保罗蓝睡莲花器官转录组SSR的分布及其序列特征分析

毛立彦; 黄秋伟; 於艳萍; 丁丽琼; 蔡元保; 黄歆怡; 覃茜; 苏群; 农晓慧; 朱天龙; 龙凌云

doi:10.19303/j.issn.1008-0384.2024.07.004

保罗蓝睡莲花器官转录组SSR的分布及其序列特征分析

doi: 10.19303/j.issn.1008-0384.2024.07.004

1.
广西壮族自治区亚热带作物研究所/广西亚热带特色水果质量安全控制重点实验室，广西　南宁　530001
2.
广西壮族自治区农业科学院花卉研究所，广西　南宁　530007
3.
广西平果华莲科技研究所，广西　平果　531400
4.
海南佛渡莲源生态农业有限公司，海南　海口　570100

基金项目: 广西壮族自治区自然科学基金青年基金项目（2023GXNSFBA026222）；广西壮族自治区农业科学院基本科研业务专项（桂农科2023YM11、桂农科2021YT152）

详细信息

作者简介:
毛立彦（1988 —），女，硕士研究生，高级农艺师，主要从事园林植物遗传育种方向研究，E-mail：2634081433@qq.com

通讯作者:
龙凌云（1985 —），男，硕士研究生，高级农艺师，主要从事园艺植物遗传育种方向研究，E-mail：longyiyun851123@163.com

中图分类号: S682.32
计量
- 文章访问数: 110
- HTML全文浏览量: 64
- PDF下载量: 25
- 被引次数: 0
出版历程
- 收稿日期: 2024-01-22
- 修回日期: 2024-05-29
- 网络出版日期: 2024-08-15
- 刊出日期: 2024-07-01

Distribution and Properties of SSR in Transcriptome of Nymphaea Paul Stetson Flowers

1.
Guangxi Subtropical Crops Research Institute/Guangxi Key Laboratory of Quality and Safety Control for Subtropical Fruits, Nanning, Guangxi　530001, China
2.
Flower Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi　530007, China
3.
Guangxi Pingguo Hualian Science and Technology Research Institute, Pingguo, Guangxi 531400
4.
Hainan Fodu Lianyuan Ecological Agriculture Co., Ltd., Haikou, Hainan　570100, China

摘要

摘要: 目的通过解析保罗蓝睡莲花转录组数据的SSR位点特征信息，开发新的与表型相关的SSR标记，为睡莲种质资源评价、新品种选育提供科学依据。方法以热带睡莲保罗蓝不同花器官部位（雌蕊、雄蕊、花瓣）的转录组数据为背景材料，用MISA软件检索序列的SSR位点，采用Excel对位点特征进行分析作图，Primer 3.0软件设计引物，TP-M13-SSR PCR筛选引物。结果在花器官转录组的39079条Unigenes中共检测到12365个SSR位点，位点发生频率为31.64%，平均每5.79 kb出现1个SSR位点。SSR位点的重复类型主要为二核苷酸重复碱基，占SSR位点总数的71.85%，优势重复类型为AG/CT，占碱基总数的61.34%；其次为三核苷酸重复碱基，为26.10%，优势重复类型是AAG/CTT，占比8.30%。SSR碱基重复次数在5～20次，序列长度为12～30 bp，平均长度18.38 bp。引物设计获得9212对引物，从中随机挑选100对进行PCR扩增验证，筛选出9对多态性好的引物，可将12份睡莲种质在遗传相似系数为0.735时聚为3支。结论热带睡莲保罗蓝花器官转录组中的SSR位点分布频率高、类型丰富，多态性较高，具较大的应用潜力。开发的9对SSR引物可将12份种质有效分开，进一步丰富了睡莲现有SSR标记，可为睡莲属植物的遗传多样性分析和分子辅助育种提供科学参考。
- 热带睡莲 /
- 转录组 /
- SSR /
- 序列特征
Abstract: Objective SSRs in the transcriptome of Nymphaea Paul Stetson flowers were studied to generate new markers for evaluating germplasms and facilitating breeding of tropical waterlilies. Method SSR loci were retrieved from the transcriptomes of floral pistils, stamens, and petals of Nymphaea Paul Stetson using MISA. Characteristics of the loci were analyzed by Excel, and primers designed by Primer 3.0 and screened by TP-M13-SSR PCR. Result There were 12365 SSR loci found in the 39079 unigenes of the transcriptome at the frequency of 31.64% averaging one SSR locus per 5.79 kb. Most of the SSR loci had dinucleotide repeat motifs comprising 71.85% of total with AG/CT being the dominant unit that made up 61.34% of the motifs. Trinucleotide repeat motifs accounted for 26.10% of the sites with AAG/CTT being dominant at 8.30%. The repeating frequency was 5–20 times with a sequence of 12–30 bp averaging 18.38 bp long. Of the 9212 pairs of primers designed, 100 were randomly selected for a validation by PCR amplification to arrive at 9 pairs with high polymorphism being used as the markers. Subsequently, the 12 germplasms were clustered into 3 branches under a genetic similarity coefficient of 0.7375. Conclusion The SSR loci in the Nymphaea Paul Stetson transcriptome were high on distribution frequency, rich in diversity, greatly polymorphic, and desirable for applications. The 9 pairs of SSR primers identified in this study extended the existing marker repertoire facilitating effective germplasm differentiation on waterlilies.
- tropical waterlily /
- transcriptome /
- SSR /
- sequence analysis

