基于cpInDel标记和cpSSR标记的柑橘属及近缘属植物亲缘关系

doi:10.3864/j.issn.0578-1752.2022.16.012

Abstract

Abstract:

【Objective】The genetic diversity analysis of the chloroplast genome was performed to reveal the genetic evolution of citrus from the cytoplasmic level, so as to provide a reference for the collection, evaluation and utilization of citrus germplasm resources. 【Method】Taking the hypervariable region of Citrus sinensis chloroplast genome as reference, De Novo assemblies were carried out with next-generation sequencing data of representative Citrus and its related genera materials. The assembled sequences were aligned to discover site-variations, and cpInDel markers were designed on the basis of variation sites. SSR sites composed of two or more nucleotide repeats were located in the chloroplast genome of sweet orange, and SSR sites with length variations were adopted to design cpSSR markers. The newly developed markers and reported cpSSR markers were used to detect genetic diversity in 48 samples of Citrus and its relatives that are representative in taxonomy and/or origin through band typing and clustering analysis. 【Result】4 cpInDel markers and 13 cpSSR markers were developed successfully and they could produce clear polymorphic bands in all tested samples. The band pattern of cpIndel was relatively simple. Single marker could distinguish a specific genus or several genera. At species and/or above taxa level, the cpInDel markers gave more convenient and accurate discrimination effect. Similar clustering results were obtained with cpInDel and cpSSR markers, and minor differences were found in the relationship between the lower-rank taxa and the classification of a few genotypes. The cytoplasmic origins of P. trifoliate hybrids were found to be different from P. trifoliata, and those of lemon, limonia and lime hybrid were different from C. medica by both markers. Wangcang zhoupigan, a representative variety of C. speciosa, had a different cytoplasmic origin from other mandarin varieties. Ziyang xiangcheng and Korean xiangcheng, both classified as C. junos, might have different cytoplasmic origin. cpSSR markers classify mandarin, sweet orange and sour orange, ichang papeda and honghe papeda into three clusters, representing C. reticulata, C. sinensis and Papeda, while cpInDel markers showed that they were closely related and classified into one cluster. Indian wild mandarin were clustered into C. medica by cpInDel, and were put into a single cluster by cpSSR. 【Conclusion】Molecular marker analysis of chloroplast genome could reveal the genetic relationship of Citrus and its related genera and discover a different relationship from the view of nuclear genome. However, if combined with markers from nuclear genome, more comprehensive and accurate indentification and phylogenetic relationship revealing should be obtained for the citrus germplasm resources.

Key words: Citrus, chloroplast genome InDel, chloroplast genome SSR, genetic evolution, germplasm

YANG Cheng,GONG GuiZhi,PENG ZhuChun,CHANG ZhenZhen,YI Xuan,HONG QiBin. Genetic Relationship Among Citrus and Its Relatives as Revealed by cpInDel and cpSSR Marker[J].Scientia Agricultura Sinica, 2022, 55(16): 3210-3223.

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Figures/Tables 7

Table 1

Forty-eight genotypes used in this study"

