Genetic Diversity of the Selected 64 Potato Germplasms Revealed by AFLP Markers  

Fang Wang1,2 , Fangdi Li1,2,3 , Jian Wang1,2 , Yun Zhou1,2 , Haihong Sun1,2
1.Institute of Biotechnology of Qinghai Academy of Agriculture and Forestry, Xining, 810016, P.R. China
2.The Qinghai-Tibet Plateau Biotechnology Key Lab of Ministry of Education, Xining, 810016, P.R. China
3.Qinghai University, Xining, 810016, P.R. China
Author    Correspondence author
Molecular Plant Breeding, 2011, Vol. 2, No. 4   doi: 10.5376/mpb.2011.02.0004
Received: 28 Dec., 2010    Accepted: 06 Jan., 2011    Published: 25 Feb., 2011
© 2011 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Wang et al., 2011, Genetic diversity of the selected 64 potato germplasms revealed by AFLP marker, Molecular Plant Breeding, Vol.2 No.4 (doi: 10.5376/mpb.2011.02.0004)

Abstract

In order to utilize the elite germplasms from the International Potato Center (CIP) for potato breeding program in China, we analyzed the genetic relationship of selected 64 potato cultivars based on the AFLP approach in this study. Twelve polymorphic primer combinations were screened out to be used for analyzing the genetic diversities among the tested materials. The number of bands generated by each selected AFLP primer pair reached about 42 in the ranged of from 33 to 56. Total of 505 reproducible bands were amplified by 12 primer, of which polymorphic bands were 388. The genetic distance were calculated among the tested 64 potato cultivars in the range from 0.041 237 1 to 0.391 732 6. The cluster analysis showed that domestic cultivars were assigned to be a group at the genetic coefficient of 0.82 and all tested potato cultivars were clustered together at the genetic similarity coefficient (GS) of 0.68. Shannon’s Information Index of examined materials was between 0.245 6 and 0.498 8 with an average of 0.374 5, as well as Nei’s Gene Diversity Index of examined materials varied from 0.151 0 to 0.325 6 with a mean of 0.235 9. In this research, we might have a conclusion that that genetic basis of domestic potato cultivars is quite narrow compared to the accessions of potato resources from CIP. Therefore, the CIP germplasms can be used for broadening the genetic basis of local potato cultivar in China.

Keywords
Potato; Potato Germplasms; Genetic Diversity; AFLP

Currently, the cultivation of potato is mainly depending on cross breeding. The selection of parent materials is the key of breeding. Determination of genetic distance and genetic relationship is one of crucial factors to be considered in matching of parents. More genetic diversity existed between the parents would breed new cultivars with resistance to disease longer-lasting. Previously, parent materials were identified by analyzing the genetic distance of morphological characteristics. Influenced by internal and external environmental factors, quantitative characters, dominant effect and other influential factors easily, the traditional method had inevitable deviations. The molecular markers due to be uneasy to be affected by environmental factors, and larger quantities, can be quickly and accurately distinguish the genetic relationship of parent materials.

