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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 920-929.doi: 10.3724/SP.J.1006.2022.14065

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Development and characterization analysis of potato SSR primers and the amplification research in colored potato materials

ZHANG Xia(), YU Zhuo, JIN Xing-Hong, YU Xiao-Xia*(), LI Jing-Wei, LI Jia-Qi   

  1. Agronomy College, Inner Mongolia Agricultural University, Hohhot 010019, Inner Mongolia, China
  • Received:2021-04-18 Accepted:2021-07-12 Online:2022-04-12 Published:2021-08-06
  • Contact: YU Xiao-Xia E-mail:1309165565@qq.com;yuxiaoxia1985@sina.com
  • Supported by:
    Inner Mongolia Major Science and Technology Project(ZDZX2018019);Animal and Plant Breeding Project for Transformation of Scientific Achievements in Inner Mongolia Agricultural University(YZGC2017006);Project of Inner Mongolia Potato Seed Industry and Technology Innovation Centre

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Abstract:

Compared with commonly cultivated potato, colored potato is rich in nutrition, especially in anthocyanins, which is the focus of breeding research in recent years. To date, the number of potato SSR primers developed is limited, especially those related to colored potato. In this study, SSR loci were analyzed using MISA software based on the whole genome. The results were as follows: a total of 218,997 SSR loci were obtained from potato genome with an average of 3.39 kb. Mononucleotide was the main repeat type, accounting for 62.05% of the total SSR, followed by dinucleotide and trinucleotide, accounting for 22.39% and 13.11%, respectively. The repeats of the six nucleotide types ranged from 5 to 746, mainly from 5 to 10, accounting for 60.9% of the total SSRs. A total of 215 motif types were obtained in all detected SSRs. The dominant motifs of the other five nucleotide repeat types were mainly A/T-containing motifs, except for hexanucleotide repeat type. The length of SSR motifs ranged from 12 bp to 20 bp, accounting for 43.19% of all SSRs. A total of 100 SSR primers were designed by Primer 5 software, among which 48 primers were preliminarily screened using parents’ genomic DNA of the colored potato, with an effective amplification rate of 48%. Then, six F2 individuals were randomly selected for PCR amplification, and 26 primers with clear and stable bands and high polymorphism were finely screened, with the average polymorphism rate of 72.52%. In summary, the number and types of SSR loci in potato genome was abundant and diverse, and the polymorphism was moderate. The developed primers were highly polymorphic, which can be used in the development of SSR markers, genetic diversity analysis, and SSR fingerprint of colored potato, providing a scientific basis for further mining genes related to anthocyanin content.

Key words: potato genome, colored potato, SSR, primer development

Table 1

Tuber phenotypic characteristics and anthocyanin contents of individual plants and parents"

材料
Material		 表皮光滑度
Epidermal smoothness		 薯形
Tuber shape		 皮色
Skin color		 肉色
Tuber flesh color		 花青素含量
Anthocyanidin (mg kg-1)
单株个体1 Individual 1 光滑 Smooth 长椭圆 Long oval 红色 Red 红中嵌白 White in red 33.84
单株个体2 Individual 2 光滑 Smooth 长圆 Long circle 紫色 Purple 紫黑色 Purple black 182.39
单株个体3 Individual 3 细纹 Fine grain 长圆 Long circle 红色 Red 浅红色 Light red 65.96
单株个体4 Individual 4 光滑 Smooth 椭圆 Oval 紫色 Purple 紫色 Purple 122.68
单株个体5 Individual 5 光滑 Smooth 长椭圆 Long oval 紫黑色 Purple black 紫中嵌白 White in purple 79.09
单株个体6 Individual 6 细纹 Fine grain 椭圆 Oval 红色 Red 黄中嵌红 Red in yellow 5.38
♂黑美人 Heimeiren 光滑 Smooth 长椭圆 Long oval 紫色 Purple 紫色 Purple 114.23
♀Red-P1 细纹 Fine grain 椭圆 Oval 红色 Red 浅红色 Light red 63.32

Fig. 1

Electrophoresis detection for genome DNA purity of six F2 individuals and their parents M: DNA marker DL2000; P1: male Heimeiren; P2: female Red-P1; 1-6: six F2 individuals."

