Genetic Diversity among Flue-cured Tobacco Cultivars Based on RAPD and AFLP Markers

AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Genetic Diversity among Flue-cured Tobacco Cultivars Based on RAPD and AFLP Markers

Han Yao ZhangI, ^*; Xiao Zhen Liu^I; Chuan Sheng He^II; Yu Ming Yang^I

^IBiotechnology Laboratory; Southwest Forestry College; White Dragon Temple; Kunming, Yunnan Province-650224; People's Republic of China

^IIThe South Center of Tobacco Breeding Research of China; Yuxi, Yunnan-653100; People's Republic of China

ABSTRACT

The aim of this work was to study the genetic diversity among flue-cured tobacco cultivars. RAPD and AFLP analyses were used to assess the genetic similarity among selected accessions of flue-cured tobacco. Seventy eight RAPD and 154 AFLP polymorphic bands were obtained and used to assess the genetic diversity among 28 tobacco accessions. The cultivar relationships were estimated through the cluster analysis (UPGMA) based on RAPD data and AFLP data. The accessions were grouped into three major clusters and these shared common ancestry clustered together.

Key words: Flue-cured tobacco, RAPD, AFLP, genetic diversity, DNA polymorphism

INTRODUCTION

The flue-cured tobacco (Nicotiana tabacum L.) is one of the most important types for the tobacco production in the world. Undoubtedly, the study of the genetic diversity of flue-cured tobacco cultivars is important not only for the germplasm conservation but also in parental choice for breeding purposes. The RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism) are genetic fingerprinting techniques suitable for the genetic evaluation of flue-cured tobacco. The techniques have been successfully used to genetically analyze many different plant species (Crochemore et al., 2003; Diniz et al., 2005; Ni et al., 2006 and Yang et al., 2006).

In this work, the RAPD and AFLP procedures were used to assess the amount of polymorphisms detected among the flue-cured tobacco cultivars and to estimate the relationships.

MATERIALS AND METHODS

Plant Materials

The seeds of the flue-cured tobacco were obtained from the germplasm collection of the South Center Tobacco Breeding Research of China in Yunnan province, southwest of China. The collection was consisted of 298 cultivars and breeding lines from different places. On the basis of results from the field trials conducted at Yuxi, Yunnan, from 1994 to 1996 (Lei et al., 1997), 28 accessions with desirable agronomic characteristics, such as large leaf size, high leaf yields, low nicotine contains, or resistance to various diseases or insects, were selected for evaluation in this study. These accessions represented the genotypes likely to be used in future flue-cured tobacco breeding efforts in south China. The name and origin of the cultivar were showed in the Table 1.

The seeds were planted in the pots and grown in the greenhouse at 28 to 32 °C. Twenty days after the germination, the shoots were harvested from 40 seedlings of each accession. The DNA was extracted from the shoots by the CTAB method (De Riek et al., 2001).

RAPD analysis

The amplification was performed in 20¼L solution containing 2μL of the 10×buffer, and 100 mM each of dNTPs, 0.4 mM primer, 25 ng genomic DNA, and 1 unit of Taq polymerase. The reaction mixture was overlaid with 40μL mineral oil.

The amplifications were carried out using a 2400 Perkin-Elmer (Perkin Elmer, USA) thermal cycler programmed for 40 cycles as follows: 30 s at 94°C, 30 s at 36 °C, 1.5 min at 72 °C, with an initial melting of 6 min at 94 °C, and a final extension of 6 min at 72 °C. The amplification products were analyzed by the electrophoresis in a 1.5 % agarose gel with 1×TAE buffer (0.004 M Tris-acetate and 0.002 M EDTA).

AFLP analysis

The AFLP analysis was performed following the manufacturer's protocol (Life Technologies). The DNA was digested simultaneously with restriction enzymes EcoRI and MseI. The selective amplifications were performed using the primer pairs listed in Table 2. The restricted genomic DNA fragments were ligated to EcoRI and MseI adapters. The primers within set EcoRI included the sequence 5'- GAC TGC GTA CCA ATT C; primers of the MseI set had the sequence 5'-GAT GAG TCC TGA GTA A. The pre and the selective amplifications were performed in a 2400 Perkin-Elmer Thermocycler. An equal volume (2¼L) of loading dye (95% v/v formamide and 0.08% w/v bromophenol blue, 20 mM EDTA) was added to each sample, which was then denatured at 95°C for 3 min and placed on the ice for 2 min before loading. The amplification products were analyzed by the electrophoresis in a 6.5% polyacrylamide gel. The electrophoresis parameters were set to 1500 V, 40.0 mA, 40.0 W, 50°C and the run time was set to 2.0 h. Separated AFLP products were visualized using silver staining as described in the Promega Silver Staining kit and gel images were saved as TIF files for analysis.

