利用Target SSR-seq技术鉴定60份柑橘种质资源

doi:10.3864/j.issn.0578-1752.2022.22.011

Abstract

Abstract:

【Background】 Bud sports mutation is a DNA mutation occurred in somatic meristem, and it often displays visible morphological and other characteristic changes different from its mother plants in branches, leaves, flowers and fruits. However, the discriminating bud sports mutation from the epigenetic variation caused by environmental conditions and cultivation measures etc. external factors was still mostly depended on the molecular fingerprint detection. 【Objective】 This study objective was to identify citrus bud sports mutant through Target-SSR sequencing technology.【Method】Firstly, the genome of clementine mandarin (Citrus reticulata Blanco) and satsuma (Citrus unshiu Macf.), as well as GSS and EST sequences of satsuma were used to scan SSRs loci with GMATA, and the highly polymorphic SSRs loci were screened out to design primers. The multiplex PCR with optimized primers were ampllified on 60 citrus bud sports mutants to construct high-thoughout sequencing library, and the amplification products were then sequenced on illumina Minseq platform. The clean sequencing short reads were mapped to reference target sequences to find differentiated SSRs loci presented in citrus bud sports mutants.【Result】 A total of 77 pairs of SSR primers were designed from highly polymorphic SSRs loci. The primer pair combinations were optimized and 18 multiplex PCR amplification products were sequenced. The target SSR-seq analysis showed that the genotyping data of SSRs could divided 60 citrus accessions into two groups corresponding to sweet orange and mandarin, and the mandarin group could be further subdivided into different citrus cultivars, such as Orah, ponkan etc. 11 SSR loci containing mostly ATT motif were found in 7 Tarroco blood orange mutants, 8 SSR loci containing mostly TAA motif were found in 2 ‘Wu Yue Hong’ mutants and 5 navel orange, 16 SSR loci containing mostly GA motif were found in 9 Bing Tang Cheng mutants, 9 SSR loci containing mostly AAT motif were found in 2 Sha Tang Ju mutants, and 15 SSR loci with mostly AAT motif were found in 4 satsuma mutants. This study showed that Target-SSR sequencing technology provided an excellent resolving approach to discriminate citrus bud mutations.【Conclusion】 In this study, an effective method for citrus bud mutant identification by using Target SSR-seq technology were established, and 60 citrus germplasm accessions could be discriminated. The precision and reliability of SSR genotyping information could be utilized in citrus germplasm resources management and variety intellectual property protection.

Key words: citrus, bud sports, Target SSR-seq

ZHU YanSong,ZHANG YaFei,CHENG Li,YANG ShengNan,ZHAO WanTong,JIANG Dong. Identification of 60 Citrus Accessions Using Target SSR-seq Technology[J].Scientia Agricultura Sinica, 2022, 55(22): 4458-4472.

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Figures/Tables 12

Table 1

Information of 60 sequencing samples"

