Estimation of diversity and combining abilities in Helianthus annuus L. under water stress and normal conditions

Field layout and data collection

The present study was performed in the research area of Department of Plant Breeding and Genetics (PBG) and the molecular studies were done in the Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, during the year 2014. The seeds of fifteen parents and fifty hybrids were sown during the kharif growing season in 2014 under normal and drought conditions. Two rows of each parent and hybrid were grown; the inter-row and inter-plant spacing was kept at 60 and 30 cm, respectively. Length of each line was kept at 2 m. A randomized complete block design with three replications was followed. Three seeds per hole were sown by hand at a depth of 3 inches. All recommended agronomic and plant protection practices for normal crop cultivation were adopted uniformly for all the parents and hybrids throughout the growing season. For stress plot (rainout shelter), irrigation was stopped after the flowering stage. Thinning was performed and one healthy plant per hole was maintained. Proper care was taken to avoid bird attack. Ten plants from each replication were tagged and guarded in both normal and drought conditions. Upon maturity, plant height (cm), head diameter (cm), achene yield/plant (g), relative water content (RWC) (Malik, 1995), excised leaf water loss (ELWL; Dhanda and Sethi, 1998), and membrane stability index (MSI; Premchandra et al., 1990) were measured for 10 guarded plants at flowering stage.

Assessment of genetic diversity using SSR markers

This experiment was conducted at CABB, University of Agriculture, Faisalabad. The DNA of parents was extracted and the SSRs were amplified using polymerase chain reaction (PCR) in a thermal cycler (Eppendorf AG22331, Hamburg) to estimate the genetic diversity present in the material. DNA of fifteen parents was extracted using the procedures stated by Doyle and Doyle (1990), with some amendments (Iqbal et al., 2011). Extracted DNA was quantitated using a spectrophotometer (Thermo Scientific Nanodrop 2000, USA). Working solution for each parental sample comprised 50 ng DNA in 1 μL double-distilled deionized water (d₃H₂O). The concentration of genomic DNA and reaction mixture were optimized for SSR analysis (Mba et al., 2001). List of primers used is presented in Table 1. Working solution of each of parental and hybrid samples containing 50 ng DNA in 1 μL d₃H₂O was prepared. The concentration of genomic DNA, 10X PCR buffer with (NH₄)₂SO₄, MgCl₂, dNTPs (dATP, dCTP, dGTP, dTTP), forward and reverse SSR primers, and Taq DNA polymerase were optimized for SSR analysis (Mba et al., 2001) Twenty pairs of forward and reverse SSR primers were obtained from Gene Link (USA) (details of primers are given in Table 1). Taq polymerase together with buffer, MgCl₂, dNTPs and gelatin were purchased from Fermentas (Germany). The master mixture for fifteen parental sample reactions against one primer was prepared through mixing of 4.3 μL d₃H₂O, 2.0 μL 10X PCR buffer, 3.0 μL dNTPs, 2.5 μL MgCl₂, 1.5 μL forward primer, 1.5 μL reverse primer and 0.2 μL Taq polymerase. For one sample reaction, 3 μL diluted DNA of each parent was mixed with 15 μL master mixture in one PCR tube. DNA amplification reactions were performed on a thermal cycler (Eppendorf AG No. 5333 00839). In the thermal cycler, at 94°C, the DNA was denatured, i.e., the two strands became separated. At 55°C, the SSR primers (forward and reverse) anneal their respective complementary sequences on the DNA strands. At 72°C, DNA polymerase extends the DNA chain by adding nucleotides to the ends of the primers. The PCR profile was as followed: the denaturation temperature of 94°C was kept for 5 min during initial cycle while it was kept for 45 s during the remaining 35 cycles. Annealing and extension temperatures were kept at 55°-60°C for 45 s and 72°C for 1 min, respectively, during 35 cycles. After completion of 35 PCR cycles, extension temperature of 72°C was provided for 7 min for a final extension.

After completion of PCR, 3.0 μL bromophenol blue was added (Mullis et al., 1994). Because of the large pore size of agarose gel, SSR fragments with minute size differences could not be separated. Therefore, to isolate such fragments and obtain distinct bands, polyacrylamide gel electrophoresis (PAGE) was performed. The amplicons generated with different SSR primers were scored as 1 or 0 for presence or absence, respectively, for all the genotypes under study. Similarity coefficient matrix among all the genotypes was computed according to Nei and Li (1979). A dendrogram based on these similarity coefficients was constructed with un-weighted pair group method of arithmetic means algorithm (Sneath and Sokal, 1973) using the NTSYSpc software package.

Statistical and biometrical analysis

All the studied traits were subjected to analysis of variance according to Steel et al. (1997). Data were further analyzed for GCA and SCA using line x tester method (Kempthorne, 1957). GCA and SCA effects were estimated.

Assessment of genetic diversity among parental material using SSRs

The electrophoretic profiles of bands amplified in parents, as assessed by SDS-PAGE, were used to construct a dendrogram. Forty-three clear bands were observed on the gel. In all the plants, on an average 3.1 alleles per locus were observed with twenty SSR primer pairs. Many other bands of low molecular weight were also observed, but owing to discrepancy in reproducibility these were not included in the results. Gel picture for primer ORS-237 amplifying SSRs from fifteen parental sunflower genotypes is given in Figure 1. A Euclidean similarity coefficient matrix was calculated for parents and a dendrogram was constructed (Figure 2). Using Ward’s method, three major lineage groups were revealed at a linkage distance of 10. Cluster 1 (linkage distance 4) further divided germplasm into two sub-clusters (1a and 1b). Sub-cluster 1a consisted of three genotypes namely A-11, A-10.8, and A-9 and sub-cluster 1b consisted of three genotypes named A-6, RL-51, and RL-52. Cluster 2 consisted of three genotypes named C-11, B-1.10, and C-3, whereas Cluster 3 consisted of two sub-clusters, 3a and 3b. Sub-cluster 3a comprised two genotypes, B-2.8 and A-35. Sub-cluster 3b comprised four genotypes, C-10, B-4, B-3, and G-60. The maximum distance was observed between A-10.8 and G-60. Darvishzadeh et al. (2010) observed 2.32 alleles per locus, and 3.5 alleles per locus were witnessed by Zhang et al. (2005) and Sujatha et al. (2008), whereas Duca et al. (2013) detected a total of 34 alleles with an average number of 3.4 alleles per locus. The contradictory results might be due to difference in the experimental conditions as well as lack of precision with ordinary gels. On the other hand, low genetic distances were observed, which indicated that the breeding material had narrow genetic base and might belong to the same geographical area. In the present study, genotypes that were susceptible to drought (used as tester) shared the same cluster whereas genotypes that were tolerant to drought (used as inbred lines) tended to cluster together. These findings were in accordance with those of Zia et al. (2014).

Figure 1

Gel picture for primers ORS-237 amplifying SSRs from 15 parental sunflower genotypes. Lane M = DNA ladder showing different sizes for amplified fragments; lanes 1 to 15 = SSRs amplified from different parental lines (lanes 1-10 are female parents; lanes 11-15 are male parents).

Figure 2

Dendrogram of 15 genotypes of sunflower based on SSRs amplified using 20 primer pairs.

Combining ability analysis