Analysis of SSR information in transcriptome and development of EST-SSR molecular markers in Pinus elliottii Engelm.

doi:10.3969/j.issn.1000-2006.201907017

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2020, Vol. 44 ›› Issue (2): 75-83.doi: 10.3969/j.issn.1000-2006.201907017

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Analysis of SSR information in transcriptome and development of EST-SSR molecular markers in Pinus elliottii Engelm.

YI Min¹(), ZHANG Lu¹, LEI Lei¹, CHENG Zishan¹, SUN Shiwu², LAI Meng¹^,^*()

1. 2011 Collaboration Innovation Center of Jiangxi Typical Trees Cultivation and Utilization, Jiangxi Agricultural University, Nanchang 330045, China
2. Baiyun Mountain Forest Farm in Qingyuan District, Ji’an 343062, China

Received:2019-07-12 Revised:2019-09-18 Online:2020-03-30 Published:2020-04-01
Contact: LAI Meng E-mail:yimin6104@163.com;laimeng21@163.com

Abstract

Abstract:

【Objective】Slash pine (Pinus elliotii Engelm.) is a high-quality resin-producing species widely distributed in southern China. Despite it being an important economically important species, genomic and transcriptomic data on this species is scarce, which has hampered its genomic studies. To date, SSR markers used in molecular studies on P. elliottii were mainly those from other related species or developed by using limited gene sequence resources from public databases, which have low polymorphism rate and high generality. In order to solve these problems, we used transcriptome data to develop EST-SSR markers for slash pine. Distribution patterns of the markers in the transcriptome sequences and their characteristics were analyzed in order to lay the foundation for molecular marker-assisted selection ofP. elliottii.【Method】The SSR loci from the transcriptome sequences were analyzed by MicroSAtellite (MISA), and statistical analyses were conducted for the distribution and characteristics of SSR loci. The parameters were set as follows: the SSRs were considered to contain mono-, di-, tri-, tetra-, penta- and hexa-nucleotides with minimum repeat numbers of 10, 6, 5, 5, 5 and 5, respectively. The 120 pairs of EST-SSR primers were designed using Primer 3. Agarose electrophoresis was used for initial check, and capillary electrophoresis was used for separation and detection of the polymorphisms in the primers. In order to study their genetic diversity, 113 samples of families were collected from three seed orchards in South America and from a seed stand in Ji’an,Jiangxi Province.【Result】A total of 79 574 unigenes with 3 818 SSR loci were detected through transcription of slash pine genes. SSR sites occurred with a frequency of 4.80%(number of SSR/number of searching sequences), with an average of one SSR per 18.27 kb. A total of 3 373 EST sequences were screened for SSRs with a frequency of 4.24%(number of sequences with SSRs/number of searching sequences). A total of 2 980 sequences contained single SSRs of different motif types, and 393 sequences contained more than two SSRs. Among the 3 818 potential EST-SSRs, six types of motifs were identified: mononucleotide (63.54%) which had the highest frequency, followed by dinucleotide (19.15%),trinucleotide (16.27%), tetranucleotide (0.52%), pentanucleotide (0.13%) and hexanucleotide repeats (0.31%). The number of repeats of the different SSR motifs varied from 5 to 22, with the exception of mononucleotides. The frequency of five repeats was the highest among all repeats (35.80%), followed by that of six repeats (29.98%) and seven repeats (14.23%). Only 2.73% of ten repeats were found. Among the dinucleotide repeats, AT/AT was the most common motif (12.86%), followed by AG/CT (4.09%) and AC/GT (2.12%). Among the trinucleotide repeats, AAT/ATT was the most common motif, accounting for 3.64% of the total trinucleotide repeats, followed by the AAG/CTT (3.20%). Among all mapped SSRs, the EST-SSR that belonged to the unknown region accounted for 24.59%. SSRs in different genomic regions (5'UTR, 3'-UTR and CDS) showed distinct patterns of distribution. At the genomic level, 3'UTRs had the highest density of SSRs, followed by 5'UTR and CDS. Among the 120 primer pairs, twenty-four pairs (containing 13 di-, 7 tri and four tetranucleotides) showed polymorphism, which accounted for 4.8% of the total number of primer pairs. Eighty-one alleles were tested from twenty-four pairs of fluorescence primers, and the number of alleles ranged from 2 to 9 with a mean value of 3.38. Polymorphic information content ranged from 0.103 to 0.726, with an average of 0.349.【Conclusion】A total of 3 818 SSRs were identified from transcriptome sequencing ofP. elliottii,with AT/AT and AAT/ATT the most common repeats.The amplified primers of the polymorphism loci were mainly dinucleotide and trinucleotide repeats.We concluded that it is feasible to develop the SSR markers based on the P. elliottii transcriptomic sequence, and our results provide new information on genetic diversity analysis and molecular marker-assisted selection of P. elliottii, as well as a basis for SSR marker development in other species.