类群 Group	编号 Code	名称 Name	类群 Group	编号 Code	名称 Name
甜橙类 C. sinensis Osbeck	1	锦秀冰糖橙 Jin Xiu Bing Tang Cheng	杂柑类 C. reticulata Blanco	31	清见 Kiyomi
	2	锦红大果冰糖橙 Jin Hong Da Guo Bing Tang Cheng		32	黄皮清见 Yellow Peel Kiyomi
	3	锦红最红冰糖橙 Jin Hong Zui Hong Bing Tang Cheng		33	不知火 Shiranui
	4	锦玉冰糖橙 Jin Yu Bing Tang Cheng		34	黄皮不知火 Yellow Peel Shiranui
	5	椭圆冰糖橙 Tuo Yuan Bing Tang Cheng		35	红美人 Beni Madonna
	6	冰糖橙仁4 Bing Tang Cheng Ren 4		36	黄皮红美人 Yellow Peel Beni Madonna
	7	冰糖橙仁5 Bing Tang Cheng Ren 5	温州蜜柑 C. unshiu Mac.	37	宫川温州蜜柑 Miyagawa unshiu
	8	品质特异5号冰糖橙 No. 5 Bing Tang Cheng		38	大分4号温州蜜柑 Oita No.4 Wase
	9	新冰30号冰糖橙 Xin Bing 30 Hao Bing Tang Cheng		39	大浦5号温州蜜柑 Ooura No.5 Wase
	10	橘湘元 Ju Xiang Yuan		40	大分1号温州蜜柑 Oita No.1 Wase
	11	塔罗科血橙1 Tarroco blood orange No. 1		41	青皮蜜橘 Qing Pi Mi Ju
	12	塔罗科血橙2 Tarroco blood orange No. 2		42	金葵早熟蜜橘 Jin Kui Early Mi Ju
	13	塔罗科血橙3 Tarroco blood orange No. 3	椪柑 C. reticulata Blanco	43	蜂洞橘椪柑 Feng Dong Ju Ponkan
	14	塔罗科血橙4 Tarroco blood orange No. 4		44	衢州椪柑 Qu Zhou Ponkan
	15	塔罗科99 Tarroco No. 99		45	兴春椪柑 Kousyun Ponkan
	16	塔罗科新系 Tarroco New Line		46	早蜜椪柑 Zao Mi Ponkan
	17	塔罗科血橙(Thermal) Tarroco blood orange Thermal		47	黔阳无核椪柑 Qian Yang Seedless Ponkan
	18	卡拉卡拉 Cara Cara		48	立山64 Li Shan 64 Ponkan
	19	晚红血橙 Wan Hong blood orange		49	椪柑 Ponkan
	20	五月红 Wu Yue Hong	沃柑芽变 C. reticulata Blanco	50	无核沃柑9号 Seedless Orah No.9
	21	五月红51-13 Wu Yue Hong 51-13		51	无核沃柑10号 Seedless Orah No.10
	22	班菲 Barfield navel orange		52	无核沃柑8号 Seedless Orah No.8
	23	赣脐4号 Gan Qi No.4		53	皱皮沃柑 Wrinkle Orah
	24	贺脐1号 He Qi No.1		54	武鸣沃柑 Wu Ming Orah
	25	纽荷尔 Newhell navel orange		55	有核沃柑 Orah
	26	奉节晚脐 Feng Jie late navel orange		56	少核沃柑 Seedless Orah
	27	桂橙1号 Gui Cheng No. 1		57	无核沃柑 Orri
	28	石棉纽荷尔 Shi Mian Newhell navel orange	沃柑杂交 C. reticulata Blanco	58	沃柑×青皮蜜橘 Orah × Qing Pi Mi Ju
砂糖橘 C. reticulata Blanco	29	砂糖橘 Sha Tang Ju		59	少核沃柑×塔罗科血橙 Seedless Orah × Tarroco blood orange
砂糖橘 C. reticulata Blanco	30	无核砂糖橘 Seedless Sha Tang Ju		60	沃柑×甘平 Orah × Kanpei

Table 1

Fig. 1

Table 2

Table 3

SSR primer sequences used in this study"