HTML全文

图 1 保罗蓝花转录组中不同重复碱基类型下的SSR分布

Figure 1. SSR distribution of repeat motif types in transcriptome of N. Paul Stetson flowers

下载: 全尺寸图片幻灯片

图 2 保罗蓝花转录组 SSR 重复片段长度分布

Figure 2. Distribution of SSR lengths in transcriptome of N. Paul Stetson flowers

下载: 全尺寸图片幻灯片

图 3 9对引物在蓝星、小花睡莲中的扩增产物荧光检测

Figure 3. Fluorescence detection map of 9 amplified primers in N. colorata and N. micrantha

下载: 全尺寸图片幻灯片

图 4 筛选的9对引物对12份睡莲种质的聚类情况

Figure 4. Clustering of 12 waterlily germplasms determined by 9 selected primer pairs

下载: 全尺寸图片幻灯片

表 1 12份睡莲种质的样品信息

Table 1. Information on 12 waterlily germplasms

编号 No.	样品名称 Sample name	来源 Source	属性 Attribute
1	喀麦隆 Nymphaea zenkeri	海南海口	原种
2	阅卿 Nymphaea Yue Qing	广西都安	园艺杂交种
3	鲁吉娜 Nymphaea rudgeana	江苏南京	原种
4	蓝星 Nymphaea colorata	海南三亚	原种
5	雪白睡莲 Nymphaea candida	海南三亚	原种
6	增殖睡莲 Nymphaea prolifera	海南三亚	原种
7	卡本 Nymphaea carpentariae	海南三亚	原种
8	小花睡莲 Nymphaea micrantha	海南三亚	原种
9	变色澳洲 Nymphaea atrans	云南大理	原种
10	小白子午莲 Nymphaea tetragona	浙江温州	原种
11	我的心 Nymphaea Wode Xin	海南海口	园艺杂交种
12	印度红 Nymphaea rubra	海南三亚	原种

下载: 导出CSV

表 2 保罗蓝花转录组SSR中重复碱基类型分析

Table 2. Type and repeat motifs of SSR loci in transcriptome of N. Paul Stetson flowers

重复碱基类型 Repeat base type	数量 Number	占总SSR比例 Proportion of total SSR/%	出现频率 Frequency of occurrence/%	分布平均距离 Mean distance/kb	平均长度 Average length/bp
单碱基重复 Mononucleotide	91	0.73	0.23	786.23	28.19
双碱基重复 Dinucleotide	8884	71.85	22.73	8.05	18.34
三碱基重复 Trinucleotide	3227	26.10	8.26	22.17	17.96
四碱基重复 Tetranucleotide	86	0.70	0.22	831.95	21.21
五碱基重复 Pentanucleotide	18	0.14	0.05	3974.85	27.50
六碱基重复 Hexanucleotide	59	0.48	0.15	1212.67	32.44
总计 Total	12365	100	31.64	5.79	18.38

