ГЕНЕТИКА, 2014, том 50, № 5, с. 570-580
ГЕНЕТИКА РАСТЕНИЙ ^
УДК 575.1:582.998.2
Estimation of Genetic Diversity Using SSR Markers in Sunflower
© 2014 Z. U. Zia1, H. A. Sadaqat1, M. H. N. Tahir1, B. Sadia2, B. S. Bushman3, D. Hole4, L. Michaels3, W. Malik5
department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan
e-mail: zia_pbg@yahoo.com 2Center of Agriculture Biotechnology and Biochemistry, University of Agriculture, Faisalabad, Pakistan 3Forage and Range Research Lab, Utah State University, Logan, UT 84322-6300 Utah, USA
4Plant Breeding/Genetics, Utah State University, Logan, UT 84322-2325 Utah, USA 5Department of Plant Breeding and Genetics, Bahauddin Zakariya University Multan, Pakistan
Received September 17, 2013
Microsatellites or simple sequence repeats (SSRs) were used for the estimation of genetic diversity among a group of 40 sunflower lines developed at the research area of Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad. Total numbers of alleles amplified by 22 polymorphic primers were 135 with an average of 6.13 alleles per locus, suggesting that SSR is a powerful technique for assessment of genetic diversity at molecular level. The expected heterozygosity (PIC) ranged from 0.17 to 0.89. The highest PIC value was observed at the locus C1779. The genetic distances ranged from 9 to 37%. The highest genetic distance was observed between the lines L50 and V3. Genetic distances were low showing lesser amount of genetic diversity among the sunflower lines.
DOI: 10.7868/S0016675814050142
Sunflower is the major oilseed crop in Pakistan. Its area and production is continuously increasing because of wide adaptability, day neutrality, short duration, quality oil and high cost to benefit ratio. Only a few hybrids and varieties are being grown all over the country year after year, of which majority of the area is under cultivation of Hysin-33 in Pakistan. It is because of low genetic variability among the existing genetic material. It is therefore very important to broaden the genetic base of sunflower for production of high yielding cultivars/hybrids. Historically different morphological and biochemical markers have been used for estimation of genetic diversity. However, DNA marker technology provides a very efficient tool for the investigation of genetic diversity [1]. Genetic diversity of sunflower germplasm has been studied using different isozymes [2, 3] and DNA markers such as restriction fragment length polymorphism (RFLP) [4—6], amplified fragment length polymorphism (AFLP) [6—9], random amplified polymorphic DNA (RAPD) [10— 12], single nucleotide polymorphism (SNP) [13], target region amplification polymorphism (TRAP) marker technique [14—16] and simple sequence repeat (SSR) [17, 18]. Microsatellites or simple sequence repeats (SSRs) are short, tandemly repeated DNA sequences that also have been isolated from the genomes of a number of plant species [19—21]. The microsatellite are one of principal classes of DNA markers being used for fingerprinting [22—24], genome mapping [25—32], phylogenetic and genetic relationship stud-
ies [33], population genetics and marker assisted breeding [34].
The main objective of this study was identification of the most diverse lines from the available germplasm. Owing to their high efficiency in polymorphism, SSRs were used for this purpose. The highly diverse lines selected as a result of this study will be used in the future breeding programs to exploit their heterotic potential.
MATERIALS AND METHODS
Plant material. The plant material consisted of 40 sunflower genotypes i.e. A12, L49, L52, L54, A30, L45, L61, V1, G32, A2, L50, L37, V3, V6, V10, L41, V12, V18, L33, L35, L38, L48, L62, V9, A27, HBRS-1, G82, A23, A1, A18, G40, A45, HBRS-5, L42, A19, A41, L44, L53, L36 and L31 having good combination of both agronomic and quality traits were collected from the Department of Plant Breeding and Genetics, University of Agriculture Faisalabad. The genetic diversity of collected plant material was estimated using SSR marker system to identify the diverse parental lines for future breeding program.
DNA extraction and polymerase chain reaction (PCR). Qiagen Mini-kit was used for extraction of the DNA from the lyophilized tissues of15 days older sunflower leaves. The quantification of the DNA was done using a Nanodrop ND-1000 Spectrophotometer. The DNA samples were diluted to a working concentration of 10 ng/^L. A set of 96 SSR primers were used for the finger printing of collect genetic material.
