Introduction

Sunflower (Helianthus annuus L.) is one of the most important oilseed crops cultivated globally for oil, confectionary and ornamental purposes. Interspecific hybridization played a vital role in introgression of the desirable traits from wild sunflowers to cultivated sunflower including cytosterility with appropriate maintainer and restorers, thus, transforming an ornamental plant to an oilseed crop with high oil productivity potential in the cultivated varieties and hybrids. Despite differences in ploidy levels (diploid, tetraploid, hexaploid), growth habit (annual, perennial), interspecific hybrids were successfully produced between cultivated sunflower and several wild species [13]. Most of these hybridization experiments resulted in complete or partial hybrids with varying chromosome number and structural rearrangements (H. mollis, H. occidentalis, H. orgyalis and H. maximiliani) [2, 10, 19, 20]. Natural hybridization between cultivated and wild forms resulting in intermediate morphologies is also reported [3, 25]. Some of the Helianthus species viz, H. decapetalus and H. strumosus have two forms of ploidy. Often difficulty is encountered in counting the accurate chromosome number in Helianthus with 34–102 chromosomes and with the lengths varying from 37.1 to 191.0 µm [13]. Hence, it is essential to use alternate methods for determining the ploidy of the parents and the resultant hybrids and for success of the introgressive breeding programmes. Aryavand et al. [1] successfully used the stomatal frequency and size to differentiate ploidy forms in Aegilops neglecta. Preliminary studies were done in our laboratory to correlate the stomata per unit area (mm2) with ploidy status but these two variables were significantly negatively correlated (−0.33814). One of the simpler alternatives is determination of ploidy using flow cytometry, which to some extent has substituted the conventional chromosome counting and densitometry as the analysis besides being fast, precise and convenient can help in analysis of large number of samples within a short time [5]. However, reliability of the results depends on the tissue being processed and the stains used as there is a possibility of nonspecific binding of fluorochrome, auto fluorescence from various pigments and interference of secondary metabolites with DNA stains leading to erroneous interpretations [7].

In wild sunflowers, in addition to differences in chromosome number and growth habit, large differences exist with regard to the leaf characteristics particularly with regard to the texture, hairiness, colour, pubescence and such characters are known to interfere in the fluorescence. A wide range of variation in DNA content (3–8.2 pg) was observed among plants of the same sunflower variety by using laser flow cytometry of propidium iodide (PI) stained nuclei [18, 22]. Price et al. [21] reported the presence of unidentified endogenous compounds in sunflower leaves that interfere with propidium iodide (PI) interaction resulting in reduced fluorescence from sunflower nuclei. Gohde et al. [12] proposed the use of DAPI fluorochrome (4,6-dimino-2-phenylindole) for flow cytometry, and since then its utilization in flow cytometry has been widely used because of its specificity and high fluorescence which is very well suited for FCM in whole cells, nuclei and chromosomes.

The coefficient of variation (CV) value was considered as an elementary criterion that reflects the quality of the applied methodology [11, 17]. Reports using DAPI instead of PI resulted in lower CV values [6, 9, 26]. Flow cytometric analysis using DAPI was done in anther culture plants of the interspecific hybrids (triploids) derived from the cross between a hexaploid species H. tuberosus with the diploid cultivated sunflower to determine the ploidy status of the hybrids and anther culture plantlets [24] and intermated F1 populations of H. annuus × H. tuberosus population [15]. The CV values of the study were in a range from 1.2 to 10 %. A CV value of less than 5 % was the acceptance criterion in plants [11]. However, the use of DAPI for estimation of ploidy might be affected by negative effect of cytosol. Some species contain cytosolic compounds (polyphenols), which interfere with DNA staining and might distort FCM measurements [7]. In sunflower, high content of polyphenols, polysaccharides and secondary metabolites resulted in low quality of nucleic acid extraction [16] which resulted in histograms of high background noise.

The present study has been undertaken with the objective of optimization of the sample preparation procedure, determine the ploidy levels of wild Helianthus species using flow cytometry and validation of ploidy in an interspecific hybrid between species of two different ploidies.

Materials and methods

Plant material

Seeds of wild Helianthus species obtained from USDA, USA, and the Institute of Field and Vegetable Crops, Novi Sad, Serbia, were established in the Helianthus species garden at the Indian Institute of Oilseeds Research, Hyderabad, India. These accessions varied in ploidy and growth habit (Table 1). Forty-three accessions of Helianthus accessions along with two accessions of cultivated sunflower (RHA 6D-1, PS 2023) were analyzed for ploidy by flow cytometry. These included one accession each of H. deserticola, H. exilis, H. hirsutus, H. laevigatus, H. multiflorous, H. occidentalis, H. resinosus, H. salicifolius; two accessions each of wild H. annuus, H. divaricatus, H. microcephalus, H. mollis; three accessions each of H. decapetalus, H. giganteus, H. grosseserratus, H. tuberosus; four accessions each of H. nuttalli, H. praecox, H. strumosus and five accessions of H. maximiliani.