下载: 导出CSV

表 3 保罗蓝花转录组中不同重复次数的SSR数量分析

Table 3. Number of SSR with different repetitions in transcriptome of N. Paul Stetson flowers

重复类型 Repeat type	SSR数量 Amount/个																		总计 Total
重复类型 Repeat type	5次	6次	7次	8次	9次	10次	11次	12次	13次	14次	15次	16次	17次	18次	19次	20次	21～29次	30～41次	总计 Total
单核苷酸 Mononucleotide	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	23	64	4	91
二核苷酸 Dinucleotide	0	2273	1518	1204	902	720	566	394	276	240	150	169	107	84	93	42	140	6	8884
三核苷酸 Trinucleotide	1798	718	333	157	79	37	35	19	19	11	7	7	4	1	2	0	0	0	3227
四核苷酸 Tetranucleotide	66	17	2	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	86
五核苷酸 Pentanucleotide	13	3	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	18
六核苷酸 Hexanucleotide	43	12	1	2	1	0	0	0	0	0	0	0	0	0	0	0	0	0	59
总计 Total	1920	3023	1854	1365	982	758	601	413	295	251	157	176	111	85	95	65	204	8	12365

下载: 导出CSV

表 4 保罗蓝转录组的SSR引物信息

Table 4. Information on SSR primers in transcriptome of N. Paul Stetson flowers

重复碱基类型 Repeat base type	符合引物设计要求的 SSR SSRs that meet primer design requirements	引物长度 Primer length/bp	退火温度 Annealing temperature/℃	上、下游引物退火温度差 Difference in annealing temperature between F and R primers/℃
单碱基重复 Mononucleotide	65	20～21	58.98～60.80	＜2
二碱基重复 Dinucleotide	5619	18～27	57.02～62.88	＜4
三碱基重复 Trinucleotide	2502	18～27	57.04～62.74	＜4
四碱基重复 Tetranucleotide	56	20～24	58.90～60.44	＜1
五碱基重复 Pentanucleotide	15	19～20	59.72～60.76	＜1
六碱基重复 Hexanucleotide	44	18～21	59.02～62.02	＜2
复合型重复 Complex repeat type	911	18～27	57.07～62.79	＜4

下载: 导出CSV

表 5 筛选出的9对SSR引物序列信息

Table 5. Information on 9 selected pairs of SSR primers

引物编号 Primer number	重复单元 Repeating motif	引物序列（5′-3′） Primer sequence	等位基因数 Na	有效等位基因数 Ne	Nei’s基因多样性指数 H	Shannon’s 信息指数 I	多态性信息指数 PIC	位点所在染色体编号 Chromosome number of the site
P1	(AG)6	F: CAAAGGCTTACAAGGTTTAACG R: CGCTTGGTTCTATTCGGAAA	3	1.13	0.11	0.22	0.55	1
P20	(GAA)11	F：TTTGCGGAATTTTATAGCCG R：AGGACATTCGCGTACTGCTT	6	1.13	0.11	0.22	0.72	3
P25	(TCA)6	F：TTCTCTGCGAGAGTGCAAGA R：CAAGCAAGCCTCTGGGTTAG	4	1.20	0.16	0.28	0.65	4
P26	(CA)6(GA)11	F：ACATGCATGATGTTCGTGCT R：TTTTGCTGTCAATCTGCGAC	4	1.11	0.10	0.21	0.60	4
P33	(GA)12	F：TGAACGCCACCAGATGTTTA R：CATGAAGCGAAGCAATCTCA	3	1.11	0.10	0.20	0.55	5
P35	(GCA)5	F：ACCAAGGTGATGGTCCAGAG R：ACCCAACGGGGAGTTAAAAG	4	1.31	0.20	0.33	0.69	5
P51	（TC）10	F：TGATCGAGAAAAATGGGGAG R：CACATGCAAGTGTCACACGA	5	1.11	0.09	0.20	0.62	7
P63	(GCA)5	F：TTGGACGCTGCAGAATTTTT R：TCCTTCCACCCATCTCTTTG	4	1.14	0.12	0.23	0.62	9
P77	(TC)11	F：CACCCTATACACGTCCCACC R：GCAGAACCCATGTTCCTGTT	5	1.12	0.11	0.21	0.64	11
平均值 Average			4.22	1.15	0.12	0.23	0.63