Data analysis

Each accession was scored 1 for the presence or 0 for the absence of each polymorphic band. The bands present in all accessions were not scored.

Only bright, clearly distinguishable bands were used in the genetic analysis. All the statistical analyses were performed by NTSYS-pc, Version 1.8. Similarity matrices (data not shown) were constructed from the binary data with Jaccard's coefficients (Jaccard, 1908). The dendrograms were generated with the unweighted pair-group method, arithmetic average (UPGMA) algorithm as described by Sneath and Sokal (1973).

RESULTS AND DISCUSSION

From the 200 primers used in RAPD analysis, 63 (31.5%) produced the amplification products that were too faint to score or could not be consistently reproduced, and 124 (62%) produced monomorphic banding patterns. Only 13 (6.5%) out of 200 primers were scored. A total of 125 bands were scored from the comparison of amplifications with 13 primers of DNAs from 28 flue-cured tobacco cultivars, with an average of 9.6 bands scored per primer (Table 2.). The polymorphic bands were 78 (62.4%), and one primer detected a mean of 6 polymorphic bands per reaction.

Fourteen selective AFLP primers were screened against all 28 accessions. Four primer pairs were not included in the final analysis because either the amplification profile was consistently too faint to score accurately (AAC/CGC) or only monomorphic amplification products were produced (AAC/CTG, ACT/CTC, ACT/CTG). The ten informative primer pairs used in the final analysis were listed in Table 2. A total of 154 fragments were analyzed using the ten pairs of primers. Five hundred and sixty-one fragments were scored in the assay performed by using the ten pairs, with an average of 56.1 fragments per pair of primers used. One hundred and fifty-four fragments were polymorphic, with an average of 15.4 per reaction, with 27.45% polymorphism.

Results from cluster analyses using RAPD or AFLP data indicated that these two marker techniques provided similar, but not identical information (data not shown). For example, Zhongyan 86 was the offspring of both Speight G-28 and Jingyehuang, in the dendrogram based on RAPD data, it was clustered with the progenies of Jingyehuang; it was grouped with the progenies of Speight G-28, in the dendrogram based on AFLP data. This observation could be related to the larger number of AFLP bands used in the analyses compared with the number of RAPD bands used.

The RAPD and AFLP data were combined to generate a dendrogram incorporating both types of the DNA marker data, the relationships among the accessions analyzed are shown in Fig. 1. The RAPD and AFLP based Jaccard's similarity coefficients ranged from 0.13 to 0.88. The accessions were grouped into three major clusters. Group I included 17.86% of the accessions; group II included 64.29% of the accessions; group III included 17.86% of the accessions.

The dendrogram did not indicate any clear pattern of division among the flue-cured tobacco accessions based on the geographic origin, as seen in some other crops (Paul et al., 1997; Spooner et al., 1996). However, those accessions that shared common ancestry clustered together. For example, the cultivars bred by Speight G-28 such as 82-3041, K149, K394, Zhongyan 14, CV73, CV85 and CV87 (Table 1) were clustered together in Group II; Jiyan 5 and Qingsheng 2 were the offsprings of the Jingyehuang, Jingyehuang was bred by Changbohuang (Table 1), they were all clustered with Jingyehuang in Group II; Yunyan 2, chunjingyan and Yunyan1 were bred by Gold Dollar (Table 1), they were clustered together in Group I; and one of the crossing parents of Yunyan 76, Yunyan 84 and Yunyan 86 was K326 (Table 1), they were also clustered together in GroupII.

ACKNOWLEDGEMENTS

The experiments in this study were carried out at the South Center Tobacco Breeding Research of China, the expenses were provided by Yunnan Tobacco Company, and the Natural Science Foundation of Yunnan (Grant No. 2007 C 216 M).

Received: November 30, 2005;

Revised: October 10, 2007;

Accepted: February 01, 2008.

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Author for correspondence

Publication Dates

• Publication in this collection
  27 Jan 2009
• Date of issue
  Dec 2008

History

• Received
  30 Nov 2005
• Reviewed
  10 Oct 2007
• Accepted
  01 Feb 2008