Introduction

The genus Stylosanthes belongs to the family Fabaceae and consists of 48 species that are naturally distributed in the tropical, subtropical, and temperate regions of the Americas, Africa, and Southeast Asia [1]. Each species has rich variations in morphology, physiology, and genetics. These abundant genetic resources establish an important foundation for improved hybrid breeding and morphological characteristics. S. guianensis (Aubl.) Sw. (2n = 20) is the most widespread Stylosanthes species and exhibits remarkable phenotypic variations [2, 3]. This species is one of the most important tropical forage legumes currently known and is native to South and Central America and Africa, where it is widely distributed, although not in the equatorial zone [2]. S. guianensis has been used successfully as a pasture legume in many parts of the tropics and subtropics [4].

A range of Stylosanthes species was introduced from Australia into tropical southern China in the early 1980s [5, 6]. Stylosanthes is well-adapted to the environment of Guangdong and Hainan province. A series of Stylosanthes cultivars has been grown by selective breeding, including S. guianensis cv. Reyan No. 2 in 1991, S. guianensis cv. Reyan No. 5 in 1999, and S. guianensis cv. Reyan No. 20 in 2010. Cross breeding is commonly used to improve the characteristics of Stylosanthes and overcome the time and labor limitations of selective breeding. Thus, the hybridity of new seedlings must be verified at an early stage to assure the uniformity and stability of the field performance and yield of the crop and optimize planting time and costs.

DNA molecular marker technology, which is based on sequence variations of specific genomic regions, provides a powerful tool for hybrid identification and seed verification. This technology has the advantages of time savings, reduced labor consumption, and higher efficiency compared with other methods [7–11].

The main DNA marker techniques currently in application are restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), inter-simple sequence repeat (ISSR), and sequence-related amplified polymorphism (SRAP). SRAP preferentially amplifies open reading frames, which are coding sequences in the genome. SRAP can disclose numerous co-dominant markers with a large amount of polymorphic loci and allows easy isolation of bands for sequencing. It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements. Core sequences, which are 13–14 bases long, where the first 10 or 11 bases starting at the 5′ end, are sequences of no specific constitution (“filler”sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by 3 selective nucleotides at the 3′ end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long [12]. This technique can generate more polymorphic fragments that reveal genetic diversity than SSR, ISSR, or RAPD markers [13]. Ferriol et al. [14] showed that the information provided by SRAP markers is more consistent with the morphological variability and evolutionary history of the morphotypes than that obtained from AFLP markers. Therefore, SRAP markers are ideal molecular markers for genotype identification, map construction, gene tagging, and genomic and cDNA fingerprinting, such as in the diversity analysis of buffalo grass [13, 15, 16]. Thus far, few studies that use SRAP markers for true hybrid identification have been reported.

Many methods for hybrids identification most focused on SSR, RAPD and AFLP molecular markers, few focused of SRAP molecular makers. Considering these advantages, the present study detects true hybrids of S. guianensis using SRAP markers. The specific goals of the study are: (1) to assess the feasibility of the SRAP technique in the study of S. guianensis and (2) to identify true S. guianensis hybrids using SRAP markers.

Materials and methods

Plant materials

Table 1 shows the 84 hybrids generated by 8 hybrid combinations of 12 parents [1,979(D) × 109(P), 1979(D) × 106(P), 3079(D) × 45(P), 3183(D) × 96(P), 3206(D) × 16(P), 3265(D) × 16(P), 3268(D) × 16(P), and 3321(D) × 109(P)] that were used in this study. The female parents were male-sterile. Each hybrids and the parents (n = 3) were grown in 20 cm diameter pots under uniform conditions in the greenhouse of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences (Hainan Island).

Table 1 Hybrid combinations and progenies of S. guianensis
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DNA extraction

Total genomic DNA was isolated according to the modified hexadecyltrimethylammonium bromide (CTAB) DNA extraction procedure described by Huang et al. [17]. The quality and quantity of genomic DNA were estimated by measuring A260/A280 using a UV spectrophotometer. The intactness of the genomic DNA was verified by gel electrophoresis [18]. The DNA concentration was adjusted to 50 ng/μL to facilitate polymerase chain reaction (PCR) amplification. DNA samples were stored at −20 °C until use.

SRAP PCR amplification

A total of 100 primer combinations, including 10 forward and 10 reverse primers, from Sheng Gong Inc. (Shanghai, China) were tested by PCR (Tables 2, 3) [12].