With developing of molecular marker technology rapidly, molecular markers have been used as a routine tool to penetrate into all areas of biological research and produce an enormous impact on the practice of crop breeding. Molecular markers were used to analysis potato genetic diversity earlier in foreign countries (Kardolus et al., 1998; McGregor, 2002; McGregor et al., 2000; John Bamberg et al., 2008; Atul Grover et al., 2009). Compared with foreign countries, molecular markers were used in Chinese potato breeding lately, but in recent years, the molecular marker has gradually applied to analysis genetic diversity of potato in China (He et al., 2007; Li et al., 2000; Liu et al., 2004; Sun et al., 2006; Li et al., 2007; Xu et al., 2007; Duan et al., 2009). AFLP (Amplified fragment length polymorphism) is a new type of DNA molecular markers which was created and continuously developed by the Netherlands scientist Zabeau and Vos in 1993. Recent years, AFLP has been thoroughly applied in potato breeding (Iovene and Barone, 2004; Tae-Ho, 2005; Riccardo et al., 2007; Björn B. D’hoop et al., 2008; Zhang et al., 2009; Li et al., 2007; Di et al., 2006). Meanwhile, it was gradually applied in genetic relationship and genetic diversity of many plants, such as Erianthus (Liu et al., 2009), Tea (Ji et al., 2009), Motherwort (Yu et al., 2009), longan (Peng et al., 2008), Naked oat (Xu et al., 2009), Jujube (Qiao et al., 2009), Cane (Lao et al., 2008), Evodia (Huang et al., 2008). The conservation of Chinese potato is little and also the genetic basis of it is narrow. Introduction and collection of potato germplasms are extremely vital, and further define the relationship between these resources and Chinese potato varieties is the premise of making full use of the introduction of resources. Give a broad overview of potato genetic diversity research in China, the results showed that Chinese potato genetic basis was narrow, and examined potato materials were limited to some domestic cultivars. In this study, the genetic diversity of International Potato Center (CIP) resources and some potato varieties in Qinghai Province were analyzed by AFLP markers. Selecting parents with genetic differences in hybridizing will be able to broaden the genetic basis, increase the heterozygosity of breeding populations, maintain a rich population polymorphic and provide parent materials with distant genetic relationship for further study of potato breeding. The aims of this study are: determine the genetic distance between the examined potato materials and to know genetic relationships of CIP resources and Chinese potato cultivars.

1 Results
1.1 Polymorphic Analysis
64 potato materials were amplified with 12 pairs of selected AFLP primer combinations. Different primer combinations had differences in the number of fragments, length of fragment and percentage of polymorphism. 12 pairs of primers amplified a total of 505 bands with a mean of 42 bands, of which 388 were polymorphic bands. Average polymorphism rate (PR) was up to 76.28%, indicating that AFLP markers can be used to analysis the genetic diversity of potato efficiently. The amplified bands were between 33 for E42M48 and 56 for E42M50 with an average of 52 per primer pair (Table 1). The Polymorphisms Rates (PR) varied from 64.29% for E42M50 to 94.44% for E36M59 (Table 1) with an average of 76.28%. The selective amplification results of 64 potato varieties in Table 1.

 
Table1 Table 1 The results of amplification of 64 potato materials

Using POPGENE 32, Shannon’s Information Index (I) and Nei’s Gene Diversity Index (H) of tested potato germplasms were obtained (Table 1). Shannon’s Information Index of materials was between 0.2456 and 0.4988 with an average of 0.3745, Nei’s Gene Diversity Index of materials varied from 0.1510 to 0.3256 with a mean of 0.2359, which dismayed all examined potato germplasms have genetic difference and rich genetic diversity.

1.2 Genetic Distance (GS)
The genetic distance (GS) of 64 potato germplasms in the range from 0.041 237 1 to 0.391 732 6, of which the smallest genetic distance is 0.041 237 1 between E87 and E95, the largest genetic distance is 0.391 732 6 between E104 and E22, E104 is the CIP collected from Peru, while E22 is from Germany. Followed by the E104 and E30, the genetic distance is 0.383 505 2. As the E series potato resource is collected from different countries (regions) by CIP so that these resources have rich polymorphism. At the GS of 0.20, the known varieties bred in China gathered in the same category, while the other CIP germplasms were grouped together, showed that there is great expansion space of the genetic background of Chinese potato varieties to some extent.