编号 Code	材料名称 Common name	学名 Scientific name
1	莽山野柑 Mangshan yegan	C. mangshanensis S. W. He & G.F.Liu
2	贺州姑婆山野柑 Hezhou guposhan yegan	C. spp
3	印度野橘 India wild mandarin	C. indica Tanaka
4	道县野橘 Daoxian yeju	C. daoxianensis S. W. He & G.F.Liu
5	印度酸橘 Cleopatra mandarin	C. reticulata var. austera
6	岑溪酸橘 Cenxi suan ju	C. sunki Hort ex Tanaka
7	茶枝柑 Chazhi gan*	C. chachiensis Hort.
8	安江红橘 Anjiang hongju	C. tangerina Hort. ex Tanaka
9	万州少核红橘 Wanzhou seedfew hongju	C. tangerina Hort. ex Tanaka
10	新生系3号椪柑 New No. 3 penggan	C. reticulata Blanco
编号 Code	材料名称 Common name	学名 Scientific name
11	克里曼丁 Clementina	C. clementina Hort
12	兴津温州蜜柑 Okitsu satsuma mandarin	C. unshiu Marc.
13	砂糖橘 Shatangju*	C. reticulata Blanco
14	永顺冰糖橘 Yongshun bingtangju	C. reticulata Blanco
15	资阳香橙 Ziyang xiangcheng	C. junos Sied. ex Tanaka
16	韩国香橙 Korean xiangcheng	C. junos Sied. ex Tanaka
17	旺苍皱皮柑 Wangcang zhoupigan	C. verrucosa Hort.
18	梨橙2号 Licheng No.2	C. sinensis (L.) Osbeck
19	塔罗科血橙 Tarocco blood orange	C. sinensis (L.) Osbeck
20	小叶酸橙 Lobular sour orange	C. aurantium L.
21	意大利酸橙 Italian sour orange	C. aurantium L.
22	尤力克柠檬 Eureka lemon*	C.limon (L.) Burm
23	里斯本柠檬 Lisbon lemon	C.limon (L.) Burm
24	来檬杂种 Lime hybrid*	C. spp
25	北京柠檬 Meyer lemon	C. meyerii Y. Tan.
26	红藜檬 Hong limeng*	C. limonia Osbeck
27	南川佛手 Nanchuan buddha's hand*	C. medica L.
28	合江佛手 Hejiang buddha's hand	C. medica L.
29	云南大香橼 Yunnan daxiangyuan*	C. medica L.
30	小香橼 Xiao xiangyuan	C. medica var. ethrog Engl.
31	白玉霜 Baiyushuang*	C. grandis (L.) Osbeck
32	红肉琯溪蜜柚 Hongrou guanximiyou*	C. grandis (L.) Osbeck
33	金刀峡宜昌橙 Jindaoxia ichang papeda	C. ichangensis Swingle
34	元江宜昌橙 Yuanjiang ichang papeda	C. ichangensis Swingle
35	宜昌橙2586 Ichang papeda 2586*	C. ichangensis Swingle
36	红河大翼橙 Honghe papeda*	C. hongheensis Y. L. D. L
37	金豆 Jindou kumquat*	F. hindsii Swingle
38	罗纹 Round kumquat	F. japonica Swingle
39	金弹 Meiwa kumquat*	F. crassifolia Swingle
40	罗浮 Nagami kumquat	F. margarita Swingle
41	四季橘 Calamindin	C. madurensis Lour
42	旺苍大叶枳 Wangcang daye trifoliate orange	P. trifoliata
43	飞龙枳 Flying dragon trifoliate orange*	P. trifoliata var. Monstrosa
44	富民枳 Fumin trifoliate orange	Poncirus polyandra S. Q. Ding et al
45	枳杂-3 Trifoliate hybrid 3	P. spp
46	卡里佐枳橙 Carrizo citrange	C. sinensis (L.) Osbeck×P. trifoliata (L.) Raf.
47	澳指檬 Australian finger lime	Microcitrus australasica (F.Muell) Swingle
48	澳沙檬 Austrlian Desert lime	Eremocitrus glauca (Lindl.) Swingle

Table 1

Table 2

Fig. 1

Table 3

Fig. 2

Fig. 3

Fig. 4

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引物名称 Primer name	正/反向引物 Forward/Reverse primer	预期产物大小 Excepted size (bp)	扩增总条带数 Amplified bands	期望杂合度 He	多态信息含量 PIC
HVar-cpINDEL1	AATCCACTCAGCCATCTC	464	8	0. 73	0.69
HVar-cpINDEL1	CCCTTTCTACGGTTYAGY	464	8	0. 73	0.69
HVar-cpINDEL2	GTGCGAACCATACCATAA	273	5	0.64	0.56
HVar-cpINDEL2	GTCAGGAGTCCTCGTAAA	273	5	0.64	0.56
HVar-cpINDEL3	AYGAAATAACTCCTCCTT	178	4	0.26	0.24
HVar-cpINDEL3	TACCACTAAACTATACCC	178	4	0.26	0.24
HVar-cpINDEL4	CSAAAGAATCGGTTAMAT	491	4	0.42	0.37
HVar-cpINDEL4	TDCGAATCCTTTTGTTTA	491	4	0.42	0.37

