Wild Perennial Glycine Species Research Articles

Evaluation of wild perennial Glycine species for resistance to soybean cyst nematode and soybean rust

AbstractThe genetic base for soybean cultivars is narrow compared to most other crop species. Twenty‐seven wild perennial Glycine species comprise the tertiary gene pool to soybean that may contain many genes of economic importance for soybean improvement. We evaluated 16 accessions of G. argyrea, G. clandestina, G. dolichocarpa, and G. tomentella for resistance to Heterodera glycines (HG), also known as the soybean cyst nematode, and to multiple isolates of Phakopsora pachyrhizi, the causal fungus of soybean rust. All 16 accessions were classified as resistant to H. glycines HG Type 2.5.7, based on number of cysts per root mass with plant introductions (PIs) 483227, 509501, 563892, and 573064 (all G. tomentella) void of any cysts indicating no reproduction by this pest. All 16 accessions had an immune reaction to one isolate of P. pachyrhizi. Regardless of isolate, no sporulating uredinia were observed on G. argyrea (PI 505151) and G. tomentella (PIs 483227, 509501, and 573064). These results demonstrate that some accessions within the perennial Glycine species harbour resistance to both H. glycines and P. pachyrhizi and would be good candidates for wide hybridization programs seeking to transfer potentially unique multiple resistance genes into soybean.

Cytogenetics and genetic introgression from wild relatives in soybean

Cultivated soybean, wild soybean, and 23 perennial Glycine species contain 2n = 40 chromosomes. Accessions of G. hirticaulis and G. tabacina have diploid (2n = 40) and tetraploid (2n = 80) chromosome numbers. In contrast, G. tomentella includes accessions with four cytotypes (2n = 38, 40, 78, or 80). Meiosis of G. max and G. soja F1 is normal and gene exchange is easy. Occasionally, accessions of the two species differ by chromosome structural changes (reciprocal translocation/paracentric inversion). The segregating populations inherit undesirable traits such as vining, lodging susceptibility, lack of complete leaf abscission, seed shattering, and small black coated seeds because of genetic drag. The introgression of useful genes from 26 wild perennial Glycine species to soybean has been restricted because of crossability barriers and requires extensive hybridization, immature seed rescue, and cytogenetic manipulations. This review paper discusses cytogenetics, gene pools of the soybean and the production of fertile G. max plants using 78-chromosome G. tomentella (PI 441001) as a paternal and maternal parent in crosses with G. max cv. ‘Dwight’ (2n = 40). Thus, reporting breaking of the intersubgeneric crossability barriers for the first time.

Characterization of Seed Storage Proteins of Several Perennial Glycine Species.

Perennial Glycine species, distant relatives of soybean, have been recognized as a potential source of new genetic diversity for soybean improvement. The subgenus Glycine includes around 30 perennial species, which are well-adapted to drought conditions and possess resistance to a number of soybean pathogens. In spite of the potential of the perennial Glycine species for soybean improvement, very little is known about their storage proteins and their relationship with cultivated soybean seed proteins. We have examined the seed protein composition of nine perennial Glycine species by one- and two-dimensional (1-D and 2-D) gel electrophoresis. The relationship between cultivated soybean and perennial soybean seed proteins was examined by immunoblot analyses using antibodies raised against G. max β-conglycinin, glycinin A3 subunit, lipoxygenase, leginsulin, Kunitz trypsin inhibitor, and Bowman-Birk protease inhibitor. Additionally, we have measured the trypsin and chymotrypsin inhibitor activities from cultivated soybean and perennial Glycine species and have found marked differences between them. Our 2-D gel and immunoblot analyses demonstrate significant differences in the protein composition and size heterogeneities of the 7S and 11S seed storage proteins of soybean and perennial Glycine species. Perennial Glycine species accumulated a 45 kDa protein that was not detected in G. max and G. soja. This unique 45 kDa protein was immunologically related to the A3 glycinin subunit of G. max. The results of our studies suggest that even though the seed proteins of wild perennial Glycine species and G. max are immunologically related, their genes have diverged from each other during the course of evolution.

Intersubgeneric hybridization between Glycine max and G. tomentella: production of F₁, amphidiploid, BC₁, BC₂, BC₃, and fertile soybean plants.