Table 2 List of the forward and reverse SRAP primers used in the present study
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Table 3 The 100 SRAP primer combinations used in the present study
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Each 25 μL PCR reaction mixture contained 80 ng of genomic DNA, 0.4 μM primer, 250 μM dNTPs, 1.5 mM MgCl2, 1.5 units of Taq polymerase, and 2.5 μL of 1 × PCR buffer. The mixtures were overlaid with 20–30 μL of mineral oil. DNA amplification was performed using a Thermal Cycler (Bio-Rad S1000 ™, USA) under the following conditions: initial denaturation at 94 °C for 4 min, followed by 5 cycles of 1 min denaturation at 94 °C, 1 min annealing at 35 °C, and 30 s elongation at 72 °C. Denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and elongation at 72 °C for 1 min were conducted in the following 30 cycles. Each cycle ended with an elongation step for 10 min at 72 °C. The amplified products were stored at 4 °C until analysis. The amplification products were separated by electrophoresis on 1.5 % (w/v) agarose gel in 1.0 × TBE buffer (0.09 mol/L Tris-H3BO3, 0.002 mol/L EDTA, pH 8.0) at a constant voltage of 100 V for approximately 1.5 h. GoldView (0.5 μg/mL) was added to facilitate UV light visualization. Molecular weights were estimated using a 100 bp DNA ladder.

Results

SRAP analysis

The primers were selected for their ability to yield clear, polymorphic, and reproducible patterns of amplification. Polymorphisms observed between male and female parents were used as markers to assess the purity of the hybrids. Only 35 of the 100 SRAP primer combinations tested generated multiple fragments between the 8 hybrid combinations of 12 parents (Table 4), 75 were rejected for failing to produce amplification or displaying monomorphic patterns. These 35 primer combinations were chosen for further analysis on the basis of their capability to facilitate good amplification. SRAP analysis results in Table 5 show that: (1) only 4 (11.43 %) of the 35 primer combinations generated male parent-special markers in hybrid combination 2 (1,979 × 109), with a total of 4 male parent-special markers; (2) 24 primer combinations (68.57 %) produced male parent-special markers in hybrid combination 4 (1,979 × 106), with a total of 40 male parent-special markers; (3) 4 primer combinations (11.43 %) produced male parent-special markers in hybrid combination 7 (3,079 × 45), with a total of 5 male parent-special markers; (4) 3 primer combinations (8.57 %) produced male parent-special markers in hybrid combination 16 (3,183 × 96), with a total of 3 male parent-special markers; (5) 4 primer combinations (11.43 %) produced male parent-special markers in hybrid combination 19 (3,206 × 16), with a total of 5 male parent-special markers; (6) 5 primer combinations (14.29 %) produced male parent-special markers in hybrid combination 21 (3,265 × 16), with a total of 9 male parent-special markers; (7) 6 primer combinations (17.14 %) produced male parent-special markers in hybrid combination 22 (3,268 × 16), with a total of 10 male parent-special markers; and (8) 3 primer combinations (8.57 %) produced male parent-special markers in hybrid combination 28 (3,321 × 109), with a total of 5 male parent-special markers.

Table 4 The 35 SRAP primer combinations used in the present study
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Table 5 Filtering results of the primer combinations
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Figure 1 shows that three primer combinations, namely, Me1-Em1, Me3-Em9, and Me4-Em2, could be used to determine hybrid purity because they exhibit polymorphic bands between the male and female parents in hybrid combination 4 (1,979 × 106).

Fig. 1
figure 1

Polymorphic bands of SRAP primer combinations between parents in hybrid combination 4 (1,979 × 106) Note: M: 50 bp ladder marker; the arrowhead indicates the polymorphic bands in the male parent; the odd numbers represent male parents, whereas the even numbers signify female parents. The primer combinations were Me1-Em1, Me1-Em5, Me1-Em8, Me3-Em1, Me3-Em2, Me3-Em3, Me3-Em9, Me4-Em2, Me4-Em4, and Me5-Em10