Table 2

Results of a genome-wide SSR locus search in potato"

项目 Item 数量 Number
检测序列的总碱基数
Total size of examined sequences (bp)		 741,585,035
检测到SSR数目
Total number of identified SSRs		 218,997
复合型SSR位点
Number of SSRs present in compound formation		 30,404
复合SSR位点间最大间隔碱基数
Maximum number of bases interrupting 2 SSRs in a compound microsatellite (bp)		 100

Table 3

Repeat type, repeat number, and proportion of SSR in potato"

重复类型
Repeat type		 重复次数Repeat number 合计
Total		 比例
Proportion (%)
5 6 7 8 9 10 >10
单核苷酸 Mononucleotide — — — — — 67,672 68,208 135,880 62.05
二核苷酸 Dinucleotide — 13,755 7439 5404 4083 3201 15,147 49,029 22.39
三核苷酸 Trinucleotide 14,007 6164 3087 1671 1057 737 1997 28,720 13.11
四核苷酸 Tetranucleotide 1312 346 140 41 35 27 55 1956 0.89
五核苷酸 Pentanucleotide 450 99 39 14 9 10 9 630 0.29
六核苷酸 Hexanucleotide 1283 584 341 169 115 73 217 2782 1.27
总计 Total 17,052 20,948 11,046 7299 5299 71,720 85,633 218,997 —
比例 Proportion (%) 7.79 9.57 5.04 3.33 2.42 32.75 39.10 — 100.00

Table 4

Types and proportions of SSR repeating units in potato genome"

重复类型
Repeat type		 基元类型数量
Number of motif types		 主要重复基元
Main repeat motif		 出现数量
Occurrence number		 占本重复基元百分比
Percentage of predominant motifs (%)
单核苷酸
Mononucleotide		 2 A/T
C/G		 124,164
11,716		 91.38
8.62
二核苷酸
Dinucleotide		 4 AT/AT
AG/CT
AC/GT
CG/CG		 39,770
5655
3574
30		 81.12
11.53
7.29
0.06
三核苷
Trinucleotide		 10 AAT/ATT
AAG/CTT
AAC/GTT
AGG/CCT
其他 Others		 12,302
8857
3751
1514
2296		 42.83
30.84
13.06
5.27
8.00
四核苷酸
Tetranucleotide		 30 AAAT/ATTT
AGAT/ATCT
ACAT/ATGT
AATT/AATT
其他 Others		 899
258
152
132
515		 45.96
13.19
7.77
6.75
26.33
五核苷酸
Pentanucleotide		 49 AATAT/ATATT
AAAAT/ATTTT
AACTC/AGTTG
AATAC/ATTGT
其他 Others		 206
91
81
49
203		 32.70
14.44
12.86
7.78
32.22
六核苷酸
Hexanucleotide		 120 AGGCCC/CCTGGG
AACTTG/AAGTTC
AGGCCT/AGGCCT
AAGAGG/CCTCTT
其他 Others		 760
552
408
264
798		 27.32
19.84
14.67
9.49
28.68

Fig. 2

Distribution of SSR length in potato genome"

Table 5

Sequences of developed SSR primers for coloured potato"

