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

摘要/Abstract

摘要：

【背景】 芽变是植物分生组织体细胞所发生的DNA突变，从而引起枝、叶、花、果及物候期、成熟期等系列表型特征的改变，其变异性状可遗传。然而由于环境条件、栽培技术等，植物可能产生彷徨变异，这种变异不可遗传。【目的】 利用高通量测序技术建立一套适用于柑橘芽变资源鉴定的方法，为柑橘种质资源的收集、保存、鉴定提供技术支撑。【方法】 为提高芽变材料的分子鉴定能力，本研究通过对‘克里曼丁’以及‘温州蜜柑’全基因组数据以及‘温州蜜柑’GSS、EST数据进行分析、比对，筛选出多态性高的位点用于深度测序。根据引物互补结构对引物进行分组后用于多重扩增检测及构建双端测序文库，构建好的文库通过Miniseq平台完成高通量测序，使用生物信息学方法对下机数据进行后续分析。【结果】 通过对大量测序数据的分析、比对，本研究共设计出77对用于柑橘芽变鉴定的SSR引物，根据引物互补结构，将引物分成18个组合。通过对60份柑橘材料进行Target SSR-seq分析，所设计引物可以将所测试的柑橘种质资源分为甜橙和宽皮柑橘，在宽皮柑橘中可细分为‘沃柑’‘椪柑’以及杂柑等栽培种，对于栽培种内芽变资源也具有较好的鉴别能力。在7个‘塔罗科’血橙芽变中发现11个SSR变异位点，2个‘五月红’芽变中发现8个SSR变异位点，5个脐橙样品中发现8个SSR变异位点，9个‘冰糖橙’品种中发现16个SSR变异位点，2个‘砂糖橘’芽变中发现9个SSR变异位点，4个‘温州蜜柑’芽变中发现15个芽变位点，8个‘沃柑’芽变中发现21个SSR变异位点，‘沃柑’杂交品种中发现11个SSR变异位点，在‘椪柑’中发现14个SSR变异位点。在SSR位点变异中，‘塔罗科’血橙以ATT基序最多，‘五月红’以TAA基序最多，脐橙以TAA基序最多，‘冰糖橙’以GA基序最多，‘砂糖橘’以AAT基序最多，‘温州蜜柑’以AAT基序最多，‘沃柑’芽变资源以AAT基序最多，‘沃柑’杂交资源以TAA、GA基序最多。【结论】 使用Target SSR-seq技术建立了一套高效的柑橘芽变鉴定方法。对所试验的60份柑橘资源具有鉴别能力，SSR基因分型信息准确、可靠，可用于柑橘种质资源管理和品种知识产权保护。

关键词: 柑橘, 芽变, Target SSR-seq

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

朱延松,张亚飞,程莉,杨胜男,赵婉彤,江东. 利用Target SSR-seq技术鉴定60份柑橘种质资源[J]. 中国农业科学, 2022, 55(22): 4458-4472.

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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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.22.011

https://www.chinaagrisci.com/CN/Y2022/V55/I22/4458

图/表 12

表1

60个测序样品信息"

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

表1

图1

表2

表3

本研究使用的SSR引物序列"

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

表3

图2

图4

图3

图5

图6

表4

在不同品系资源中可扩增具有SSR位点差异的引物"

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

表4

表5

图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