Key words: Pinus elliottii Engelm. (slash pine), transcriptome, SSR, primer, EST-SSR moleculcy marlcer, mono-nucleotide, di-nucleotides, tri-nucleotides

CLC Number:

S722.8
Q319

YI Min, ZHANG Lu, LEI Lei, CHENG Zishan, SUN Shiwu, LAI Meng. Analysis of SSR information in transcriptome and development of EST-SSR molecular markers in Pinus elliottii Engelm.[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(2): 75-83.

Figures/Tables 8

Table 1

Table 2

Table 3

Fig.1

Table 4

Fig.2

Table 5

Table 6

The information of polymorphism primer"

引物编号 primer No.	引物命名 name	重复基序 repeat motifs	扩增片段大小 amplification fragment size	正向引物序列 forward primer sequence (5'-3')	反向引物序列 reverse primer sequence (5'-3')	多态性条带数 polymorphic band number	多态信息含量(PIC) polymorphism information content
1	Pe103802	(AT)7	141	GGATGATCAGGGCATGAAAT	CATAAAAGTTGGCACCACCA	9	0.679
4	Pe111209	(AC)6	256	AGCTATGTGGACTATGGCGG	CTGTGAAGTGTGAAGCCCAG	3	0.382
8	Pe114582	(TA)6	275	ATACCTAGGCAGATGCCCCT	TTAGGCTGGACAACCCAAAC	2	0.384
16	Pe117651	(GA)6	262	GTTAGCCTCCCTCACCGATT	CACAAACCTTGGTGTTGCAC	2	0.118
19	Pe119033	(AT)6	267	TTCTTGATACATCGGGGCAT	AAACCTGTTCAAATCCTCACAA	3	0.168
28	Pe102606	(AAT)5	162	TTGTTTCACTGCACTCCAGC	GGCATTTTAATGCGGTTGTT	2	0.203
30	Pe103288	(ATT)5	274	GAGGGAAGCTTATCCCGAAC	TCCTTTCATGAGGAGGCACT	6	0.726
32	Pe106732	(AGG)5	163	CGGTGGAAGATTTAGGTCCA	GAAAAACAGCGGCAGAAAAG	4	0.103
38	Pe109562	(CCT)5	209	CAGGAGCCGAAGTCTGAGTC	TTCTGGCCCTCCTCTCTACA	2	0.320
57	Pe139538	(TTAG)5	280	TGAAAGGTGGAGATCCTTGG	AGGTCTGAGAGCATGGAGGA	4	0.400
58	Pe140688	(GATG)5	264	ATGAACGCTTTAGTTCCCCC	GTGATGCGAGATGTGCAGTT	2	0.112
60	Pe146453	(CATT)5	257	CTGATCCACCCTCATCTGCT	GGGAGCAACCAGAACAACAT	3	0.468
61	Pe110222	(TTG)5	277	TCTGTAACTTGGACTGGCCC	CAGCCACAGTAGGTGCAACA	2	0.176
62	Pe111574	(CAC)6	170	ACGGTGTTCCACAGATGGAT	AGAAGATGCAGGAGCCTGAG	2	0.203
63	Pe111823	(CAT)6	181	ATCCGCTCTAGTTCTGCCAA	CAAGTGCGGGCAATCTTTAT	2	0.238
66	Pe137956	(AAAC)5	240	TGGGGTTAGGCTCTCTGCTA	GAATATCCGAATTGCGAGGA	2	0.118
70	Pe106199	(TA)6	257	CCATAGGCCACAGTGAGGTT	TTCTTGGACATTGACCCTCC	2	0.169
79	Pe110432	(CT)7	196	TGGACCTTCCAAACCAAAAG	GACGAGAAGGAGCCGTGTAG	2	0.189
80	Pe112948	(AC)6	263	ACGTTGATCTTTGTGTGCCA	TGAACATCCTCCCTCATTCC	2	0.201
81	Pe113019	(AG)7	240	ATCTAGCGATCCCGGAAGTT	ACCACCTTCTTCCTCCCATT	3	0.590
92	Pe113428	(CT)6	271	ATGCATGGGTTTAGCCTGTC	CCCTGTTTGACTCCTTCCAG	4	0.391
94	Pe114582	(TA)6	275	ATACCTAGGCAGATGCCCCT	TTAGGCTGGACAACCCAAAC	5	0.652
99	Pe115123	(AG)9	275	AAAACCCCAGCTAGGGAAGA	TCTATTGGCTCTTCGCATGT	6	0.672
116	Pe115359	(AT)6	197	AGGGCCAAAACACATCAAAG	ACAACCCTTGGAATGACTGC	7	0.709

