Introduction

Glycine is a genus of leguminous plants, which includes the cultivated soybean (Glycine max [L.] Merr.), the wild annual soybean (Glycine soja Sieb. & Zucc.), as well as a number of perennial species (Kollipara et al. 1995; Hymowitz 2004). Phytophthora sojae Kaufm. & Gerd. is an oomycete pathogen of soybeans, causing root and stem rot in older plants, and damping-off of seedlings. Annual worldwide losses to Phytophthora root and stem rot can reach US $1–2 billion (Wrather et al. 2001; Wrather and Koenning 2006). The disease is managed through the deployment of single genes (Rps genes) that confer resistance to P. sojae. Currently fourteen Rps alleles have been reported at eight different loci. The Rps8 locus was identified in the South Korean landrace PI 399073 (Dorrance and Schmitthenner 2000), and was assigned to the soybean molecular linkage group (MLG) F (Gordon et al. 2006) corresponding to chromosome 13 of G. max.

Multiple mapping populations were developed in recent years by crossing PI 399073 and the cultivar Williams, which is considered as the universal susceptible genotype to P. sojae. These segregating populations were advanced for several generations for the purpose of the identification of molecular markers for the development of breeding material carrying the Rps8 locus, and to assist in the cloning of the gene. Each population was phenotyped for resistance to P. sojae and genotyped with a set of markers on chromosome 13. The resistance phenotype was associated with markers in the Rps8 locus region (Ortega et al. 2010). However, the expected phenotypic and genotypic segregation ratios were always highly skewed (Ortega et al. 2008, 2010). In each of the populations derived from Williams × PI 399073 crosses, an excess of Rps8/Rps8 homozygous RILs and Rps8/rps8 heterozygous RILs were obtained at the expense of the rps8/rps8 homozygous RILs.

Segregation distortion is not uncommon in mapping populations, and is one of the several factors that influence the precision of genetic mapping. It has been shown that both the genetic distance between markers and the order of the markers on linkage groups could be affected by this phenomenon (Lorieux et al. 1995a, b). In soybean, 18 regions on ten different linkage groups where high levels of segregation distortion occur were previously described (Yamanaka et al. 2001), including the soybean root fluorescence locus Fr1 on chromosome 9 (MLG K) (Jin et al. 1999). The mechanisms involved in segregation distortion are not well understood. However gametophytic factors, competition among gametes, or the abortion of gametes have all been proposed (Lu et al. 2000, 2002; Lyttle 1991; Matsushita et al. 2003).

Continued development of virulent pathotypes of P. sojae that can infect plants with Rps resistance genes (Grau et al. 2004) and the limited number of effective Rps genes that are currently deployed in US cultivars, makes the quest for novel sources of resistance a high priority. Sources of resistance to host-specific plant pathogens are usually found in the regions of greatest differentiation of host species (Leppik 1970). In the case of Phytophthora root and stem rot, sources of resistance have been identified in both Chinese and South Korean germplasm (Dorrance and Schmitthenner 2000; Kyle et al. 1998; Lohnes et al. 1996). However, phenomena like segregation distortion can complicate the development of resistant cultivars when genes are introgressed from novel sources of resistance. In the case of Rps8, segregation distortion in populations derived from PI 399073 represents a major challenge to fine map and clone this gene. Identifying the mechanisms that contribute to the segregation distortion of Rps8 is crucial for the identification of breeding strategies that will expedite the use of this gene for the management of Phytophthora root and stem rot of soybeans.

In plant development, microsporogenesis is the cellular division that produces haploid microspores which develop into pollen grains, and this is also an important process where meiotic abnormalities may be detected. A large number of microspores are produced in each anther, making them a feasible target for the evaluation of the different meiotic phases. Normal microsporogenesis in soybean has been described (Albertsen and Palmer 1979), and serves as a useful guide for the evaluation of abnormalities. Structural changes in chromosomes that effect the production of normal gametes, such as inversions and translocations (Mahama et al. 1999; Palmer et al. 2000) have also been previously described in soybean.