编号 Code	正向序列 Forward	反向序列 Reverse
>MK1	AATGACGACGACAACGATGA	TGGAATCGGAATGGATTGTT
>MK2	TGAAATTGACGATGGAGAGAAG	ATGGCAACTTCTCCAGCAAA
>MK3	CGATGGAGAGAAGTAACGAAGAA	GGTCCAAAATCAACAAATGG
>MK4	CGAAGAAATTGCACAAGAGAGA	GGTCCAAAATCAACAAATGG
>MK5	GAAACCTTGCTGCGCTTTT	CACCTTGATGTAGAAAGCATGA
>MK6	TTTACAGCCCTAAGCGGAAA	CGAAAATGCCCTTTGTTGAT
>MK7	CTGGGGCTCACATAAATCGT	GATTTGTCCGCCAATCAAGT
>MK8	GCATTGCAGCACTTTTGTCAT	GATTTGTCCGCCAATCAAGT
>MK9	GCATTGCAGCACTTTTGTCAT	CAAACTCAACTAACGGATGGTAAG
>MK10	ACAACAATGGGGTCAAGCAC	CCACTGTCAAACTCCAAAATGA
>MK11	AATGGGGTCAAGCACATTTT	CAAAGGAAACACGAATTCAACC
>MK12	GGTGCAACACATCTCAAAGC	TTTGTCGAAGATGGTCGATT
>MK13	CAGCTGTTCTGAGAGACTTTTCTT	TCCGTACTGTTTGGGATTACG
>MK14	CCTTCTCGAATGAAAACATCCT	TCCGTACTGTTTGGGATTACG
>MK15	GATGAAGTGGGCAGTTACGTT	TTGGGCTTGACATAAAAGCA
>MK16	AGCTACCCACCTCGTTGAAT	ACCAGTACCACTGCTTACTCTTTT
>MK17	CCCACCTCGTTGAATCTCTC	ACCAGTACCACTGCTTACTCTTTT
>MK18	CAATGATACACCCCAGAGCA	ACCAGTACCACTGCTTACTCTTTT
>MK19	GGGTTGGGAATGTGAATGAA	CCTAGGGGTGGGCATATTTT
>MK20	GTGCATCACACCACAGCTTC	TGGCATGAGGGTAGTCAATTTT
>MK21	TCGATGTCATGTGTTATGCTTG	AAGTAACACGGGATGGTTGC
>MK22	TGTGTTATGCTTGTTAGACACCTG	AAGTAACACGGGATGGTTGC
>MK23	AGGCATGAGAGAGAGTTGACTT	CGAGGTTATCATCCGAATCC
>MK24	AAATGCAATGTGGGCTATGC	TTCTGGAAGTGAGCCACAAG
>MK25	GCAATGTGGGCTATGCATTT	CCTTCCCTGAATTCGAGCAT
>MK26	GGTGATGATGAATGCGTGAA	TCTCGTCTGCTAATCGCATC
>MK27	GATGAATGCGTGAAATCGAC	CGTCTGCTAATCGCATCTTTC
>MK28	CGTAAGGCTCGCTATGTCCT	AATCGATCGGCTGGACTAAA
>MK29	CCACCGTCGAGGTCATTTAT	AAATCGATCGGCTGGACTAA
>MK30	GGGCAGTGGCAATAAGAATG	ATGGGCTTCAACACACATGA
>MK31	TCAGCACCACAAGTCAATCC	ACATGGGTGGAGAGCAAACT
>MK32	TCAGCACCACAAGTCAATCC	GAAATCTGGGGTTCAAATCG
>MK33	CATTCTTTCCCTCCACTCCA	AACCCAATGCTTGCTTTTGT
>MK34	ATGAGGCTTGCGAGGTTTA	CAAGGCCAGGTGGACAAATA
>MK35	TGAGGCTTGCGAGGTTTAT	CAAATACCAGAGCCAAATCTCC
>MK36	GGCTGTTGCCGCATTAGTTA	ACAAGGCTCCAGCGACAG
>MK37	GCCAAAGCCCAAACTCAATA	ACGAGAGCCAACCTGACATC
编号 Code	正向序列 Forward	反向序列 Reverse
>MK38	TTGCTTTTGTACTCCTTTAGGC	ACGAGAGCCAACCTGACATC