The PCR was carried out in a final volume of 10 |L containing 3.7 |L d3H2O, 2.0 |L DNA template (10 ng/|L), 1 |L dNTPs (2 mM), 1 |L 10x PCR buffer (w/MgCl2), 0.2 |L jumpstar Taq DNA polymerase, 1 |L of each of the forward and reverse primers (10 mM) and 0.1 |L fdctp (10 mM). The amplification of DNA samples was carried out in a thermocy-cler (ABI). Touchdown' PCR [35] was used to reduce spurious amplification. The initial denaturation step was performed at 94°C for 5 min, followed by 5 cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 60 s. The annealing temperature was decreased 1°C per cycle in subsequent cycles until reaching 60°C (the base annealing temperature varied from 54 to 60°C depending on the primer pairs. Products were subsequently amplified for 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s with a final extension for 20 min.
Markers with non-overlapping fragments were multiplexed and visualized separately on ABI-3730 DNA Analyzer. A total volume of 12.15 |L was prepared for DNA analyzer containing a 2 | L of PCR product, 10 |L of formadide and 0.15 |L of Liz500 size standard. This mixture was loaded in the ther-mocycler and denatured for 5 minutes at 95°C. It was chilled on ice and then analyzed on ABI-3730 DNA Analyzer (Applied Biosystem).
Data analysis. The results from DNA analyzer were converted into a compatible format using a program Genescan and then visualized in Genographer 1.6 for scoring. The bands at each locus were scored as present (1) or absent (0) and generated a 0, I matrix. Genetic Distances (GD) and Principal Co-Ordinate were calculated using GenaLex software. The genetic relationship among genotypes was also estimated by constructing a dendrogram through unweighed pair-group method with arithmetic means [UPGMA; 36] using a computer program PAUP.
RESULTS
Genetic divergence among the genotypes based on SSR markers. A total of 96 SSR primer pairs were used for the estimation of genetic diversity among 40 parental lines, out ofwhich only 22 primers were found to be polymorphic (Table 1). Total numbers of alleles amplified by 22 polymorphic primers were 135 with an average of 6.13 alleles per locus. The number of amplified products varied from two (C0306, C3797, C3464 and C3258) to 13 (ORS691). The results depicted that total polymorphism was 45.19% and it ranged from 25% to 100%. PIC values (expected heterozygosity) for polymorphic primers ranged from 0.17 (C2293) to 0.89 (C1779). The average value ofpolymorphic information content (PIC) for all the 22 polymorphic primers was 0.59 (Table 2). Only four out of 22 primers showed 100 percent polymorphism (Fig. 1,a,b,c,d).
Genetic similarity
The results of similarity matrix revealed a low genetic diversity among all the parental lines. Overall, the values for genetic distances ranged from 9% to 37%. The highest genetic distance (37%) was observed between the genotypes i.e. L50 and V3 followed by 36% among genotypes L50 and V1 and A12 and A2. Minimum genetic distance (6%) was observed between V6 and L61 (Table 3).
Cluster analysis
Unweighted pair-group method using arithmetic averages (UPGMA) cluster analysis was used to construct the dendrogram (Fig. 2). The constructed dendrogram divided 40 genotypes into two main clusters (A and B). Cluster A had three subclusters i.e. 1, 2 and 3 whereas; Cluster B had four subclusters i.e. 4, 5, 6 and 7. Three lines were in subcluster-1 (L31, L41, L48), five in subcluster-2 (L36, L37, L54, V01, L62), three in subcluster-3 (A12, L50, V09), six in subclus-ter-4 (A30, L61, V06, V10, V03, V18), five in subclus-ter-5 (A02, L33, A41, L35, L38), five in subcluster-6 (A27, HBRS-5, L42, L45, V12) and eight in subclut-er-7 (A01, A18, A45, A23, G82, L44, G40, HBRS-1).
DISCUSSION
Molecular markers are considered to be a versatile tool for studying genetic diversity and variability among different plant species. The combining ability analysis is widely used for the identification of best parents for hybridization programs [37]. However, this procedure is laborious and time consuming. The molecular markers provide an alternative tool for the identification of potential inbred lines for development of elite hybrids. The major advantage of DNA markers is that these are not affected by environmental conditions [38, 39]. The microsatellites (SSR) are being used widely nowadays for the exploitation of variability among parental lines due to their polymorphic and multiallelic nature. High number of alleles per locus (6.13) was also observed in this study which is greater than that of 2.32 alleles per locus observed by [40] and 3.5 alleles per locus by [41]. As sunflower is a highly cross pollinated crop therefore, a high number of alleles per locus could be a result of the natural out-crossing among the parental material and also due to having a broad genetic base [42]. These results were closer to those ofYu et al. [17] who observed 5.57 alleles per locus on an average basis. The high number of alleles also could be a result of the experimental conditions because this experiment was performed on an automated system and gels were analyzed on ABI DNA analyzer 3730 followed by Genscan and Geno-grapher which is highly precise than the conventional gels or PAGE ones. All the markers cannot be used for routine genotyping, genetic diversity and variety identification. The strength of a mark
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