To produce pollen grains, male gametogenesis starts with the division of a diploid sporophyte that gives rise to both the tapetum and pollen mother cells (PMCs) (McCormick 1993). The later cells undergo meiosis and give rise to tetrad cells that are released as microspores when the callose is degraded by the enzyme callase produced by the tapetum. The microspores undergo mitosis to generate pollen grains containing the larger vegetative cell and the small generative cell. The irregularities reported in soybean male meiosis include: chromosome associations, abnormal spindles, precocious chromosome migration, chromosome stickiness, chromosome fragments, laggards, bridges, micronuclei, cytokinesis failure, and production of microcytes (Bione et al. 2000, 2002, 2005; Kumar and Rai 2006; Palmer et al. 2000). In general, abnormal male meiosis tends to have an outcome of partial or total pollen sterility. This is especially important in soybean, as this genus self fertilizes and genotypic factors associated with those pollen grains would not be passed to the next generation.

Due to the high level of segregation distortion across the numerous crosses of PI399073 and G. max cultivars, a comparative study of Rps8/rps8 lines from two parental combinations, Williams × PI 399073 and PI 399073 × PI 408211B was initiated. Our objective was to determine if male gametogenesis abnormalities occur in Rps8/rps8 lines which originated from populations with a high degree of segregation distortion as well as examine populations where this locus segregates in a true Mendelian fashion. For each genotype, the targets were meiosis I and II, microspores, and mature pollen grains.

Materials and methods

Plant material

A BC4F2:3 population consisting of 30 lines was generated by backcrossing the region containing the Rps8 locus from PI 399073 into cultivar Williams (recurrent parent). BC4 seeds were advanced by single-seed-descent to generate F2:3 seeds. In a previous study, the population was phenotyped for resistance to P. sojae through hypocotyl inoculation. P. sojae isolate OH25, virulent to plants carrying the Rps genes 1a, 1b, 1c, 1k, and 7, was used to identify lines carrying the resistance locus Rps8 from PI 399073 in the BC4F2:3. In addition, a F4:5 population consisting of 152 recombinant inbred lines (RILs) was generated by crossing PI 399073 and PI 408211B, F2 seeds were advanced by single-seed-descent. The phenotypic data for disease resistance was obtained by inoculation with P. sojae isolate BUTMU. This isolate has a compatible interaction (susceptible response) with the Rps genes in PI 408211B, and an incompatible interaction (resistance response) in PI 399073. Lines with a heterozygous phenotype were genotyped with 72 SSR and SNP markers in the Rps8 region, and four lines were selected from each population for this study, these lines were heterozygous for the resistance phenotype and molecular markers located between the SSRs Satt114 and Satt362 (Table 1).

Table 1 Genotype of the Rps8 region on the selected BC4F2:3 lines and F4:5 RILs
Full size table

Seeds from each heterozygous line and parental genotype were planted in 2-l pots of sterilized soil mixture. Based on earlier genotypic data, recombinant inbred lines (RILs) 1403, 1404, 1412, and 1413 from the BC4F2:3; and RILs 37, 50, 58, and 143 from the F4:5 populations were selected for this experiment (Ortega et al. 2010). Plants were grown at 27°C with 14 h daylight period (with supplemental lighting provided); watered twice every day, and supplemented with 100 ppm 20N:20P:20K greenhouse fertilizer. Studies were from January to March 2009.

DNA isolation and genotyping

Genomic DNA was extracted using a modification of the protocol described by Keim et al. (1988). Cotyledons from 3 week-old seedlings were collected in 10 × 10 cm2 reclosable plastic bags (Uline, Inc., Philadelphia, PA) and stored at 4°C until processing. Tissue was ground in CTAB buffer: 100 mM Tris–HCl pH 8.0, 1.4 mM NaCl, 2.0% CTAB (hexadecyltrimethyl-ammonium bromide), and 20 mM EDTA pH 8.0. One milliliter of the extraction buffer was added to the plastic bag, and the tissue was macerated using a hand-held roller (BIO-RAD, Hercules, CA). The suspension was transferred to a 2.0 ml tube and incubated at 65°C for 1 h, and mixed vigorously every 15 min. The sample was cooled to 27°C, and an equal volume of 24:1 (v/v) chloroform-isoamyl alcohol (Sigma Chemical Co., St. Louis, MO) was added. An emulsion formed after inverting the tubes several times, followed by centrifugation at 10,000 rpm for 10 min in a table-top microcentrifuge. The supernatant was transferred to a 2.0 ml tube, and the DNA was precipitated from the solution by adding 99% isopropyl alcohol and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the DNA pellet was washed with 70% ethyl alcohol. The DNA pellet was air dried overnight and resuspended in 500 μl of TE buffer (10 mM, 1 mM EDTA pH 8.0). RNA was removed by treatment with 2.0 μl of 5 mg/ml Ribonuclease A (Sigma–Aldrich, St. Louis, MO) were added to the reactions and incubated at 37°C for 1 h. DNA was quantified by spectrophotometry using NanoDrop (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following the manufacturer instructions, and the samples were stored at −20°C.

