. According to this principle, chromosome deletion was usually used to map genes in a specific chromosome or chromosome fragment. In this study, a Brassica auto-allohexaploid (B^cB^cC^cC^cC^oC^o) was synthesized by the hybridization between B. carinata (2n = 34, B^cB^cC^cC^c) and B. oleracea (C^oC^o, 2n = 18). Two young hexaploid plants were differentiated from the callus of the same immature embryo during embryo rescue on MS medium with colchicine and hormone. However, while one plant showed purple leaves and stems and light-yellow flowers as its maternal parent B. carinata, the other plant had green leaves and stems and white flowers like the paternal parent B. oleracea. Cytological observation, fluorescence in situ hybridization (FISH), and genome re-sequencing analysis showed that a large fragment of chromosome B04 was absent in green plant. Transcriptome analysis showed DFR gene in the deletion region and MYB90 on the reference genome B03 chromosome may be the key genes controlling the purple color formation on leaves and stems, while Z-ISO and CRTISO2 were two genes within deletion region determining the formation of yellow flowers in hexaploids.

Key words: Brassica, hexaploids, chromosome deletion, anthocyanin, carotenoids

邱杰, 王泰, 蔡博伟, 段圣省, 徐林珊, 陈晓迪, 王晶, 葛贤宏, 李再云. 芸薹属同源异源六倍体(B^cB^cC^cC^cC^oC^o)染色体片段缺失的鉴定及其在色泽形成基因定位中的应用[J]. 作物学报, 2023, 49(11): 2902-2912.

QIU Jie, WANG Tai, CAI Bo-Wei, DUAN Sheng-Xing, XU Lin-Shan, CHEN Xiao-Di, WANG Jing, GE Xian-Hong, LI Zai-Yun. Identification of chromosome deletion in synthesized Brassica auto-allohexaploids and its application in mapping genes of pigment synthesis[J]. Acta Agronomica Sinica, 2023, 49(11): 2902-2912.

图/表 9

图1

附表1

图2

图3

图4

图5

图6

表1

2种六倍体叶片中花青素合成调控基因的表达分析"

基因号 Gene ID	基因名称 Gene name	基因位置Location	基因表达量Gene expression value (FPKM)						P-adj值 P-adj value
基因号 Gene ID	基因名称 Gene name	基因位置Location	绿1 Green 1	绿2 Green 2	绿3 Green 3	紫1 Purple 1	紫2 Purple 2	紫3 Purple 3	P-adj值 P-adj value
BjuB042150	PAL1	B01	59	43	65	8	9	9	7.13E-20
Bol025522	PAL1	C04	309	235	402	686	885	1275	3.83E-24
Bol037689	PAL1	C04	389	288	495	970	1161	1770	6.05E-27
BjuB041653	PAL1	B06	149	104	199	246	324	521	6.47E-11
Bol005411	PAL2	C06	106	113	116	397	498	626	2.32E-53
Bol004610	C4H	C03	180	141	234	327	339	520	2.47E-13
Bol033347	C4H	C04	72	119	83	3	2	21	3.43E-07
BjuB026422	C4H	B01	4	2	2	181	202	218	8.87E-08
Bol026623	4CL5	C03	120	110	160	187	253	276	4.19E-12
Bol012584	4CL3	C06	294	259	302	451	507	601	3.29E-16
BjuB043976	4CL3	B03	28	9	16	96	113	156	1.62E-16
Bol043396	CHS	C09	789	301	1009	1448	1855	1824	3.57E-07
Bol034259	CHS	C03	217	70	295	2867	3304	4032	1.32E-36
BjuB012368	CHS	B05	645	410	790	1597	1797	1863	1.76E-19
BjuB012366	CHS	B05	55	28	70	122	197	157	2.22E-10
BjuB014803	CHS	B08	65	16	90	202	260	362	5.99E-10
Bol044343	CHI	C08	50	19	57	123	144	287	7.54E-11
BjuB044274	F3'H	B02	2	2	0	20	18	40	2.55E-07
Bol043829	F3'H	C09	9	14	25	614	701	1177	9.51E-88
BjuB001305	DFR	B04^*	0	0	0	682	887	827	1.60E-25
Bol042059	ANS(LDOX)	C07	89	96	90	420	474	492	4.66E-45
BjuB044852	ANS (LDOX)	B02	78	66	96	184	225	348	9.32E-33
BjuB014115	ANS (LDOX)	B05	4	10	6	474	405	424	5.45E-55
Bol014986	ANS (LDOX)	C01	10	15	11	2981	3408	3924	3.28E-210
Bol027055	UGT75C1	C08	10	17	7	2500	3037	3696	2.17E-192
Bol038805	UGT79B1	C09	0	0	2	236	245	294	7.62E-18
BjuB035464	TT19	B02	36	25	26	76	62	102	8.82E-08
BjuB013191	TT19	B05	452	383	574	1012	1158	1550	2.87E-34
Bol021325	TT19	C02	15	11	17	655	750	1075	6.61E-113
Bol019821	TT19	C09	4	4	3	533	696	884	1.44E-65
BjuB046545	MYB12	B06	8	17	12	38	46	76	1.15E-08
Bol016164	MYBL2	C06	45	39	68	445	532	343	2.83E-32
BjuB043935	MYB90 (PAP2)	B03	0	0	0	4052	4792	4563	4.12E-36
BjuB007698	MYB111	B07	15	16	14	39	47	92	1.79E-08
BjuB004115	TT8	B08	7	9	11	43	52	65	1.26E-12
Bol012528	MYB90 (PAP2)	C06	6	9	12	111	89	99	1.01E-20

