Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (1): 37-45.doi: 10.11983/CBB18019

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Identification of 21 New Camellia Hybrid Varieties by Fluorescence-labelled Simple Sequence Repeat Markers

Tao Naiqi1,Zhang Bin2,3,Liu Xinkai4,Zhou Heda2,Zhong Naisheng4,Yan Danfeng4,Zhang Min1,Gao Jiyin4,Zhang Wenju1,*()   

  1. 1 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai 200433, China
    2 Shanghai Xingyuan Agricultural Experiment Center, Shanghai 201417, China
    3 Shanghai Agricultural Development and Promotion Center, Shanghai 200335, China
    4 Palm Eco-Town Development Co, Ltd, Guangzhou 510627, China
  • Received:2018-01-18 Accepted:2018-04-28 Online:2019-07-31 Published:2019-01-01
  • Contact: Zhang Wenju E-mail:wjzhang@fudan.edu.cn

Abstract:

In this study, 12 highly polymorphic simple sequence repeat (SSR) loci were selected from Camellia azalea transcriptome data and previous studies, and the genotypes of 21 new Camellia hybrid varieties were analyzed by fluorescence capillary electrophoresis. Repeatable and distinct amplification products could be obtained at 12 SSR loci from all samples, and 21 new varieties could be identified by the specific genotype of 12 SSR loci. Significantly, for the 21 new varieties, the number of discrepant loci was 2 to 10 among varieties from the same parental combination but 5 to 12 among varieties from different combinations. Hence, each of the 21 varieties was marked by a unique group of molecular markers, which can be used to identify these varieties accurately. These markers are very useful for identifying and protecting Camellia varieties, especially for those propagated that are produced by grafting or cutting.

Key words: Camellia, fluorescence labelled SSR, new variety protection, variety identification

Table 1

Information of 21 new Camellia hybrid varieties in this study"

Cross combination No. New variety
No.
Parental types of cross
combinations
ZH-01 SJ-01 Var. B (♀)×Var. A (♂)
SJ-02 Var. B (♀)×Var. A (♂)
SJ-03 Var. B (♀)×Var. A (♂)
SJ-04 Var. B (♀)×Var. A (♂)
ZH-02 SJ-05 Var. C (♀)×Var. A (♂)
SJ-06 Var. C (♀)×Var. A (♂)
SJ-07 Var. C (♀)×Var. A (♂)
SJ-08 Var. C (♀)×Var. A (♂)
ZH-03 SJ-09 Var. D (♀)×Var. A (♂)
SJ-10 Var. D (♀)×Var. A (♂)
SJ-11 Var. D (♀)×Var. A (♂)
ZH-04 SJ-12 Var. E (♀)×Var. A (♂)
ZH-05 SJ-13 Var. F (♀)×Var. A (♂)
ZH-06 SJ-14 Var. A (♀)×Var. G (♂)
SJ-15 Var. A (♀)×Var. G (♂)
SJ-16 Var. A (♀)×Var. G (♂)
ZH-07 SJ-17 Var. A (♀)×Var. H (♂)
SJ-18 Var. A (♀)×Var. H (♂)
ZH-08 SJ-19 Var. A (♀)×Var. I (♂)
ZH-09 SJ-20 Var. A (♀)×Var. J (♂)
ZH-10 SJ-21 Var. A (♀)×Var. K (♂)

Table 2

Information of 12 primer pairs in this study"