下载: 导出CSV

参考文献(34)

[1]	李淑娟, 尉倩, 陈尘, 等. 中国睡莲属植物育种研究进展 [J]. 植物遗传资源学报, 2019, 20(4):829−835. LI S J, YU Q, CHEN C, et al. Breeding progress of waterlilies in China [J]. Journal of Plant Genetic Resources, 2019, 20(4): 829−835. (in Chinese)
[2]	毛立彦, 龙凌云, 黄秋伟, 等. 基于SRAP分子标记的147份睡莲属植物遗传多样性分析 [J]. 南方农业学报, 2023, 54(2):454−466. MAO L Y, LONG L Y, HUANG Q W, et al. Genetic diversity analysis of 147 Nymphaea Linn. plants based on SRAP molecular marker [J]. Journal of Southern Agriculture, 2023, 54(2): 454−466. (in Chinese)
[3]	KALIA R K, RAI M K, KALIA S, et al. Microsatellite markers: An overview of the recent progress in plants [J]. Euphytica, 2011, 177(3): 309−334. doi: 10.1007/s10681-010-0286-9
[4]	苏群, 杨亚涵, 田敏, 等. 睡莲种质资源遗传多样性分析及DNA指纹图谱构建 [J]. 热带作物学报, 2020, 41(2):258−266. SU Q, YANG Y H, TIAN M, et al. Genetic diversity analysis and DNA fingerprinting construction of waterlily germplasm resources [J]. Chinese Journal of Tropical Crops, 2020, 41(2): 258−266. (in Chinese)
[5]	POCZAI P, MÁTYÁS K K, SZABÓ I, et al. Genetic variability of thermal Nymphaea (Nymphaeaceae) populations based on ISSR markers: Implications on relationships, hybridization, and conservation [J]. Plant Molecular Biology Reporter, 2011, 29(4): 906−918. doi: 10.1007/s11105-011-0302-9
[6]	苏群, 王虹妍, 卢家仕, 等. 睡莲的SSR引物对及合成方法和应用: CN113832254A[P]. 2021-12-24.
[7]	PARVEEN S, SINGH N, ADIT A, et al. Contrasting reproductive strategies of two Nymphaea species affect existing natural genetic diversity as assessed by microsatellite markers: Implications for conservation and wetlands restoration [J]. Frontiers in Plant Science, 2022, 13: 773572. doi: 10.3389/fpls.2022.773572
[8]	QIAN Z H, MUNYWOKI J M, WANG Q F, et al. Molecular identification of African Nymphaea species (water lily) based on ITS, trnT-trnF and rpl16 [J]. Plants, 2022, 11(18): 2431. doi: 10.3390/plants11182431
[9]	LIU G, XIE Y J, ZHANG D Q, et al. Analysis of SSR loci and development of SSR primers in Eucalyptus [J]. Journal of Forestry Research, 2018, 29(2): 273−282. doi: 10.1007/s11676-017-0434-3
[10]	OLIVEIRA DE OLIVEIRA L, CARLOS BEISE D, DAMIAN DOS SANTOS D, et al. Molecular markers in Carya illinoinensis (Juglandaceae): From genetic characterization to molecular breeding [J]. The Journal of Horticultural Science and Biotechnology, 2021, 96(5): 560−569. doi: 10.1080/14620316.2021.1892534
[11]	JIANG M, YAN S, REN W C, et al. Genetic diversity of the Chinese medicinal plant Astragali Radix based on transcriptome-derived SSR markers [J]. Electronic Journal of Biotechnology, 2023, 62: 13−20. doi: 10.1016/j.ejbt.2022.12.001
[12]	SHI Z Y, ZHAO W Q, LI Z A, et al. Development and validation of SSR markers related to flower color based on full-length transcriptome sequencing in Chrysanthemum [J]. Scientific Reports, 2022, 12(1): 22310. doi: 10.1038/s41598-022-26664-3
