Choose your language:
Open search

Identification of Camellia nitidissima Chi Hybrids by ISSR

Fan Zhengqi, Li Jiyuan, Li Xinlei, Yin Hengfu, Xiao Zheng, Sun Yingkun
Research Institute of Subtropical Forestry, CAF, Fuyang, Zhejiang 311400, China

Introduction

Camellia nitidissima Chi, with golden yellow flowers, is a rare germplasm resource in the world, and become a precious parent to breed the golden flower camellia cultivars.

The crossbreeding is a main way to obtain hybrids of C. nitidissima. The early identification for hybrids is very necessary to improve breeding efficiency because of its long period of crossbreeding procedure. Molecular marker technology was widely used in identifying hybrids nowadays. ISSR (Inter Simpie Sequence Repeat) is believed as a popular molecular marker technology with advantages of distinctness, simple operation, rich polymorphism, high stability and lower cost. This technology has been applied to identify hybrids of Ticum aestivum L.[1],Brassica napus L.[2], Oryza sativa L.[3], Camellia sinensis O.ktze.[4], Lycoris radiate (L'Her.) Herb. [5],,Paeonia suffruticosa[6], Linlium oriental hybird [7], Armeniaca cathayana D. L.[8]. In this study, the genetic relationship among four hybrids related to C. nitidissima and their parents was analysis by ISSR, which will provide molecular evidence and a fast method of hybrid identification from C. nitidissima.

1 Material and Methods

1.1 Experimental Material

Leaves of C. nitidissima Chi and hybrids were collected from Nanning Golden Camellia Park, and Research Institute of Subtropical Forestry as well as Jinhua Moxian Camellia Nursery in Zhejiang Province, China. The details are given in table 1.

Table1 The origin of hybrids of Camellia nitidissima and their parents

No.

Name

Origin

P1

Camellia nitidissima Chi

Nanning Golden Camellia Park

P2

Camellia tunghinensis Chi

Nanning Golden Camellia Park

P3

Camellia sasanqua Linn. ‘Xiaomeigui’

Fuyang, Zhejiang

P4

Camellia japonica Linn.‘Wubao’

Jinhua, Zhejiang

G1

‘Dongyue’(Camellia nitidissima Chi×Camellia sasanqua Linn. ‘Xiaomeigui’)×Camellia nitidissima Chi

Nanning Golden Camellia Park

G2

Camellia nitidissima Chi×Camellia sasanqua Linn. ‘Xiaomeigui’

Nanning Golden Camellia Park

G3

‘Jinbei Danxin’(Camellia nitidissima Chi×Camellia japonica Linn. ‘Wubao’)

Nanning Golden Camellia Park

G4

Camellia tunghinensis Chi×Camellia nitidissima Chi

Nanning Golden Camellia Park

Leaves were treated in liquid nitrogen and saved in a refrigerator at -80℃. The reagents for PCR amplification were purchased from TaKaRa Company. 20 primers were selected according to the reference[9] and were synthesized by Dingguo biological engineering and technology services limited company in China.

1.2 Genome DNA extraction and PCR amplification

DNA were extracted by CTAB (slightly improved)[10], and then quality was tested for DNA by way of 1 percent of agarose gel electrophoresis. DNA concentrations were determined by Nanodrop ND-2000.

The ISSR reaction used in this study was 20 ul. The appropriate concentration of PCR system was summarized as followings: Mg2+ 1.5mmol/L, dNTP 0.2mmol/L, primers 0.5μmol/L, Taq 2.5U, buffer 2μl and 12.7 μl sterilized water. Amplification reactions program: 94℃, degeneration, 5min; one cycles; 94℃, degeneration, 40S, specific temperature annealing 45S (different primers with different annealing temperature); 72℃, extension, 1.5min; 40cycles; 72℃,extension, 7min; termination reaction, preservation at 4℃. Products of ISSR-PCR were electrophoresed in the 2% of agarose gel electrophoresis for 90min. Voltage was 5v/cm and taken photos by FR-200A gel imager.

1.3 Data analysis

Difference of samples amplified belts can be observed, and the bands were examined by artificial methods. For the same primers in electrophoresis maps, each marker which produced bands in some samples recorded as “1”, no band recorded as “0” no matter whether bands are strong or weak, and bivariate matrix was established.

The UPGMA cluster analysis based on genetic similarity coefficient was carried out by NTSYSpc2.10e[11], and the relationship tree will be obtained.