No. Primer sequences (5′-3′) Repeat motifs Ta (°C) References
478 F: CAACACCACCAACAAGA (AAAGG)4 53 Liu et al., 2008
R: GATATGAGATCCGTCCC
SSR2 F: TATTGCCTACGACCATTTCCA (GA)14 56 Kaundun and Matsumoto, 2002
R: TTTGAGTTCGTTGCCTTCTCT
CamsinM11 F: GCATCATTCCACCACTCACC (CA)12 60 Freeman et al., 2004
R: GTCATCAAACCAGTGGCTCA
CamSSR01 F: CCAACAAGAATCAGGAAGAG (AAT)6 54 In this study
R: ATCCAACGGTGGTAGACGAG
CamSSR02 F: AGTTCCGCCTCCAGTTTGAC (ACG)7 54 In this study
R: GGACCGAGAGGTAACAGTGG
CamSSR03 F: GCCACTACCCTCTTTACACC (CAC)7 55 In this study
R: TTCTCTTCCTCTTTCTTCCC
CamSSR04 F: ATGTGTTGAGTAGCGAGCGT (AT)10 56 In this study
R: TTGTCCATCTTTATGTAGGG
CamSSR05 F: GCAAACACCAACTGATTACC (TA)10 56 In this study
R: TTCCATACAACTCAACCAAA
CamSSR06 F: GGTTTGGAAAAAGGACACGC (GCC)7 58 In this study
R: AATCTGCCTCTGGTAGTCCG
CamSSR07 F: TCTCATCCCCATCTTTATCC (TCC)7 58 In this study
R: GTTCCCTGCTGCTGTTGTTA
CamSSR08 F: TCACCAGTCACTTTCCCTCC (AC)10 58 In this study
R: CCACCAAAAGGCACAATACC
CamSSR09 F: CATCATCCATCAAACCGTCC (AT)10 58 In this study
R: GAAGGCACATTGGTTCTGGG

Figure 1

SSR peak map of 3 new Camellia hybrid varieties by the CamSSR03 primer (A) Variety SJ-02; (B) Variety SJ-18; (C) Variety SJ-21"

Table 3

The number of alleles and polymorphism of 12 SSR loci"

Primers Na Ne PIC
478 7.00 4.12 0.72
SSR2 6.00 2.63 0.58
CamsinM11 4.00 1.85 0.43
CamSSR01 3.00 1.48 0.29
CamSSR02 4.00 2.97 0.62
CamSSR03 6.00 4.96 0.77
CamSSR04 6.00 2.99 0.62
CamSSR05 4.00 2.82 0.58
CamSSR06 4.00 1.76 0.41
CamSSR07 4.00 1.58 0.35
CamSSR08 6.00 4.96 0.77
CamSSR09 5.00 2.32 0.49
(mean) 4.92 2.87 0.55

Table 4

The encode standard of genotypes at 12 SSR loci in this study"

Code of pattern Cam-
SSR08
Cam-
SSR04
478 SSR2 Cam-
SSR03
Cam-
SSR09
Cam-
SSR06
Cam-
sinM11
Cam-
SSR01
Cam-
SSR07
Cam-
SSR05
Cam-
SSR02
1 125/131 165/167 113/115 217 278/313 209/215 176/185 171 188/200 206 234/240 271/274
2 125/135 165/171 113/129 217/219 278/321 211/217 179/182 173 195 206/212 234/242 271/277
3 127 167 115 217/225 278/322 213/215 179/185 177 195/200 206/215 236/240 271/283
4 127/131 167/171 115/129 217/227 278/323 215 182/185 181 200 206/221
5 129 167/173 117/129 217/231 307/321 215/217 185
6 129/131 169/171 121/129 217/235 307/322 217
7 129/133 171 123/129 219/235 307/323
8 129/135 171/173 123/133
9 131/133 171/175
A 133
B 133/135

"

Table 6

Molecular identity code of 21 new Camellia hybrid varieties"

No. Molecular identity
code
No. Molecular identity
code
SJ-01 192315514322 SJ-12 393121414432
SJ-02 291315514322 SJ-13 485225334121
SJ-03 122516514322 SJ-14 546224111121
SJ-04 897336114322 SJ-15 545254111121
SJ-05 975265414113 SJ-16 646254114121
SJ-06 913235513113 SJ-17 353661513121
SJ-07 A73135522123 SJ-18 445664323113
SJ-08 A35165424121 SJ-19 934175513111
SJ-09 973663214212 SJ-20 563424544123
SJ-10 972646514212 SJ-21 748772514411
SJ-11 B61664544212
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