引物名称 Primer name	正/反向引物 Forward primer/Reverse primer	预期产物大小 Excepted size (bp)	扩增总条带数 Amplified bands	期望杂合度 He	多态信息含量 PIC
cpSSRY1	TATCTCCGTGTCAACCAA	180	9	0.81	0.78
cpSSRY1	GGGCAACCCATTCTTATT	180	9	0.81	0.78
cpSSRY2	CAAATCCTATTGGACGCA	291	6	0.58	0.54
cpSSRY2	TCAGGAACAAAAGGAACG	291	6	0.58	0.54
cpSSRY3	TTCTGAACCGGCCCTTGC	247	2	0.19	0.17
cpSSRY3	ACGGGTCAATAAAGCATAGCAG	247	2	0.19	0.17
cpSSRY4	CCGCTCAATCAACGACTT	227	5	0.73	0.68
cpSSRY4	TAATCGAAAGGCTGCTCT	227	5	0.73	0.68
cpSSRY5	GGTGACACTCGCCGCTAT	170	2	0.48	0.36
cpSSRY5	TGAACCGATGACTTACGC	170	2	0.48	0.36
cpSSRY6	GGGATACACGACAGAAGG	160	6	0.78	0.74
cpSSRY6	AGATGGTGCGATTTGATT	160	6	0.78	0.74
cpSSRY7	GACTCCGCAAATTCACCA	249	4	0.48	0.38
cpSSRY7	TCCGAGCAGAACATCAAACT	249	4	0.48	0.38
cpSSRY8	ATTTGAACCCGTGACATT	349	2	0.04	0.04
cpSSRY8	AGGCCCACTTGTATTTGT	349	2	0.04	0.04
cpSSRY9	TTTTACGAACTGAAGCCCTTAT	278	4	0.30	0.27
cpSSRY9	AGGGATGCGACCCGTTTT	278	4	0.30	0.27
cpSSRY10	AATCTGGGTTCTTCTACTTC	188	4	0.47	0.43
cpSSRY10	TCTATTTCGTACCCTTCG	188	4	0.47	0.43
cpSSRY11	AATAGGTATCTAAGGGTG	229	4	0.33	0.31
cpSSRY11	TAGTTTTACGGTTCATTTCATTATC	229	4	0.33	0.31
cpSSRY12	TTCCCTTTTGTAATGTTTTG	205	9	0.83	0.80
cpSSRY12	GTTTATGGTGGGGTGATG	205	9	0.83	0.80
cpSSRY13	ATCTATTTCGTACCCTTCG	189	7	0.81	0.77
cpSSRY13	AATCTGGGTTCTTCTACTT	189	7	0.81	0.77
cpSSR1	AACGGAAAGAGAGGGATTCG	161	12	0.82	0.80
cpSSR1	ACGGGCTTTTTCAAGCATTA	161	12	0.82	0.80
cpSSR2	TCGTATTCTCGAACCCCTTTT	243	10	0.78	0.75
cpSSR2	ATAAATTGCATGGCCGTACC	243	10	0.78	0.75
cpSSRH4	GCTATCCGCCAAGGTAAA	223	9	0.67	0.63
cpSSRH4	GGTTCAAATCCCGTCTCC	223	9	0.67	0.63
cpSSR5	TGATCCCACAAACAAAGGAA	247	5	0.70	0.63
cpSSR5	TTTGAGTCTGGGGAAAAGGA	247	5	0.70	0.63
cpSSR6	TCAAATGGGTTTGAGGTTGA	197	7	0.77	0.72
cpSSR6	GGCGTCCAAAATGCCTATAA	197	7	0.77	0.72
cpSSR21	CAACGAGTCGCACACTAAGC	228	5	0.76	0.70
cpSSR21	TACGGGGTATTGGGAATCAA	228	5	0.76	0.70

Genetic Relationship Among Citrus and Its Relatives as Revealed by cpInDel and cpSSR Marker

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