This paper describes methods for unlocking genetic treasure from wild perennial Glycine species of Australia for soybean improvement. The genetic resources of the ca. 26 species of the genus Glycine subgenus Glycine have not been exploited to broaden the genetic base of soybean (Glycine max; 2n = 40). The objectives of this study were to develop methods for producing F1, amphidiploid, BC1, BC2, BC3, and fertile soybean plants from crosses of soybean and the genus Glycine subgenus Glycine species, in order to utilize the subgenus Glycine germplasm in soybean breeding. Soybean cultivars were hybridized with six accessions of 78-chromosome G. tomentella as well as one accession each of 40-chromosome G. tomentella, G. argyrea and G. latifolia. They were chosen because they exhibit resistance to soybean rust. We were successful in producing fertile soybean from soybean cv. 'Dwight' and 78-chromosome G. tomentella accession PI 441001, while other hybrids were discontinued either at F1 or amphidiploid stage. The F1 seeds aborted prior to reaching maturity, so developing seeds from 19 to 21 day old pods were cultured aseptically in various media formulations. Seed maturation and multiple embryo generation media were developed. F1 plants with shoots and roots (2n = 59) were transplanted to pots in greenhouse. Amphidiploid (2n = 118) plants were backcrossed to 'Dwight'. BC1 (2n = 79) plants were propagated through in vitro and 43 mature BC2F1 seeds were harvested. Fifteen surviving BC2F1 plants were morphologically distinct, sterile, and had chromosome numbers ranging 2n = 56-59. Chromosome numbers of the BC3F1 plants ranged 2n = 40-49. Derived fertile soybeans were first planted in the field in 2008 and are being evaluated for yield, resistance to pathogens and pests and tolerance to salt through material transfer agreement.

Multicellular Secretory Trichome Development on Soybean and RelatedGlycineGynoecia

Multicellular glandular trichomes form on gynoecia of wild annual Glycine species, annual soybean cultivars, and wild perennial Glycine species. These trichomes occur from the ovary base to the style base and, in perennial species, along the style as well. Trichomes form at least 2 d before anthesis, and new trichomes develop throughout flowering and also on young seed pods. Trichome structure is similar in all taxa examined, usually five to seven linearly arranged cells. Stalk cells with callose walls become highly vacuolate, and their cytoplasms have reduced numbers of Golgi bodies and endoplasmic reticulum. During secretion, two to four distal cells develop dense cytoplasms containing both prominent Golgi bodies with large vesicles and endoplasmic reticulum with enlarged lumens. Both organelles are involved in forming carbohydrate/protein secretory products. Internal secretion begins with products exuding between the plasmalemma and the primary wall of each distal cell. Secretion progresses between the primary wall and the outermost cuticle that entirely covers the trichome, which is thicker and more highly modified than a normal cuticle. This cuticle separates from cell walls in secretion regions. Products are exuded outside, occur on the inner surface of trichome and gynoecium, and are more obvious in perennial than in annual taxa. The composition and function of products are unknown.

A four-nucleotide base-pair deletion in the coding region of the Bowman-Birk protease inhibitor gene prevents its accumulation in the seeds of Glycine microphylla PI440956.

The Bowman-Birk protease inhibitor (BBI), an abundant soybean [ Glycine max (L.) Merr.] seed protein, is a major antinutritional factor. Nulls for the major soybean BBI have been reported in several of the wild perennial Glycine species including G. microphylla (Benth.) Tind PI440956. This perennial Glycine species does not accumulate the major BBI and the molecular basis for the absence of the major BBI in this plant introduction (PI) line is not known. We have cloned the BBI gene from G. microphylla PI440956, G. microphylla PI505188, and G. max cv. Jefferson and determined its nucleotide sequences. Analysis of the G. microphyllla PI505188 and G. max cv. Jefferson nucleotide sequences revealed a complete open-reading frame encoding the BBI. In contrast, the BBI coding region of G. microphylla PI440956 contained a frameshift mutation that resulted in the introduction of a stop codon at the amino terminal region of the protein. Reverse transcription-polymerase chain reaction analysis revealed that the BBI gene was expressed in developing seeds of G. microphylla PI505188 and G. max cv. Jefferson, but not in developing seeds of G. microphylla PI440956. In contrast, a BBI-related isoinhibitor gene was expressed at similar levels in all three Glycine species. Our results suggest that the frameshift mutation in the BBI coding region is responsible for the absence of BBI in the seeds of G. microphylla PI440956.