To verify that each RIL carried heterozygous genotypes, each line was screened with eight SSR markers across the Rps8 region. The markers Satt114, Satt334, Satt362 located on chromosome 13 (MLG F) on the soybean consensus map (Cregan et al. 1999); AC15916, 98FA16; and F420_18, F336-01, and F336_18 developed from sequences from Williams82 BAC clones and mapped to chromosome 13 were used for genotyping. Template DNA was diluted to 50 ng/μl in TE buffer and stored at −20°C. The PCR amplification was done in a 12.5 μl reaction mixture containing 1× Green Go Taq Flexi Buffer (Promega, Madison, WI), 2 mM MgCl2 (Promega), 200 mM of each deoxynucletide (Promega), 200 nM of each primer, 1 U Go Taq DNA polymerase (Promega), and 50 ng of genomic DNA. All PCR reactions were carried out on a DNA Engine Tetrad 2 Peltier Thermal Cycler (BioRad, Hercules, CA). The thermal conditions were 94°C for 5 min; ten cycles of touch-down PCR: 94°C for 45 s, 60–50°C (decreasing 1°C per cycle) for 45 s, and 72°C for 1 min; followed by 24 cycles with annealing temperature of 50°C; and final extension at 72°C for 10 min. PCR products were analyzed on 4.0% agarose 3:1 HRB (Amresco, Solon, OH). Agarose was dissolved in 1× RapidRun agarose buffer (USB, Cleveland, Ohio), pre-stained with 0.5 μg/ml of ethidium bromide (Sigma–Aldrich, St. Louis, MO) and cast in 20 × 25 cm trays (Fisher Scientific, Pittsburgh, PA). Ten microliter amplicons were electrophoresed in 1× RapidRun agarose buffer for 25 min at 250 V. Electrophoresed gels were visualized and digitally photographed.

Pollen viability and germination

Open flowers were collected between 9:00 and 11:00 a.m. during the first 3 weeks of flowering. Three flowers were collected from each plant, three times a week. Each set of anthers was dissected and dusted onto pollen germination medium (Gwata et al. 2003) and incubated at 27°C for 18 h. A minimum of 100 pollen grains per anther were observed for germination under a S6D Stereozoom microscope (Leica Microsystems Inc., Deerfield, Illinois, USA). A grain was classified as germinated if a recognizable pollen tube, at least 20 μm long was present. Pollen viability was assessed with Lugol’s solution (Electron Microscopy Sciences, Hatfield, PA), consisting of 5% iodine and 10% potassium iodide, and this staining detects starch content. The same set of anthers used to determine percent germination were placed in 100 μl of Lugol’s solution on a 25.4 × 76.2 mm slide (Becton–Dickinson Labware, Franklin Lakes, NJ). The slide was covered with a 22 × 40 mm cover glass (Daigger, Vernon Hills, IL) and visualized under a binocular DME light microscope with the 20× magnification objective (Leica Microsystems Inc., Deerfield, IL). Pollen grains which were stained dark brown to black were considered viable.

Cytological analysis of male meiosis

In this study acetic carmine staining was used for visualization of the chromosomes (Schreiber 1954). Immature flower bud clusters were collected between 9:00 and 12:00 a.m., the stage reported for meiotic analysis in soybeans (Bione et al. 2003; Mahama et al. 1999; Palmer et al. 2000). Flower buds from single plants were placed in 2.0 ml microcentrifuge tubes containing 1.5 ml of formalin-aceto-alcohol mixture (Ricca Chemical Company, Arlington, TX) for fixation. The samples were incubated at 27°C for 24 h and stored at 4°C until assayed.