The alleles (Na), effective number of alleles (Ne), expected heterozygosity (H)  and Shannon’s information index(I) were analyzed with PopGene Version 1.31[12].

2 Result and Analysis

2.1 The results of ISSR amplification and polymorphism

11 primers were screened from 20 primers according to the stability, repeatability and polymorphism of bands. 199 bands were amplified, of which 195 bands are polymorphic. The percentage of polymorphism bands was 97.99%.The number of bands from each primer ranged from 3 to 13 bands with an average of 9.5 bands. The size of product ranged from 100 to 2000 bp.

amplification results amplification results

Fig.1 The amplification results of C. nitidissima hybrids based on the primer
UBC843(left) and UBC818(right); P1-P4, Parents; G1-G4, hybrids

2.2 The genetic similarity coefficient analysis between hybrids of C. nitidissima and their parents

The results of the genetic similarity coefficient were displayed in Table 2. The genetic similarity coefficient is 0.759 between C. nitidissima and C.tunghinensis in parents which are all from Sect. Chrysantha of Genus Camellia, higher than these between others parents from C. japonica (Sect.Camellia) and and C.sasanqua (Sect.Oleifera ) respectively, which ranged from 0.508 to 0.598. The genetic similarity coefficient between C.nitidissima and its hybrids is ranged from 0.618 to 0.769.

In hybrids, ‘Dongyue’, the first released yellow peony form cultivar in China, reached the highest in the genetic similarity coefficient with its father C.sasanqua, while that of the hybrid G2 is the higher with its father C.sansanqua. The genetic similarity coefficient between G1 and G2 is 0.789, the highest in all data because of relationship between a hybrid and a mother parent. G3 has the higher value with its father P4 than mother C.nitidissima, while the value of G4 and its mother is higher than its father even if this two data is all high. In conclusions from the results of the genetic similarity coefficient, G2 is believed as a hybrid of C.nitidissima and C.sasanqua. G1 is the hybrid by hybrid of C.nitidissima and C.sasanqua backcross with C.nitidissima, farther than G2 in genetic distance with C.sasanqua. It is confirmed that G3 is the hybrid of C.nitidissima and C.japonica while G4 is the one of C.tunghinensis and C.nitidissima by the genetic similarity coefficient.

Table 2 The genetic similarity coefficient of hybrids of C. nitidissima and their parents

 

P1

P2

P3

P4

G1

G2

G3

G4

P1

1.000

 

 

 

 

 

 

 

P2

0.759

1.000

 

 

 

 

 

 

P3

0.558

0.558

1.000

 

 

 

 

 

P4

0.538

0.508

0.598

1.000

 

 

 

 

G1

0.618

0.578

0.658

0.608

1.000

 

 

 

G2

0.618

0.568

0.668

0.608

0.789

1.000

 

 

G3

0.623

0.513

0.482

0.663

0.563

0.543

1.000

 

G4

0.769

0.759

0.508

0.527

0.578

0.558

0.513

1.000

2.3 The clustering analysis results of hybrids of C. nitidissima and their parents

According to the matrix of genetic distance, clustering analysis was carried out by way of UPGMA. The relationship tree is showed in Fig 2. The relationship of G1 and G2 is closest, and they form into a group with C.sasanqua firstly following C.nitidissiam. G3 was classified into a group with its father firstly, and then its mother, while G4 goes to a group with its mother C.nitidissima firstly following its father C.tunghinensis. We will know from Fig 2, the parents of G4 are C.nitissima and C.tunghinensis, G3 are from C.nitidissima and C.japonica, and G1 from G2 are C.nitidissima and C.sasanqua.

Dendrogram Fig.2 The clustering analysis results of hybrids of Camellia nitidissima and their parents

2.4 The results of genetic variation of the parents and hybrids of C. nitidissima

The results of genetic variations in the parents and hybrids of C. nitidissima is displayed in Table 3. Ne between G1 and G2 is 1.8384 and 1.8602, H is 0.4500 and 0.5151, I is 0.6346 and 0.6948, respectively. These parameters are higher than those of their two parents, 1.7850 of Ne, 0.4397 of H and 6316 of I. Those of G3 and G4 are also higher than their parents. The result indicates the hybrids are improved significantly in Ne, H and I.