Genomic relationships in the genusGlycine (Fabaceae: Phaseoleae): use of a monoclonal antibody to the soybean Bowman‐Blrk inhibitor as a genome marker

We investigated the use of a monoclonal antibody (MAb 238) to the soybean Bowman‐Birk inhibitor (BBI) to verify and understand the intergenomic relationships among the wild perennial Glycine species. Competitive enzyme linked immunosorbent assay and western blot screening studies revealed that the accessions of B‐genome (G. latifolia, G. microphylla, and G. tabacina, 2n = 40) and C‐genome (G. curvata and G. cyrtoloba) species did not contain the MAb 238 crossreactive proteins (BBI‐nulls). By contrast, all the A‐genome (G. argyrea, G. canescens, G. clandestina, and G. latrobeana), E‐genome (G. tomentella, 2n = 38), and F‐genome (G. falcata) species, G. arenaria (genome unknown), and the polyploid (2n = 78,80) G. tomentella accessions were BBI‐positive. The D‐genome G. tomentella (2n = 40) and tetraploid G. tabacina (2n = 80) contained both BBI‐null and BBI‐positive type accessions. Among the recently described species, G. hirticaulis (2n = 40), G. lactovirens, and G. pindanica contained the MAb 238 crossreactive proteins while G. albicans did not. Glycine hirticaulis, G. pindanica, and G. tomentella (2n = 38) displayed highly similar MAb 238 crossreactive isoelectric focusing banding patterns, indicating that they are genomically close to each other. Glycine hirticaulis was found to have both diploid (2n = 40) and tetraploid (2n = 80) cytotypes. We demonstrated that the MAb 238 was specific to the trypsin inhibitor domain of the BBI. The MAb 238 clearly reflected all the previously established relationships in the genus Glycine, validating its use as a genome marker.

Genomic Relationships in the Genus Glycine (Fabaceae: Phaseoleae): Use of a Monoclonal Antibody to the Soybean Bowman-Birk Inhibitor as a Genome Marker

We investigated the use of a monoclonal antibody (MAb 238) to the soybean Bowman-Birk inhibitor (BBI) to verify and understand the intergenomic relationships among the wild perennial Glycine species. Competitive enzyme linked immunosorbent assay and western blot screening studies revealed that the accessions of B-genome (G. latifolia, G. microphylla, and G. tabacina, 2n = 40) and C-genome (G. curvata and G. cyrtoloba) species did not contain the MAb 238 crossreactive proteins (BBI-nulls). By contrast, all the A-genome (G. argyrea, G. canescens, G. clandestina, and G. latrobeana), E-genome (G. tomentella, 2n = 38), and F-genome (G. falcata) species, G. arenaria (genome unknown), and the polyploid (2n = 78,80) G. tomentella accessions were BBI-positive. The D-genome G. tomentella (2n = 40) and tetraploid G. tabacina (2n = 80) contained both BBI-null and BBI-positive type accessions. Among the recently described species, G. hirticaulis (2n = 40), G. lactovirens, and G. pindanica contained the MAb 238 crossreactive proteins while G. albicans did not. Glycine hirticaulis, G. pindanica, and G. tomentella (2n = 38) displayed highly similar MAb 238 crossreactive isoelectric focusing banding patterns, indicating that they are genomically close to each other. Glycine hirticaulis was found to have both diploid (2n = 40) and tetraploid (2n = 80) cytotypes. We demonstrated that the MAb 238 was specific to the trypsin inhibitor domain of the BBI. The MAb 238 clearly reflected all the previously established relationships in the genus Glycine, validating its use as a genome marker.

Backcross (BC2‐BC4)‐Derived Fertile Plants from Glycine max and G. tomentella Intersubgeneric Hybrids

Wild perennial relatives of the soybean [Glycine max (L.) Merr.] have not been utilized to broaden the genetic base of the crop. This study provides information on production, morphology, cytology, and breeding behavior of backcross (BC2 to BC4)‐derived fertile plants from soybean (2n = 40, genome GG) and G. tomentella Hayata (2n = 78, genome DDEE). The main hurdle was to obtain BC~ plants (G. max cv. Altona) × (G. tomentella, Pl 483218)] → F1, 2n = 59, genome GDE → colchicine treatment (CT) → 2n = 118, GGDDEE × soybean cv. Clark 63 → BC1, 2,n = 76 (expected 2n = 79). Three sterile BC2 plants (2n = 58, 2n = 56, 2n = 55) were produced. Procedures for immature seed‐rescue were utilized to obtain F, amphiploid, BC1 and BC2 plants. Soybean cv. Clark 63 was used as the recurrent parent in the backcrossing programs. The range of chromosome number among BC3 plants was 2n = 41 to 2n = 52, and among BC4 plants was 2n = 40 to 64. A hypertriploid (2n = 64) in BC4 plant originated after fertilization of an unreduced egg (n = 44) by a normal haploid (n = 20) male gamete. Phenotypes of BC3 and BC4 plants were dissimilar but generally resembled closely to the soybean cv. Clark 63. We expect to isolate a series of aneuploid lines from BC3 to BC6 generations that may open up the path to introgress desired traits from wild perennial Glycine species to the cultivated soybean.