1.3 Cluster Analysis
UPGMA cluster analysis was performed with the SAHN procedure of NTSYS-pcVersion2.1 (Figure 1).

 
Figure 1 Phenogram of UPGMA cluster analysis based on among 64 potato germplasms

Cluster results showed that the genetic similarity coefficient (GS) of examined potato materials varied from 0.68 to 0.96. The dendrogram of genetic relationship of 64 potato germplasms was obtained by UPGMA cluster analysis (Figure 1). At GS of 0.76, all materials can be divided into six major categories, a category group VI and five small groups of I, II, III, IV, V. Category I consists of two anti-virus varieties E104 and E107 introduced from CIP; Category II has a CIP varieties anti-virus E35 which was introduced from Peru; Category III consists of Longshu5 and B4, Longshu5 was a new hybrid cultivar derived from cultivar Xiaobaihua as the female parent, and innovation 119-8 as male parent selection by Gansu Academy of Agricultural Sciences grain in 1988. Category IV included anti-virus variety E32; Category V consists of two cultivars E31 and E22; Category VI contains 56 species, accounting for 87.5% of tested potato materials. At the GS of 0.75, category VI divided into four sub-categories, including a large class d and three small sub-categories a, b and c. d is a major sub-category which contains 52 cultivars, at the GS of 0.78, d was further divided into four groups, group d1 consists of four cultivars with resistance to virus E73, E43, E62 and E37; group d2 included five anti-virus varieties E49, E41, E90, E83, E33 and a domestic variety B5; group d3 included a variety E80; group d4 was a large group of including 41 cultivars, of which 14 were Chinese cultivars, accounting for 34.1% of this group, 27 were CIP varieties.

The cluster results showed that most of Chinese cultivars gathered together, such as Qingshu168, Gaoyuan4, Gaoyuan5, B1, B2, Xiaobaihua, Leshu1, Qingshu328, Qingshu2 and Qingyin5 were grouped in the 5th of the d subgroup group d4; cultivars with same resistance were grouped together, such as cultivars with resistance to virus E19, E41, E49, E38, E52, E60, E80, E87, E94 and E95 gathered in group d4 of sub-category d.

Genetic diversity analysis, genetic distance and cluster analysis genetic all showed that Chinese potato varieties have closer genetic differences and have quite narrow genetic base, CIP potato resources and Chinese potato cultivars have greater genetic diversity. In potato breeding of China, CIP potato resources can be taken advantage to broaden the genetic background of potato cultivars.

2 Discussions
2.1 The Effectiveness of AFLP Markers
In this study, 12 pairs of primer combinations with rich markers, high polymorphism were selected for analyzing the genetic diversities among the tested 64 potato cultivars. The selected primer combinations could completely separated all materials(Figure 2), indicating that the AFLP approach is accurate and reliable. AFLP markers have been widely used in analyzing the genetic diversity of germplasms and genetic relationship because of its rich information, high polymorphism and stability (Lucchini et al., 2003).

 
Figure 2 The results of selective amplification products in 6% denaturing

The study of Zhou (1998) showed that when the number of markers 20 below, the information obtained is not very reliable, as the number of locus increases, the amount of information generated and reliability have increased, and when the number of loci is reached or exceeded 70, the reliability of the information provided was stable. In this study, 505 AFLP loci were obtained, its stability and reliability of the information are sufficient for analyzing the genetic diversity of potato. In the test, cultivar Qingshu328 is pre-cultivated name of Qingshu2, processing cultivar F18 and Shepody are the same species, processing F1 and anti-virus E19 are the same species, in the dendrogram Qingshu2 and Qingshu328 clustered together, their GS of 0.91, F18 and Shepody clustered together, the GS of 0.90, F1 and E19 clustered together, their GS of 0.89, these results not only showed the differences between individual of same species, but dismayed that the AFLP markers is accurate and reliable; E104 and E107 have highest GS up to 0.97, indicating E104 and E107 have similar parents or the source of parents is relative, the two materials need to be identified because of its may be same cultivars drawn from different countries with different names.

2.2 Genetic Uniqueness of Potato
In breeding, select hybrid parents with genetic differences for hybridization can obtain high heterosis. It is particularly important for potato to broaden the narrow genetic base. The traditional breeding approach chose the hybrid parents according to phenotype of plant accompany of many limitations. The reproduction of potato belongs to vegetative organs. Tetraploid and highly heterozygous are the main characteristic. The separation of their offspring is much more complicated than the diploid plants. The effect of selection of parent mterials was affected by the composition of gene loci, genetic inheritance of traits and the interaction of environmental conditions, which results in potato breeding extremely difficult. According to statistics, probability of breeding a new variety is 1/200000, probability of breeding a new significant variety is one millionth. Therefore, it is most crucial to select excellent parent meterials in potato breeding.