Anthers were dissected and transferred to 0.2 ml tubes containing 0.75% acetic carmine (Carolina Biological Supply Company, Burlington, NC). Carmine was dissolved in 45% acetic acid, and it served the double purpose of fixation and staining; acetic acid penetrates membranes rapidly, and carmine is insoluble in chromatin. The staining was enhanced by adding 2 μl of 10% w/v ferric chloride solution (Sigma Chemical Co., St. Louis, MO). Dissected anthers were incubated at 70°C for 8 h and maintained at 27°C for another 24 h. The anthers were blotted on Kimwipes (Kimberly-Clarke, Roswell, GA) and placed on a 25.4 × 76.2 mm slide (Becton–Dickinson Labware, Franklin Lakes, NJ) containing 100 μl of mounting media (Rattenbury 1956). The slide was covered with a 22 × 40 mm cover glass (Daigger, Vernon Hills, IL). The slides were placed on the dissecting scope and each anther was crushed, by applying pressure on the cover glass with a dissecting needle, until the anther wall broke and meiotic cells were released. The preparations were sealed with nail polish.

The preparations were viewed under a binocular DME light microscope (Leica Microsystems Inc., Deerfield, Illinois, USA) at 1000× magnification, and photographed using a Nikon digital sight DS-SM camera and DS-L1 computer (Nikon Corp., Japan).

Results

Pollen viability

One plant from each line and parents was used for evaluation of pollen viability, this was done so that it would be possible detect variation for this parameter between the different collection times, and at the same time leave enough immature flowers for the cytogenetic studies. A mean of 90% of the pollen grains stained dark brown with Lugol’s solution indicating viability (data not shown). In addition, there was no significant difference among plants for pollen germination on the same sampling day, but a significant (P = 0.05) difference was found between sampling days for the same plant (Fig. 1). Pollen collected from flowers produced on the first week of the reproductive stage, independently of the plant evaluated, had germination percentages lower than 55%. The percentage of pollen grains that germinated increased in the second week, and was maintained above 85% during the third week of flowering.

Fig. 1
figure 1

Pollen germination percentage for one plant from each line which was evaluated from the beginning of the reproductive phase (R1). Three open flowers were collected 3 days per week for 3 week. The percentage of germinated grains was determined after 18-h incubation in the medium described by Gwata et al. (2003)

Full size image

Cytogenetics

During the microsporogenesis process in PI 399073 and RILs from the BC4F2:3 Williams × PI399073 several meiotic abnormalities were observed from the stained anthers collected from immature flower clusters. Pollen abnormalities were not found in PI 408211B nor in Williams. For the Williams × PI 399073 BC4F2:3 derived plants, there were 255 abnormal meiotic cells from the 371 meiotic cells evaluated; this number was higher than the observed in any other genotype. For the PI 399073 × PI 408211B F4:5 lines, only 9 of 396 cells exhibited abnormalities (Table 2). During meiosis I, the following abnormalities were observed: extra nucleolus (Fig. 2a) in the pollen mother cells at prophase I, chromosome fragments that were not part of the metaphase plate (Fig. 2b–d), laggards present between the two chromosomes sets at anaphase I, and micronuclei formed between the two nuclei at telophase I (Fig. 2f). In the fixed flower buds of the genotypes, anthers at meiosis I were identified more frequently than anthers at meiosis II. Similar meiotic abnormalities were also observed in meiosis II.

Table 2 Pollen meiotic abnormalities in PI 399073, PI 408211B, Williams, and selected Rps8/rps8 heterozygous RILs from: Williams × PI 399073 BC4F2:3, and PI 399073 × PI 408211B F4:5 crosses
Full size table
Fig. 2
figure 2

Meiotic irregularities observed in Williams × PI 399073 BC4F2:3 Lines. a Prophase I, micronucleus. b, c Metaphase I, chromosome fragments. d Metaphase I, chromosome not aligned at the metaphase plate. e Late anaphase I, laggards. f Telophase I, micronucleous. g Metaphase II, Chromosome fragments. h, i Coenocytic tetrad, binucleate cells. j Tetrad cells, micronuclei. k multinucleate microspore. l Dyad, microcyte