Table 1 Ploidy of Helianthus accessions as determined by flow cytometry (FCM)
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Ploidy determination

Preparation of samples for ploidy determination in a PA-I flow cytometry (Partec, Monster, Germany) was done as per the manufacturer’s instructions. Approximately 0.5 cm2 of freshly harvested leaf tissue material was chopped with a sharp razor blade in a Petri dish in 400 µl of nuclei extraction buffer (Partec cystain UV precise P). As the CV was high, modifications such as use of 1 % PVP (polyvinylpyrrolidone) or 15 mM β-mercaptoethanol in nuclei extraction buffer and grinding in liquid nitrogen were done. As leaves of some species (H. tuberosus, H. resinosus) had tough texture, leaf samples were processed using the tissue processor to release nuclei into the lysis solution according to Cousin et al. [4]. Also, the histograms of fresh and frozen tissues (10, 20, 30 days after freezing) were studied. The nuclei suspension was filtered through a cell trics disposable filter (50 µm) into the sample tube. Nuclei were stained for 1 min with 1.6 ml of DAPI staining buffer (Partec Cystain UV precise P). Flow cytometry analysis was done with UV excitation (by mercury arc lamp) of the ploidy instrument with fluorescence excitation below λ = 420 nm and blue emission for DAPI at wavelength between λ = 435 and λ = 500 nm. The sample processing was done on ice and maintained in cold conditions till analysis. The gain of the instrument was calibrated with trout erythrocytes to give a CV of less than 1.0 %. Analysis of samples processed on a particular day was completed on the same day. The start and end samples and every 10th sample included cultivated sunflower RHA 6D-1 (2n = 2x = 34) as control. In each sample, 5000–10,000 nuclei were counted and a minimum of three measurements were made for each sample. The chromosome number of the diploid cultivated sunflower RHA 6D-1 was checked by direct counting on root-tip cells (with the kind help of Dr. Zhao Liu, USDA-ARS, Fargo, ND) (Fig. 1), which served as reference for the 1C measurements and based on previous reports for sunflower [13, 23], the 1C for the reference was taken as 3.4 pg.

Fig. 1
figure 1

Chromosome counting of RHA 6D-1(H. annuus, 2n = 34)

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Confirmation of hybridity of the interspecific hybrid of H. annuus (2n = 2x) and H. hirsutus (2n = 4x) by ploidy analysis and molecular analysis

To validate the ploidy results, a interspecific hybrid (2n = 3x) between H. annuus (2n = 2x) and H. hirsutus (2n = 4x) was characterized using flow cytometry and microsatellite markers. Total genomic DNA of the parents (H. annuus and H. hirsutus) was extracted by following the standard CTAB method with minor modifications [8]. The PCR amplification reaction (15 µl) consisted of 25 ng of DNA, 1X PCR buffer (10 mM Tris pH 9.0, 50 mM KCl, and 1.5 mM MgCl2), 7.5 mM of each of the four dNTPs, 3.75 pM each of forward and reverse primers of the SSR markers ORS 610 and ORS 1114 and 0.6 U of Taq DNA polymerase (Bangalore Genei, India). PCR amplifications were performed in a GeneAmp 9700 Thermal Cycler (Perkin Elmer Applied Biosystems) with an initial denaturation at 94 °C for 2 min followed by 35 cycles at 94 °C for 45 s, 58 °C for 45 s and 72 °C for 1 min with a final extension at 72 °C for 5 min. The SSRs used were selected based on the parental polymorphism from 56 primers. The PCR products were separated on 3.0 % agarose gel in 1x TAE buffer.

Results

Optimization

Release of phenolics and oxidation was observed in some of the samples. Hence, representative material of 2 genotypes was chopped in extraction buffer with and without 1 % PVP. The addition of 1 % PVP to the chopping procedure produces isolated nuclei free of cytoplasm and sunflower inhibitors and makes the DNA accessible to DAPI dye. The addition of PVP used in lysis buffer had a measurable effect on reducing the inhibition and showed distinct peaks (Fig. 2). The peaks measured with a coefficient of variation (CV) less than 5 %, which was the acceptance criterion in ploidy determination (Table 2). Inclusion of β-mercaptoethanol in the nuclei isolation buffer failed to show significant improvement in peak quality or the CV. There were no peaks in samples ground in liquid nitrogen while fluorescence was negligible in frozen samples and samples processed with tissue homogenizer.

Fig. 2
figure 2

Representative histograms obtained with different sample preparation methods in sunflower. a and b. histograms of PS 2023 and RHA 6D-1, respectively with chopping without polyvinylpyrrolidone, c and d. chopping with inclusion of 1 % polyvinylpyrrolidone

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Table 2 Effect of incorporation of PVP in preparing nuclear suspension of sunflower for flow cytometry
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Ploidy analysis

All the 43 Helianthus accessions subjected to flow cytometric analysis with the optimized protocol gave histograms with single peak. Most of the accessions gave narrow peaks with a CV ranging from 4 to 10. FCM histograms obtained were with good resolution (Fig. 3). Based on 1C DNA content, the 45 sunflower accessions analyzed were separated into three different ploidy groups (Table 1). Thirty-five of the accessions were diploid with 1C value varying between 2.3 and 4.9 with a mean of 3.67 pg; four were tetraploid with 1C content ranging from 5.6 to 7.5 with a mean of 6.73 pg; six were hexaploid with the 1C level varying from 8.3 to 11.2 with a mean of 10.07 pg. Accessions belonging to species like H. decapetalus and H. strumosus were reported with different ploidy levels. Ploidy analysis differentiated the 4X and 6X accessions of H. strumosus while all the H. decapetalus accessions used in the study were diploid.