引物名称
Primer name		 序列
Primer sequence (5'-3')		 重复基元
Repeat motif		 退火温度
Tm (℃)		 多态性比率
Percentage of polymorphic loci (%)
Z-1 F: CGACGCCAAAGTTAGCCA
R: GTCCCCAGAAGCCAAGAG		 (GTT)7 57 50.00
Z-2 F: TTTCTGTGCTTCTTGCTCCTC
R: GTATCGCCCTCAGCCTTG		 (TTC)6 57 100.00
Z-3 F: GAAGCGAGAAAAGCAGCAC
R: CCAGGGCAAAGGAAAACA		 (TTC)6 57 66.67
Z-4 F: CTCCGTTTCCCAAGCCCTAA
R: GTCACCGTCCTCGTCATCG		 (CCA)5 58 42.86
Z-5 F: GGAAATGGTCTGAAATGC
R: TGGTGGTGGACTACTTGG		 (ACA)8 51 77.78
Z-6 F: CTCCCACCACTCCCTTAT
R: TCGTCATCACCATCTTCG		 (TGA)5 54 85.71
Z-7 F: CACATTTGCCCACTCCTG
R: CATCCCCTCTTCCACCTC		 (GAT)8 55 71.43
Z-8 F: AGCGTGAAATGAATGGAC
R: CAAATACGAGGCGAAACT		 (AT)10 51 100.00
Z-9 F: CCTTGAATCCGAGCAGAAAT
R: TGGCACTCCACAGGGAATAG		 (CAG)5 58 66.67
Z-10 F: TGCTGCTCCCTATTCTAT
R: AAATGACTGCCAATCTGA		 (CTG)7 48 85.71
Z-11 F: TCGTGGAGAAGAATGAGA
R: TGACCAGGGTAAATGACTA		 (ATT)5 48 66.67
引物名称
Primer name		 序列
Primer sequence (5'-3')		 重复基元
Repeat motif		 退火温度
Tm (℃)		 多态性比率
Percentage of polymorphic loci (%)
Z-12 F: AAGAGTTGGAAGAGCGTGAG
R: GGTAGGGTAAGGTCTATGTGC		 (ACA)5 54 83.33
Z-13 F: TCAATGGAGACGACGACT
R: AGAGGATGGATTCCGATG		 (CTC)5 52 71.43
Z-14 F: ACGCATCGCCTAAATCACTG
R: TCATTCGCATTCCCAAACTC		 (GGC)5 54 71.43
Z-15 F: TATGTGGCGAAGTGAGTG
R: AAATGACGGGAGAAGGAC		 (TA)9 51 77.78
Z-16 F: ATGATTCCGTTGCTGTCT
R: ATTGCTGATTCCTGTTCC		 (TC)15 50 66.67
Z-17 F: ACATTTTGGTTGTGACTTGG
R: ATGGGTTTTGAGATTTGAGG		 (CTT)6 54 81.82
Z-18 F: CCGTGGCTCTGAGATTTA
R: CGTGGGACTACACTGGATA		 (AGT)5 51 93.33
Z-19 F: TCCAGCACTTAGACCAACC
R: CTCCGCATCTCGTCATAC		 (ATG)6 52 57.14
Z-20 F: GGGTCTTCCCGAACAAAC
R: TACTTCCAAAACCGCCTC		 (TGT)6 54 100.00
Z-21 F: TTCTAACTCCGCCCCTAC
R: CAAGACTTTTCCACCACC		 (TC)16 52 64.29
Z-22 F: CGAAGAGGTAGTGATAAGGC
R: GTCAGCAAGGTTCAGGGA		 (TTG)5 53 50.00
Z-23 F: CTTAGTGCTGGCGATGAT
R: TGGGCTTGCTCTTTGTCT		 (AGA)6 53 57.14
Z-24 F: TTGTGGGATTTCACTGGG
R: TGGAAGACGGGAATGGTA		 (TGT)5 54 77.78
Z-25 F: TGGGAAGAAACGAAGAAACA
R: GTTACATGAGTCAGGCTAGG		 (CTC)5 55 70.00
Z-26 F: TAATTGTGACTCCTCCTCCT
R: CAGGCTAGGTCGTTTTGATA		 (TA)9 54 50.00

Fig. 3

Amplification of partial SSR primers in parents and six F2 individuals M: 100 bp DNA marker; P1: male Heimeiren; P2: female Red-P1; 1-6: six F2 individuals. The red arrow is the marker of partial polymorphic bands."

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