Full size image

The types of abnormalities of the male gametes were determined by observation of the microspores formed after meiosis II (Table 2). Flowers in the same cluster were each in a different stage of microsporogenesis, thus the characteristics of microspores and pollen grains were noted for each genotype (Table 3). In PI 399073, a mix of dyads, triads, and pentads were found (Fig. 3). In this parental genotype, tetrads comprised only 67% of the meiotic products; and 63% of them had micronuclei in at least one of the microspores. For Williams and PI 408211B, only tetrads were observed. In selected lines from the Williams × PI 399073 BC4F2:3, microspores containing micronuclei were common (Fig. 2j, k), and ‘triads’ containing two microspores and a microcyte were also observed (Fig. 2i). This type of triad was only observed in the anthers from the Rps8/rps8 Williams × PI 399073 BC4F2:3 RILs. Uninoculated microspores with thick cell walls were formed in the non dehiscent anthers from PI 399073, PI 408211B, the Rps8/rps8 Williams × PI 399073 BC4F2:3 RILs, and the Rps8/rps8 PI 399073 × PI 408211B F4:5 RILs (Fig. 4a, b, e, f). In contrast, in Williams, the majority of the microspores were in the binucleate stage, after mitosis I (Fig. 4c); germinated pollen grains were also common inside the non dehiscent anthers of cultivar Williams (Fig. 4d). A few non-viable pollen grains, not stained with acetic carmine, were observed in the immature anthers of all the genotypes evaluated (Fig. 4b). Pollen grains from the anthers of Rps8/rps8 BC4F2:3 RILs were one-third the size of an average pollen grain and contained one or more micronuclei, the cytoplasm in these grains was not darkly stained but a cell wall like structure was observed (Fig. 4e). These small grains corresponded to 87% of the sterile pollen found in the Rps8/rps8 BC4F2:3 RILs, and may be the product of the microcytes formed in earlier stages. However, these grains were not detected when dehiscent anthers were used for evaluation of pollen viability and germination, indicating that these small grains may collapse and degrade before anthesis.

Table 3 Characteristics of the meiotic products and pollen grains from PI 399073, PI 408211B, Williams, and selected Rps8/rps8 heterozygous RILs from: Williams × PI 399073 BC4F2:3, and PI 399073 × PI 408211B F4:5 crosses
Full size table
Fig. 3
figure 3

Meiotic products in PI 399073: a multinucleate dyad, b multinucleate tetrad, c multinucleated triad, d mononucleated pentad

Full size image
Fig. 4
figure 4

Microspores characteristics among parental genotypes and one RIL: a PI 408211B, mononucleated grains; b PI 399073, sterile grain; c Williams, binucleated grain; d Williams, grain germinating inside the anther; e Line 1403 (BC4F2:3), microcyte grain and f Line 37 (F4:5), mononucleated grains

Full size image

The meiotic abnormalities and meiotic products observed for the BC4F2:3 Rps8/rps8 heterozygous lines were also identified on the anthers of BC4F2:3 Rps8/rps8 heterozygous plants that were grown in a preliminary study, during the summer of 2008 (June–August). The plants evaluated on the preliminary study included four lines from the same BC4F2:3 population evaluated on this study, including RIL 1412, and six lines from another BC4F2:3.

Discussion

In this study the male gametogenesis in Rps8/rps8 heterozygous lines and their parents was evaluated. When pollen from the same anthers was studied in selected heterozygous RILs and parental genotypes in the first week of flowering, most of the grains stained with Lugol’s solution, indicated that they were viable. However, a low percentage of pollen germinated under the test conditions across all genotypes. The cause of poor pollen germination during the first week of flowering in this study is unknown. Previously in soybeans, temperature, UV radiation, and CO2 levels have been shown to affect pollen morphology and germination (Koti et al. 2005). Cytogenetic staining techniques were effective for the visualization of meiotic chromosomes and detection of abnormalities during their separation during haploidization. The products of male gametogenesis were also subjected to analysis, and two methods were employed to determine the percentage of viable pollen in each genotype. The frequency of meiotic abnormalities was higher in Rps8/rps8 heterozygous lines from a Williams × PI 399073 BC4F2:3 population with a high degree of segregation distortion in the Rps8 region, than in Rps8/rps8 heterozygous lines from a PI 399073 × PI 408211B F4:5 population in which the Rps8 region segregated normally in a Mendelian fashion.