2.3 Genetic Diversity of Chinese potato
Analysis showed that CIP potato resources and Chinese potato varieties with lower genetic similarity and genetic difference. At the GS of 0.82, most of Chinese cultivars grouped together, showing that Chinese cultivars have high genetic similarity and narrow genetic base. This result is consistent with results researched befor (Di et al., 2006; Xu and Jin, 2008; Duan et al., 2009). Meanwhile, the 64 potato materials, of which 47 were introduced from the CIP, these potato resources are collected from all over the world, such as Peru, Colombia, Mexico, Bolivia, Germany, India and other countries, the other 17 potato cultivars were from Qinghai, Gansu, Shanxi provinces, which formed the basis of genetic diversity of examined potato germplasms to some extent. The introduction of the potato CIP resources can be fully used for potato breeding in China.

3 Materials and Method
3.1 Plant Materials
In this study, 64 potato individuals were examined, among which 47 germplasms are introduced from CIP with resistance to virus, processing resources and 17 are Chinese cultivars (Table 2).

 
Table 2 Materials

3.2 DNA Extraction
The genomic DNA of potato was extracted by optimized CTAB method. The concentration and quality of extracted DNA were detected by the spectrophotometer and 1% agarose gel electrophoresis. The purified DNA (20 ng/ul) was preserved at -20℃.

3.3 AFLP Analysis
AFLP analysis system was the optimized protocol of Li (2010). Restriction enzyme Mseâ…  and EcoRâ…  were purchased from NEB (New England Biolabs), Mseâ… and EcoRâ…  adaptors and primers were synthesized by Shanghai Health Engineering Biological Engineering Technology Services Limited (Sangon). T4 DNA ligase was purchased from Fermentas. Taq DNA polymerase, dNTPs, DL2000 and 100 bp DNA Ladder was purchased from Tiangen Bio-technology (Beijing) Co., Ltd.

12 pairs of primer combinations were E42M50, E36M54, E42M48, E38M48, E35M62, E42M59, E36M48, E39M54, E39M59, E36M61, E36M59, E39M49 (Table 3).

 
Table 3 The Sequence of primers and adapters

3.4 AFLP Data Analysis
Clear bands on the AFLP profile were counted as “1”, no band counted as “0”, statistics from each pair of primers of all bands and polymorphic bands, the results were inputted Microsoft Excel Table.

Percentage of polymorphic bands (P): P=(K/N)×100%, of which K is the number of polymorphic bands, N is the number of total bands. Using NTSYS-pcVersion2.1, according to GS=2Nij (Ni+Nj), the genetic similarity coefficient (GS) was calculated: Ni is number of bands of ith cultivar, Nj is number of bands of jth cultivar, Nij is number of bands of i and j cultivars. Based on the GS of materials, dendrogram of 64 potato germplasms were obtained by non-weighted group average method (Unweighted Pair-group Average Method, UPGAM, Sneath and Sokal, 1973). Utilizing POPGENE 32 calculated Shannon’s Information Index (I) and Nei’s Gene Diversity Index (H) of examined materials.

Authors’ contributions
FW and FDL analyzed drafted the manuscript. JW and YZ worked on the collection of potato, helped with the materials of the paper. FDL and HHS obtained and analyzed the AFLP data and was involved in the writing. FW conceived the overall study, performed the experiment designs and took part to the data analysis and to the writing. All authors read and approved the final manuscript.

Acknowledgements
This research was supported by the earmarked fund for Modern Agro-industry Technology Research System (nycytx-15) and Innovation Foundation of Qinghai Academy of Agriculture and Forestry.

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