Fig. 3
figure 3

Histograms of relative fluorescence (relative nuclear DNA content) obtained from fresh leaves of Helianthus species

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Hybrid confirmation using SSR primers and Flow cytometry

The hybridity of the interspecific hybrid (H. annuus × H. hirsutus) was confirmed with SSR primer which produced bands of both the parents and the polymorphism observed with ORS 1114 is presented in Fig. 4. Further confirmation of hybridity was done by flow cytometric estimation of ploidy of both the parents and hybrid. The mean value of the peak was 91 for H. annuus (diploid), 182 for H. hirsutus and 131 for the F1 hybrid indicating the triploid nature of the hybrid.

Fig. 4
figure 4

Hybridity confirmation of an interspecific hybrid developed from two Helianthus species with different ploidy levels. P1. Histogram of diploid H. annuus (2n = 2x), P2. Histogram of the tetraploid H. hirsutus (2n = 4x), H. Histogram of the triploid F1 (2n = 3x), S. Hybridity confirmation of F1 with SSR marker, ORS 1114, P1 (H. annuus, 2n = 34) and P2 (H. hirsutus, 2n = 68) are the parents, H is the hybrid between H. annuus and H. hirsutus, M represents 100 bp marker

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Discussion

Chromosome counting and identification in wild Helianthus species and their derivatives is laborious due to high number (2n = 2x, 4x, 6x = 34, 68, 102), small size, poor spread resulting in inconsistencies. Interspecific hybridization requires accurate identification of not only the parental material but also the resultant hybrids and the subsequent derivatives through backcrossing.

It is reported that sunflower leaves have unidentified compounds which lead to low fluorescence with propidium iodide (PI) staining of nuclei [21]. In this present investigation, flow cytometric analysis has been carried out in Partec Ploidy analyzer in which the kits are based on the DAPI fluorochrome (4,6-dimino-2-phenylindole). Flow cytometric analysis with the optimized protocol gave histograms with single peak. Most of the species gave narrow peaks with a CV of less than 10. The addition of 1 % PVP for extraction of nuclei might have reduced the oxidation of polyphenolics and resulted in good yield and purity of nucleic acids [16]. The procedure yielded FCM histograms of the nuclear DNA content with good resolution and allowed detection of small increase of DNA content variability. Failure to get histograms or low fluorescence in samples ground in liquid nitrogen, tissue homogenizer and frozen samples could be due to the damage to the nuclei or poor release of intact nuclei.

In the present study, the 1C values reported for sunflower are in the range of values reported for cultivated sunflower. The 1C values were between 3.0 and 3.98 in different sunflower inbreds [18] and 3.1–3.19 in a natural populations of sunflower collected from different sites [22].

Well spread metaphase plates are possible in cultivated sunflower (Fig. 1) but accurate determination of the chromosome number is difficult in sunflower and the interspecific hybrids because of the existence of different ploidy levels (2n = 34, 68, 102) [14]. Chromosome counting is laborious, time consuming and is often a challenge to get good spread of metaphase chromosomes. Flow cytometry allows ploidy level determination with speed and precision. In the present study, ploidy levels of 45 sunflower accessions of cultivated and wild sunflowers were determined accurately. The species with existence of two ploidy levels (DEC-01, DEC-1887, DEC-1992, STR-15, STR-216, STR-1532 and STR-1934) were easily distinguished through ploidy analysis. Most of the ploidy levels represented in the present study corroborates the findings from chromosome counts [13].

Studies of interspecies hybridization in sunflower have included wide crosses between annual sunflower and perennial species. The F1 hybrids are confirmed either by cytological examination or using molecular markers [2, 14, 20]. The present investigation had successfully proved the hybridity and ploidy status of F1 hybrid between H. annuus (2n = 2x) and H. hirsutus (2n = 4x). This method of ploidy determination by flow cytometry is rapid and accurate and can be utilized in conjunction with molecular markers for characterization of interspecific hybrids and their derivatives. FCM has become the method of choice because of it is convenience, rapidity, reliability and ploidy can be determined at initial stage of growth of the interspecific hybrids.

Conclusions

In the present investigation, the ploidy analysis procedure for sunflower has been optimized and used for determination of ploidy of different Helianthus species. Further, the utility of the technique was checked through confirmation of interspecific hybrid between H. annuus × H. hirsutus with different chromosome numbers using sunflower specific SSR primers and flow cytometry. Thus, the technique of flow cytometry with minor modifications could be employed to confirm the ploidy status of wild Helianthus species and also the interspecific hybrids between Helianthus species from different ploidy levels reliably and can be successfully used in introgressive breeding programmes in sunflower.