Chromosome elimination affects the correct separation of chromosomes during cellular division and has been attributed to: chromosome fragmentation, micronucleous formation and chromatin degradation (Subrahmanyam and Kasha 1973; Thomas 1988); lagging chromosomes (laggards), bridges, chromosomes non-congregated at the metaphase plate, and failure of chromosome migration to the poles during anaphase (Bennett et al. 1976). Chromosome elimination has also been reported during microsporogenesis (Adamowski et al. 1998). In our study, the meiotic abnormalities observed during microsporogenesis, accompanied by the presence of microspores and microcytes containing micronuclei, indicates that chromatin elimination may be a potential mechanism influencing segregation distortion in these lines. This mechanism does not seem to have noticeable effects on pollen viability, because there was no correlation between meiotic abnormalities and the percentage of stained pollen or germinated pollen at later flowering dates. However there was a correlation between the frequency of meiotic abnormalities and the presence of microcytes. In particular, RILs from the Williams × PI 399073 BC4F2:3 where a high level of abnormalities were detected, the fertility of the mature pollen was not affected, indicating that the loss or gain of the micronuclei may not have a serious effect on the pollen grain viability.

Many mechanisms of chromosome elimination have been described (Singh 1993), however the process involved in the elimination of micronuclei as microcytes is still obscure. The elimination of micronuclei from microspores in oat (Avena sativa L.) was reported by Baptista-Giacomelli et al. (2000). A micronucleus reaches the microspore wall and separate from it by forming a bud, then the formed microcyte give rise to a sterile pollen grain; this process has not been described in any other species to date. Partial genome elimination through micronuclei and the production of aneuploid gametes was described in plants from a natural population of G. max (Kumar and Rai 2006). In this study tetrads containing quiescent micronuclei were also present, and pollen viability was not affected. The process that gave rise to the microcytes in the Williams × PI 399073 BC4F2:3 is not clear, although a similar mechanism is suspected since micronuclei in the tetrads were located close to the wall (Fig. 3j), and it is unlikely that the cell wall in the small pollen grains could have originated using the limited genetic material inside them. The maintenance of micronuclei within the microspores could be the result of low efficiency in the elimination process.

The identification of different types of meiotic products in PI 399073 and RILs of the Williams × PI 399073 BC4F2:3 was striking. Although these abnormal dyads, triads, tetrad, and pentads do not appear to have an effect on pollen viability, this phenomenon may not be uncommon as similar types of meiotic products have been observed in other species including a pentaploid accession of Brachiaria brizantha (Risso-Pascotto et al. 2003). In the microsporogenesis stage, micronuclei were formed and some remained inside the microspores, while others were eliminated as microcytes in a similar mechanism to the described by Baptista-Giacomelli et al. (2000). The dyads and triads formed in B. brizantha were produced by failure in cytokinesis, these microspores developed into 2 N pollen through reinstitution of nucleus. It is possible that a similar process occurs in PI 399073 as this type of pollen was observed in anthers of this landrace. This type of meiotic behavior could limit the breeding potential of a particular genotype if the progeny exhibits these meiotic abnormalities. Precocious pollen germination in soybean genotypes was previously described by Kaur et al. (2005) as a strategy that might facilitate a high degree of selfing and interfere in hybridization efforts. This could explain the production of pods from partially open flowers observed in the cultivar Williams.

These findings are limited to the heterozygous plants that were evaluated in this study, thus the mechanism behind the meiotic abnormalities in PI 399073 and its progeny, and what role if any these abnormalities play specifically in the high degree of segregation distortion at the Rps8 locus still needs to be explored. In this study, the meiotically abnormal lines originated from a cross where PI 399073 was used as pollen donor, whereas in the meiotically normal lines PI 399073 was the pollen recipient; it is unknown if abnormalities in megasporogenesis are occurring, if the ovules were affected, this could explain why the differences were found between these two populations. Unfortunately lines from reciprocal crosses were not available at the time of this study. In the future, if these lines are available they can be used to determine if the meiotic abnormalities depend on the genotype used as donor parent or on the geographic/genetic distance between the parents. Future studies will focus on the Rps8 region and its association with the chromosome fragments, laggards, micronuclei and microcytes. This will only be possible if BAC clones from PI 399073 and Williams 82 located in this region are identified and fully characterized.