[13]	叶鹏, 李显煌, 唐军荣, 等. 云南金花茶转录组SSR的分布及其序列特征 [J]. 中南林业科技大学学报, 2019, 39(9):86−91. YE P, LI X H, TANG J R, et al. Distribution and characteristics of SSR in transcriptome of Camellia fascicularis [J]. Journal of Central South University of Forestry & Technology, 2019, 39(9): 86−91. (in Chinese)
[14]	辛静, 辛雅萱, 董章宏, 等. 云南火焰兰转录组SSR分布及其序列特征分析 [J]. 南方农业学报, 2020, 51(7):1634−1641. XIN J, XIN Y X, DONG Z H, et al. Distribution and sequence characteristics of SSR in transcriptome of Renanthera imschootiana Rolfe [J]. Journal of Southern Agriculture, 2020, 51(7): 1634−1641. (in Chinese)
[15]	ZHANG L S, YANG X N, QI X N, et al. Characterizing the transcriptome and microsatellite markers for almond (Amygdalus communis L. ) using the Illumina sequencing platform [J]. Hereditas, 2017, 155: 14.
[16]	JIANG B, XIE D S, LIU W R, et al. De novo assembly and characterization of the transcriptome, and development of SSR markers in wax gourd (Benicasa hispida) [J]. PLoS One, 2013, 8(8): e71054. doi: 10.1371/journal.pone.0071054
[17]	TEMNYKH S, DECLERCK G, LUKASHOVA A, et al. Computational and experimental analysis of microsatellites in rice (Oryza sativa L. ): Frequency, length variation, transposon associations, and genetic marker potential [J]. Genome Research, 2001, 11(8): 1441−1452. doi: 10.1101/gr.184001
[18]	梁燕, 韩传明, 孙超, 等. 基于SSR标记的核桃种质资源遗传多样性与遗传结构分析 [J]. 北方园艺, 2022, (9):47−54. LIANG Y, HAN C M, SUN C, et al. Genetic diversity and genetic structure analysis of walnut germplasm resources based on SSR markers [J]. Northern Horticulture, 2022(9): 47−54. (in Chinese)
[19]	CRISTANCHO M, ESCOBAR C. Transferability of SSR markers from related Uredinales species to the coffee rust Hemileia vastatrix [J]. Genetics and Molecular Research: GMR, 2008, 7(4): 1186−1192. doi: 10.4238/vol7-4gmr493
[20]	苏群, 田敏, 刘俊, 等. 基于生物信息学的睡莲SSR位点特征分析 [J]. 西南农业学报, 2021, 34(10):2076−2083. SU Q, TIAN M, LIU J, et al. SSR loci characteristic analysis of water lily based on bio-informatics methodology [J]. Southwest China Journal of Agricultural Sciences, 2021, 34(10): 2076−2083. (in Chinese)
[21]	张华丽, 丛日晨, 王茂良, 等. 基于万寿菊转录组测序的SSR标记开发 [J]. 园艺学报, 2018, 45(1):159−167. ZHANG H L, CONG R C, WANG M L, et al. Development of SSR molecular markers based on transcriptome sequencing of Tagetes erecta [J]. Acta Horticulturae Sinica, 2018, 45(1): 159−167. (in Chinese)
[22]	杜晓华, 杨雅萍, 朱小佩, 等. 三色堇转录组SSR分析及分子标记开发 [J]. 园艺学报, 2019, 46(4):797−806. DU X H, YANG Y P, ZHU X P, et al. Development of genic-SSR markers by transcriptome sequencing in Viola × wittrockiana [J]. Acta Horticulturae Sinica, 2019, 46(4): 797−806. (in Chinese)
[23]	郭聪, 陈燕, 王莹, 等. 美国红枫转录组SSR序列分析 [J]. 中南林业科技大学学报, 2021, 41(7):132−141. GUO C, CHEN Y, WANG Y, et al. Sequence analysis of SSR in transcriptome of American red maple [J]. Journal of Central South University of Forestry & Technology, 2021, 41(7): 132−141. (in Chinese)