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Hybrid identification

Only the primer combinations that amplified male parent-specific bands of each hybrid were considered to verify hybrid purity. Table 6 shows the following: (1) Primer combination Me1Em8 verifies that 2-01 is a true hybrid whereas 2-07 is not a true hybrid in hybrid combination 2 (1,979 × 109). (2) Primer combination Me4-Em2 can verify hybrid purity in hybrid combination four (1,979 × 106) because it exhibits two bands (950 and 1,450 bp) in the progenies, which are specific to the respective male parents in all cases. The 9 progenies of hybrid combination 4 (1,979 × 106) have 1 or 2 male-special bands except for 4-08. Thus, these progenies may be verified as true hybrids (4-01, 4-02, 4-03, 4-09, 4-11, 4-12, 4-13, 4-15, 4-16). Hybrid 4-08 is not a pure hybrid (Fig. 2). (3) Two progenies are pure hybrids (7-01, 7-18), whereas 4 progenies (7-03, 7-08, 7-17, 7-19) are not true hybrids in hybrid combination 7 (3,079 × 45). Hybrid purity may be verified by Me5Em10. (4) Four progenies in hybrid combination 7 (3,079 × 45) are pure hybrids (16-04, 16-06, 16-07, 16-08), as verified by Me1Em8. (5) Only 19-07 is a true hybrid in hybrid combination 19 (3,206 × 16). All other progenies are not true hybrids (19-06, 19-07, 19-08, 19-09, 19-10, 19-11, 19-12, 19-13, 19-14, 19-15,19-16), as verified by Me5Em10. (6) Twenty-eight progenies of hybrid combination 21 (3,265 × 16) are true hybrids (21-01, 21-02, 21-03, 21-04, 21-05, 21-14, 21-16, 21-19, 21-20, 21-22, 21-23, 21-28, 21-29, 21-31, 21-32, 21-37, 21-40, 21-41, 21-44, 21-46, 21-51, 21-52, 21-53, 21-54, 21-60, 21-69, 21-70, 21-71); these findings may be verified by Me9Em1. (7) Eighteen progenies in hybrid combination 22 (3,268 × 16) are true hybrids (22-01, 22-03, 22-04, 22-06, 22-07, 22-09, 22-10, 22-11, 22-12, 22-13, 22-14, 22-15, 22-16, 22-17, 22-18, 22-19, 22-20, 22-21), as verified by Me5Em10, Me10Em7, Me8Em1, and Me4Em2. (8) Five progenies in hybrid combination 28(3,321 × 109) are true hybrids (28-01, 28-03, 28-04, 28-05, 29-07), as verified by Me5Em10 and Me8Em9.

Table 6 Hybrid identification results
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Fig. 2
figure 2

SRAP patterns obtained from hybrid combination 4 (1,979 × 106), as determined by the Me4-Em2 primer combination. Note: M: standard DNA (50 bp ladder marker). Lanes 1–10: fragments from hybrids; the arrowhead indicates male parent-specific markers. The hybrids obtained include 4-01, 4-02, 4-03, 4-08, 4-09, 4-11, 4-12, 4-13, 4-15, 4-16

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Discussion

Identification of hybrid authenticity is important in cross-breeding. The rapid and accurate identification of hybrid authenticity is an important basis of cross-breeding. However, not all types of markers are suitable and or feasible for identification and characterization of the hybrids. Traditionally, grow-out trails (GOTs) has been widely applied to hybrid identification in a variety of crops, which involve growing a representative sample of the seed followed by analysis of several morphological characteristics in different developmental stages. However, GOTs is time-consuming and costly, and requires extensive use of land. Furthermore, GOTs is easily affected by growing conditions which making the determination difficult [19]. Isozyme electrophoresis technologies also have been employed for hybrid identification, but the limitation of this method is their failure to detect polymorphisms in closely related lines. Thus, more sensitive methods are necessary to test and determine hybrid purity. This has shifted the focus to DNA-based molecular markers.

With the advantages of time- and resource-saving, less labor-consumption and more precision, molecular markers are becoming vital tools for cultivar identification and seed quality control in many crops. Recently, several molecular markers, such as SSR [19–21], RAPD [10, 22, 23], ISSR [24, 25], and AFLP [26] have been extensively used for the identification of hybrids. However, studies that use SRAP for true hybrid identification have rarely been reported. The present study developed a method to identify S. guianensis hybrids. The authenticity of 84 progenies of 8 hybrid combinations of S. guianensis was identified by SRAP markers. Sixty-eight (80.95 %) progenies exhibited male parent-specific bands and were thus identified as true hybrids. These true hybrids could be used for map construction and cross-breeding in S. Guianensis.

This study is the first to apply DNA markers to identify the purity of S. guianensis hybrids. A new, simple, and low-cost method was presented in this study. The proposed method may be used in S. guianensis breeding programs worldwide. Our experimental results indicate that SRAP technology is a powerful and efficient approach for hybrid identification. Several studies that employ the SRAP technique to assess the genetic diversity of several crops, such as Pennisetum purpureum Schumach [27], Lactuca sp. [28], Salvia miltiorrhiza Bge [29], and Cynodon Radiatus [30], have been published. SRAP markers offer several advantages over other available molecule techniques, including production of highly specific polymorphic fragments and low requirements of DNA amount and quality.

The SRAP profiles of 84 hybrids and their respective parents show that the SRAP marker technique has good potential application in the identification of S. guianensis hybrids. Potential primers depicting distinct and good polymorphic markers may be used for the identification, registration, and protection of S. guianensis.

Conclusions

This study demonstrates the feasibility of applying SRAP markers to indentify the authenticity of hybrid progenies. A total of 84 progenies were determined. Sixty-eight (80.95 %) of these progenies that exhibited male parent-specific bands were identified as true hybrids. The remaining 16 progenies were self-hybrids because of the absence of male parent-specific bands. These true hybrids will be used for map construction and cross-breeding in S. Guianensis.