>MK39	TTGCTTTTGTACTCCTTTAGGC	CCCAATGCTTTTCCATTTTC
>MK40	AATTAAATGCACGGCAAGGA	CAAATCTCCACTTGCAGACG
>MK41	ATTAAATGCACGGCAAGGAG	CAAATCTCCACTTGCAGACG
>MK42	ATTAAATGCACGGCAAGGAG	AGGCATAGATGATCCACCTCA
>MK43	GTGCCACTACTGTTACGTTCTTTT	TTAGTTCATGGGTGAATGGTG
>MK44	GCCTTCATTTCTTGGTTGATG	ATGGAAAATTGGGGAAAAGC
>MK45	TCATTGGCACCATCATCATT	ATGGAAAATTGGGGAAAAGC
>MK46	TATCATTGGCACCATCATCA	GGCCACGTCATTCAAGAAAT
>MK47	TCATTGGCACCATCATCATT	AGTAGGCCACGTCATTCAAG
>MK48	TCATTGGCACCATCATCATT	TTGAAATAGTAGGCCACGTCA
>MK49	TCATTGGCACCATCATCATT	GGAAAATTCCCTAATCCTGAGA
>MK50	AAAAGGTCATGTGCATCCAA	ATTGGCAGCATGCAAGATA
>MK51	AAAAGGTCATGTGCATCCAA	TGGTGGCAGTAGTGTTGTAGTAA
>MK52	TCTTGCATGCTGCCAATAA	TGGTGGCAGTAGTGTTGTAGTAA
>MK53	GTTTTCTATTCGGCCATCCA	CGAAGGCAATTTGGGATGTA
>MK54	CCAGCATGCATATGGCTAGA	GGGCCAAATATTATAACGAAGG
>MK55	CCAGCATGCATATGGCTAGA	CGAAGGCAATTTGGGATGTA
>MK56	CCAGGTGAATCCAAACAGTACA	TATGCATTCGTCGTGATGGT
>MK57	CACCCATTAATATTGACTTCCTTGC	TTTACACCGTGGGAGGTTCT
>MK58	TACATCAAGCAAGCCACGAG	AGTGAGCAGGGAGCTCAAAA
>MK59	TACATCAAGCAAGCCACGAG	TGCATACGTCAGTAGAAAAGATGAG
>MK60	TGATTCCAAGACGCCTCCTA	ACGTGTACCGTTGAAGTGGA
>MK61	TTCAATACCCCAAACGTAACC	AGGATGGCTACGATGTCTGAA
>MK62	ACCCGAACCGAATTTTAACC	TTTTCTCAGACCTGATTCACCA
>MK63	GCGCTCACCCCTAATGTAAA	TTTTCTCAGACCTGATTCACCA
>MK64	ACAGCAAGGAAGGGGAAGAT	TGGGTTCAGGGATTTTATCAG
>MK65	CAACTATGCTACGCGTTTATTTGAC	CAGCTTATTGATGAACCTGCAA
>MK66	CAACCCCATTGTTAGGTAATTG	GCTCAGCAACAGCAACTGG
>MK67	CCGCAACAAAATCAAGTCAA	GGGTCTCTAGAAAACTTTCAACCA
>MK68	AAAATGGCCACAATGAGCTT	GGTCAATTTGGAGGTTCTTCTT
>MK69	TCTCAATCCCACAAATTAGGC	TTTTGACACCCCGTAACAACT
>MK70	TCTCAATCCCACAAATTAGGC	GAGAAGTCTACAAAATGGAACCTCA
>MK71	CGTGGCCCAACCTCAATTA	TCCATGTTTGCTGATGTAGGA
>MK72	ACGAAATCCTGGGAGGAAAG	TCCATGTTTGCTGATGTAGGA
>MK73	TGATCACACAACATGAGACCAG	TGAGCCTGATAATCCCTCCA
>MK74	GCGGCGGAATTTATACCTCT	ATTGGTAATTGCTGGGCATC
>MK75	GCGGCGGAATTTATACCTCT	AGCACCCTTACTTGCTGTGA
>MK76	GAGAAGTTGCCTGGTGATCG	GCGAATGGTCAGTTTTGCAC
>MK77	GAGAAGTTGCCTGGTGATCG	GAAATTTTGCCGCGAATG