Table 6

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来源 source	家系数 number of family
佐治亚州种子园自由授粉家系 open-pollinated families of seed orchard in Georgia State	12
密西西比州种子园自由授粉家系 open-pollinated families of seed orchard in Mississippi State	48
佛罗里达州种子园自由授粉家系 open-pollinated families of seed orchard in Florida State	48
吉安市林科所母树林优良单株混合种子 mixed seeds from plus-trees in the parent-stand of Ji’an Forestry Institute	5

项目 item	数值 value
搜索序列总数 number of searching sequences	79 574
搜索序列总长度/bp length of searching sequences	69 735 997
SSR总数 number of SSR	3 818
含SSR位点的序列数 number of sequences with SSRs	3 373
含1个以上SSR位点的序列数 number of sequences with more than one SSRs	393
SSR发生频率/% occurrence frequency of SSR	4.24
unigene出现频率/% occurrence frequency of unigene	4.80
平均距离/kb average distance	18.27

重复类型 type of repeat	重复单元类型数 number of type	SSR数目 number of SSR	比例/% proportion	频率/% frequency	平均距离/kb average distance	平均长度/bp average length
单核苷酸mononucleotide	4	2 426	63.54	3.05	28.75	11.51
二核苷酸dinucleotide	11	731	19.15	0.92	95.40	14.39
三核苷酸trinucleotide	55	621	16.27	0.78	112.30	15.95
四核苷酸tetranucleotide	20	20	0.52	0.03	3 486.80	22.20
五核苷酸pentanucleotide	5	5	0.13	0.01	13 947.20	25.00
六核苷酸hexanucleotide	13	13	0.31	0.02	5 811.33	33.23
总计 total	108	3 818	100	4.80	18.27	20.38

重复类型 type of repeat	单元长度/bp motif length	数量 number	比例/% proportion	重复类型 type of repeat	单元长度/bp motif length	数量 number	比例/% proportion
单核苷酸 mononucleotide	10	1 093	45.05	二核苷酸 dinucleotide	20	28	3.83
	11	505	20.82		22	8	1.09
	12	251	10.35		24	1	0.14
	13	171	7.05	三核苷酸 trinucleotide	15	470	75.68
	14	136	5.61		18	110	17.71
	15	114	4.70		21	37	5.96
	16	94	3.87		24	4	0.64
	17	43	1.77	四核苷酸 tetranucleotide	20	16	80.00
	18	14	0.58		24	2	10.00
	19	1	0.04		32	1	5.00
	20	3	0.12		44	1	5.00
	22	1	0.04	五核苷酸pentanucleotide	25	5	100.00
二核苷酸 dinucleotide	12	301	41.18	六核苷酸 hexanucleotide	30	8	61.54
	14	161	22.02		36	4	30.77
	16	149	20.38		38	1	7.69
	18	85	11.63

重复类型 type of repeat	3'UTR	5'UTR	CDS	未知 unknow
单核苷酸 mononucleotide	946	793	103	584
二核苷酸 dinucleotide	274	230	33	196
三核苷酸 trinucleotide	114	142	216	149
四核苷酸 tetranucleotide	8	6	0	6
五核苷酸 pentanucleotide	2	2	0	1
六核苷酸 hexanucleotide	2	3	5	3
总计total	1 346	1 176	357	939

Analysis of SSR information in transcriptome and development of EST-SSR molecular markers in Pinus elliottii Engelm.

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