[24]	张震, 许彦明, 陈永忠, 等. 油茶转录组测序与SSR特征分析 [J]. 西南林业大学学报, 2018, 38(6):63−68. ZHANG Z, XU Y M, CHEN Y Z, et al. Transcriptome sequencing and analysis of SSR characteristics of Camellia oleifera [J]. Journal of Southwest Forestry University (Natural Sciences), 2018, 38(6): 63−68. (in Chinese)
[25]	李娜, 姚民, 梅兰菊, 等. 基于山桐子转录组序列的SSR分子标记开发 [J]. 应用与环境生物学报, 2017, 23(5):952−958. LI N, YAO M, MEI L J, et al. Development of SSR molecular markers based on transcriptome sequencing of Idesia polycarpa Maxim [J]. Chinese Journal of Applied and Environmental Biology, 2017, 23(5): 952−958. (in Chinese)
[26]	VARSHNEY R K, GRANER A, SORRELLS M E. Genic microsatellite markers in plants: Features and applications [J]. Trends in Biotechnology, 2005, 23(1): 48−55. doi: 10.1016/j.tibtech.2004.11.005
[27]	YANG Q W, JIANG Y J, WANG Y P, et al. SSR loci analysis in transcriptome and molecular marker development in Polygonatum sibiricum [J]. BioMed Research International, 2022, 2022: 4237913.
[28]	杨彬, 许蔷薇, 牛明月, 等. 云锦杜鹃转录组SSR分析及其分子标记开发 [J]. 核农学报, 2018, 32(12):2335−2345. YANG B, XU Q W, NIU M Y, et al. Analysis of SSR information in transcriptome and development of SSR molecular markers in Rhododendron fortunei [J]. Journal of Nuclear Agricultural Sciences, 2018, 32(12): 2335−2345. (in Chinese)
[29]	郭俊, 朱婕, 谢尚潜, 等. 油梨转录组SSR分子标记开发与种质资源亲缘关系分析 [J]. 园艺学报, 2020, 47(8):1552−1564. GUO J, ZHU J, XIE S Q, et al. Development of SSR molecular markers based on transcriptome and analysis of genetic relationship of germplasm resources in avocado [J]. Acta Horticulturae Sinica, 2020, 47(8): 1552−1564. (in Chinese)
[30]	蔡金峰, 杨晓明, 郁万文, 等. 基于苦楝转录组测序的SSR分子标记开发 [J]. 林业科学, 2021, 57(6):85−92. CAI J F, YANG X M, YU W W, et al. Development of SSR molecular markers based on transcriptome sequencing of Melia azedarach [J]. Scientia Silvae Sinicae, 2021, 57(6): 85−92. (in Chinese)
[31]	郝广婧, 祁银燕, 张得芳, 等. 基于转录组的黑果枸杞SSR分布特征分析及引物设计 [J]. 分子植物育种, 2019, 17(13):4342−4350. HAO G J, QI Y Y, ZHANG D F, et al. Analysis of SSR distribution characteristics and primer design of Lycium ruthenicum Murr. based on transcriptome [J]. Molecular Plant Breeding, 2019, 17(13): 4342−4350. (in Chinese)
[32]	HARR B, SCHLÖTTERER C. Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation [J]. Genetics, 2000, 155(3): 1213−1220. doi: 10.1093/genetics/155.3.1213
[33]	ZHANG L S, CHEN F, ZHANG X T, et al. The water lily genome and the early evolution of flowering plants [J]. Nature, 2020, 577(7788): 79−84. doi: 10.1038/s41586-019-1852-5
[34]	刘思思, 乔中全, 曾慧杰, 等. 灰毡毛忍冬转录组SSR位点分析及EST-SSR标记开发 [J]. 分子植物育种, 2021, 19(9):3015−3021. LIU S S, QIAO Z Q, ZENG H J, et al. Analysis on SSR loci in transcriptome and development of EST-SSR molecular markers in Lonicera macranthoides [J]. Molecular Plant Breeding, 2021, 19(9): 3015−3021. (in Chinese)