Table 3

Fig. 2

Fig. 4

Fig. 3

Fig. 5

Fig. 6

Table 4

Primers with SSR site differences can be amplified in different strain resources"

品系 Strain	编号 Code	资源名称 Name	可扩增具有SSR位点差异的引物 Primers with SSR site differences can be amplified
塔罗科血橙 Tarroco Blood Orange	11	塔罗科血橙1 Tarroco blood orange No.1	mk16、mk16F/mk23F、mk23F/mk53R、mk34、mk35、mk42、mk44、mk53、mk56、mk57、mk67
	12	塔罗科血橙2 Tarroco blood orange No. 2
	13	塔罗科血橙3 Tarroco blood orange No. 3
	14	塔罗科血橙4 Tarroco blood orange No. 4
	15	塔罗科99 Tarroco 99
	16	塔罗科新系 Tarroco New Line
	17	塔罗科血橙(Thermal) Thermal blood orange Tarroco
五月红 Wu Yue Hong	20	五月红 Wu Yue Hong	mk23F/mk53R、mk53、mk16F/mk23F、mk44、mk56、mk66、mk34、mk23F/mk53R
五月红 Wu Yue Hong	21	五月红51-13 Wu Yue Hong 51-13
脐橙 Navel orange	23	赣脐4号 Gan Qi No.4	mk16F/mk23F、mk23F/mk53R、mk23F/mk53R、mk34、mk44、mk53、mk56、mk66
	24	贺脐1号 He Qi No.1
	25	纽荷尔 Newhell navel orange
	26	奉节晚脐 Feng Jie late navel orange
	27	桂橙1号 Gui Cheng No. 1
冰糖橙 Bing Tang Cheng	1	锦秀冰糖橙 Jin Xiu Bing Tang Cheng	mk2、mk5、mk16、mk16F/mk23F、mk23、mk23F/mk53R、mk32、mk33、mk35、mk42、mk44、mk54F/mk71R、mk59、mk60、mk62、mk66
	2	锦红大果冰糖橙 Jin Hong Da Guo Bing Tang Cheng
	3	锦红最红冰糖橙 Jin Hong Zui Hong Bing Tang Cheng
	4	锦玉冰糖橙 Jin Yu Bing Tang Cheng
	5	椭圆冰糖橙 Tuo Yuan Bing Tang Cheng
	6	冰糖橙仁4 Bing Tang Cheng Ren 4
	7	冰糖橙仁5 Bing Tang Cheng Ren 5
	8	品质特异5号冰糖橙 No. 5 Bing Tang Cheng
	9	新冰30号冰糖橙 Xin Bing No.30 Bing Tang Cheng
砂糖橘 Sha Tang Ju	29	砂糖橘 Sha Tang Ju	mk5、mk6F/mk26F、mk23F/mk53R、mk34、mk35、mk42、mk44、mk56、mk66
砂糖橘 Sha Tang Ju	30	无核砂糖橘 Seedless Sha Tang Ju	mk5、mk6F/mk26F、mk23F/mk53R、mk34、mk35、mk42、mk44、mk56、mk66
温州蜜柑 Satsuma	37	宫川温州蜜柑 Miyagawa unshiu	mk2、mk5、mk6F/mk26F、mk16F/mk23F、mk23F/mk53R、mk23F/mk53R、mk32、mk34、mk35、mk42、mk56、mk59、mk62、mk66、mk76
	38	大分4号温州蜜柑 Oita No.4 Wase
	39	大浦5号温州蜜柑 Ooura N0.5 Wase
	40	大分1号温州蜜柑 Oita No.4Wase
沃柑芽变品种 Budding variety of Orah	50	无核沃柑9号 Seedless Orah No.9	mk2、MK5、mk11R/mk62R、mk16、mk16F/mk23F、mk23F/mk53R、mk32、mk33F/mk38F、MK35、mk42、mk44、mk46F/mk47R、mk53F/mk54R、mk56、mk59、mk62、mk64、mk66、mk6F/mk26F、MK71、mk73F/mk75R
	51	无核沃柑10号 Seedless Orah No.10
	52	无核沃柑8号 Seedless Orah No.8
	53	皱皮沃柑 Wrinkle Orah
	54	武鸣沃柑 Wu Ming Orah
	55	有核沃柑 Orah
	56	少核沃柑 Seedless Orah
	57	无核沃柑 Orri
品系 Strain	编号 Code	资源名称 Name	可扩增具有SSR位点差异的引物 Primers with SSR site differences can be amplified
沃柑杂交品种 Hybrid variety of Orah	58	沃柑×青皮蜜橘 Orah × Qing Pi Mi Ju	mk2、mk5、mk16、mk18、mk23F/mk53R、mk32、mk34、mk62、mk65、mk66、73F/75R
	59	少核沃柑×塔罗科血橙 Seedless Orah × Tarroco blood orange
	60	沃柑×甘平 Orah × Kanpei
椪柑 Ponkan	43	蜂洞橘椪柑 Feng Dong Ju Ponkan	mk2、mk5、mk23、mk23F/mk53R、mk32、mk34、mk35、mk44、mk46F/mk48R、mk53、mk56、mk59、mk62、mk68
	44	衢州椪柑 Qu Zhou Ponkan
	45	兴春椪柑 Kousyun Ponkan
	46	早蜜椪柑 Zao Mi Ponkan
	47	黔阳无核椪柑 Qian Yang Seedless Ponkan
	48	立山64 Li Shan 64 Ponkan
	49	椪柑 Ponkan

Table 4

Table 5

Fig. 7

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类群 Group	位点 Site	引物编号 Primer code	引物序列 Sequence	目标产物大小 Production length (bp)
冰糖橙 Bing Tang Cheng	Scaffold_7 20854883	Neast_1-F	TGCTTGCTTTTGTACTCCTTCT	848
		Neast_1-R	TCGACACTTACGTACGTTGC	848
		Test_1-F	GCTTGGCTGGATCCTACAAA	187
		Test_1-R	ATCTGAGCGGTGCCATATCA	187
沃柑 Orah	Scaffold_7 346954	Neast_2-F	TGGCAGTTGTTAACCCAAGC	646
		Neast_2-R	GCCTTTCGCGCCAACAATTT	646
		Test_2-F	CAACTGTGATGCGTATTTCCG	309
		Test_2-R	GCACAGTCAACTAACGGTCA	309

类群 Group	染色体 CHROM	位置 POS	参考基因 REF	比对基因 ALT	变异信息 INFO
冰糖橙Bing Tang Cheng	Scaffold_7	20854883	A	AAATAATACTAACAAT	11011
沃柑Orah	Scaffold_7	346954	T	TAATAATAATA,TAATAATAATAATA	11001

Identification of 60 Citrus Accessions Using Target SSR-seq Technology

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