Table 3 The results of genetic variations in the parents and hybrids of C. nitidissima

Combination

Na

Ne

H

I

P1/P3

2.0000

1.7850

0.4397

0.6316

G1=P1×P3

2.0000

1.8384

0.4500

0.6346

G2=(P1×P3)×P1

2.0000

1.8602

0.5151

0.6948

P1/P4

2.0000

1.7369

0.4234

0.6142

G3=P1×P4

2.0000

1.9400

0.4845

0.6776

P1/P2

2.0000

1.8072

0.4467

0.6388

G4=P2×P1

2.0000

1.8857

0.4697

0.6625

3 Discussions

It is important that hybrids authenticity is identified in the early stage in selecting hybrids of C. nitidissima crossbreeding. The early to know whether it is true hybrid offspring and predict their properties more paternal or maternal, assist to determine the hybrids whether to continue training to reduce breeding costs. In this study, the bands of C. nitidissima can be amplified from four hybrids using the most popular primers, meanwhile ones of their fathers also exist. The high genetic similarity and the close relationship between hybrids and their parents indicates that they indeed are the hybrid offsprings of C. nitidissima and another parent is C. tunghinensis Chi, or C. sasanqua Linn.’Xiaomeigui’ and or C. japonica Linn. ’Wubao’. The relationship between the hybrids is closer than those between the parents.

‘Dongyue’  ‘Jinbei Danxin’
‘Dongyue’ ‘Jinbei Danxin’

The yellow peony form camellia cultivar ‘Dongyue’ was obtained by backcross of the yellow parent of C.nitidissima, but no yellow camellia cultivars have been produced between one yellow parent and one non-yellow parent. This indicated that biosynthesis pathway producing yellow substances may be interfered by non-yellow camellia parent, but can be recovered by backcross of one yellow parent. Therefore, backcross by one yellow camellia parent will be a very helpful to breed golden camellias in future.

Acknowledgments:

This work was supported by the National Key Twelfth-Five Science and Technology Program (2012BAD01B0703), International Cooperation Project of China (2011DFA30490), and Zhejiang Key Flower Breeding Program (2012C12909-6).

References

[1]      Zhan K,Sun H,Gao X,et al. Relationship between the genetic distances based on molecular markers and heterosis in hybrid wheat. Journal of Triticeae Crops, 2006, 26(2): 27-31.
[2]      Shen J, Fu T, Yang G. Relationship between hybrid performance and genetic diversity based on SSR and ISSR in Brassica napus L.. Scientia Agricultura Sinica, 2004, 37(4): 477-483.
[3]      Hu H, Xing S, Guo J, et al. Identification of three-line hybrid rice and its parents by RAPD and ISSR primer. Journal of Zhanjiang Ocean University, 2006, 26(3): 86-89.
[4]      Hou Y, He Q, Liang G, et al. ISSR analysis if the hybrid descendants of tea Camellias. Journal of Southwest Agricultural University, 2006, 28(2): 267-270.
[5]      Zhang L, Gao Y,Zhu Y,et al. An inter simple sequence repeats(ISSR) reaction system for Lycoris(Amaryllidaceae). Journal of Zhejiang Forestry College, 2007, 24(2): 156-161.
[6]      Suo Z, Zhang H, Zhang Z, et al. DNA molecular evidences of the hybrids between Paeonia rockii and P.suffruticosa based on ISSR markers. Acta Botanica Yunnanica, 2005, 27(1): 42-48.
[7]      Wu X, Cui G, Wu L, et al. Identification of ISSR in Lily hybrids. Acta Horticulturae Sinica, 2009, 36(5): 749-754.
[8]      Liu M, Fu D, Tian M, et al. Analysis on genetic variation of Armeniaca cathayana F1 hybrids by ISSR. Journal of Central South University of Forestry & Technology, 2011, 31(10): 100-104.
[9]      Lin L, Li J, Ni S, et al. The influence of insular geographical isolation on population genetic structure of Camellia japonica. Forest Research, 2012, 25(3): 378-384.
[10]  Zhou Y, Ge S, Wang X, et al. Systematic and evolutionary botany molecular markers. Beijing, Science Press, 2001, 43-83.
[11]  Rohlf F J. NTSYS-pc: Numerical taxonomy and muhivariate analysis system. Version (1.08). NY, USA, Exeter Softwate Seauket, 1993.
[12]  Ye F C, Yang R C, Boyle T B J, et al. POPGENE, the user friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Alberta, Canada, 1997.

 
 

Web design by Tribal Systems