表1

表2

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引物名称Primer name	连锁群Linkage group	距离 Distance from top	正向引物序列 Forward sequence (5°-3°)	反向引物序列 Reverse sequence (5°-3°)	片段大小 Expected product size (bp)
sJ4933	J11	27.8	TGCAATCAGATCCGACTCAG	CTTGCAAATATTCAGGCCGT	335-341
sJ3891	J11	61.4	TAAACTTGTTGCAGGGGGTT	GTTTGATTGCGTGGCAGTAA	100-110
sJ3302RI	J12	10.0	AAATAACCTGCGACGGTGAC	GGGGCAAAAGGAACTTGAG	392-409
sB4817R	J12	65.8	AGCTTTTCGGTGGGTAAAGA	GCCAGTTAGCGAGTCTTTGG	250-364
sB1822	J13	17.5	TCGTTTATCCCGCGTTTATC	GCACGTTTCTTGCAGACTGA	250-267
sB1752	J13	61.5	AATGGACTCGTCCGTTATGC	TGCGTCTACCACAGACGAAG	406-422
sB2131	J14	0	GTTGCGGTCTCAAGAAGAGG	CCGCTTTAAAAATGCAGCTC	311-321
sA0306	J14	16.8	TGGGGTGGGTCTTAAATGAA	CCGTTTTAAGCCATGGAAGA	362-396
sB0372	J14	20.1	CACACTGCCATCTCTCTCCA	ACGCCGGTTGATATTAGCAC	233-245
sB2141AI	J14	26.8	CCCAAATCCGTGGCTACTTA	TATGATTGGTGGATGGCAAA	381-389
sB1935A	J14	40.4	CCTGCATGAAGGAACGTTTT	AATTCATGGAAACGGCTGTC	257-260
sJ8033	J14	47.7	TTTACGGTTTGGTTTGGCTC	GCTTGACGGCTGAAGAAAAT	150-225
sJ3874I	J15	9.9	ACAACATTTTCCATTCCCCA	ATGAGGTTTCATCCCGACAG	164-185
sB3872	J15	60.9	ACAGAAGCATTAGGCGAGGA	TCCATATAATCACCGCACGA	176-183
sJ7104	J16	35.5	TCCGCTGTCACGATTATGAA	GCTGCAGGGAAACTTGAGAC	322-337
sJ3640I	J16	82.9	CCACTCTCAATCTTCGAGCC	TGGTTGGTTTCAAGACAAAAGA	327-346
sJ13133	J17	25.8	AACCAAACCAAACCAAGTCG	GGCATCGATCAAGTGACCTT	294-300
sJ4633	J17	64.4	CCCGGAAAATAAATGAAAACAA	AACCTGAACGCCATTTTCAC	307-312
sJ34121	J18	24.8	TGCTTAAGTCCATCAATGCG	ACTGATCGAAATGGACTCGG	320-340
sB5162	J18	62.9	CACGGCTCTCAACTCTTTTG	CTGATGAGCTCGAAACCGAT	274-316