Characterization of trypsin and chymotrypsin inhibitors in the wild perennial Glycine species

Seven wild perennial species were used for characterizing the seed protease inhibitors in the genus Glycine Willd. subgenus Glycine to determine the presence and examine the variability and expression patterns. Seeds of all the species contained trypsin and chymotrypsin inhibitors. There were highly significant variations among the wild perennial species in the electrophoretic profiles of trypsin and chymotrypsin inhibitors, migration patterns of anti-KTI and anti-BBI (mAB 238)immunocrossreactive proteins, and trypsin and chymotrypsin inhibitor activities of seeds

Examination of 2,4-D tolerance in perennial Glycine species

High levels of tolerance to 2,4-D [(2,4-dichlorophenoxy)acetic acid] were expressed at the cell and whole plant level by several accessions of wild perennial Glycine species. To determine the mechanism(s) responsible for tolerance, heterotrophic cell suspension cultures of soybean [ Glycine max (L.) Merr.] and four perennial Glycine accessions were used to investigate[ 14C]2,4-D uptake and metabolism. Uptake of 2,4-D was similar between soybean and the perennials. However, soybean metabolized only 7% of the absorbed 2,4-D at 2 and 10 μ M concentrations. The four perennial accessions metabolized 2,4-D at least 79% at 10 μ M and 64% at 50 μ M 2,4-D. The primary metabolite produced by the tolerant perennial lines was the glycoside conjugate of 4-OH-2,5-D, as determined by high-performance liquid chromatography cochromatography and a gas chromatography-mass spectrometer. 2,4-D-sensitive soybean and the least tolerant perennial accession produced high quantities of amino acid conjugates of 2,4-D. Varying levels of 2,4-D tolerance among the perennial Glycine accessions may also be influenced by differential sensitivity of each accession to 2,4-D and/or metabolites.

Restriction fragment length polymorphism (RFLP) of wild perennial relatives of soybean

Total DNA from callus tissue of 28 accessions representing seven wild perennial Glycine species was compared using recombinant genomic probes derived from G. max, the soybean. Using two probes, we show that this molecular approach both confirms and extends the model for the taxonomic relationships between the species derived from morphological and cytogenetic data, and that it provides clear evidence that RFLP analysis of genomic sequences has the potential for revealing the derivation of the member species of the wild perennial Glycine taxon. Although, in this preliminary report, the sample size for each species is small, it is clear that the greatest between-accession variation occurs in G. tabacina (B2B2) and G. clandestine (A1A1), suggesting that these may be the taxa from which further speciation occurred in the subgenus.

Variation in Flowering Response to Photoperiod in Perennial Glycine Species

The wild perennial Glycine spp. offer a potential source of new germplasm for the improvement of soybean [Glycine max (L.) Merr.], but information is needed on useful characteristics in this material. The objective of this study was to determine the flowering response of the wild perennial Glycine species to different daylengths. Twenty‐five accessions of G. tomentella Hayata, one of G. arenaria Tind., and one of G. tabacina (Labill.) Benth. were grown under photoperiods of 8, 12, and 16 h at day/night temperature regimes of 28/24 and 24/20 °C in a phytotron. The accessions represent the latitudinal range within each of five diploid (D1–D5) and five tetraploid (T1–T5) isozymically distinct groups of G. tomentella, and include two previously allied accessions that now have been placed in separate species. The 12‐h daylength was most conducive to cleistogamous and chasmogamous flower development while the 16‐h daylength suppressed flowering. Plants generally took longer to reach comparable phenologicai stages of growth in the cooler temperature regime, but the initial flowers appeared at similar nodes in both warm and cool temperatures. Eight accessions flowered in all three daylengths. The accessions from North Queensland in the D4 (G1300, G1780) and T2 (G1769, G1146) isozyme groups were the least sensitive since they produced both flower types in all photoperiods tested and may be of value in soybean germplasm development.

Interspecific hybrids between the soybean and wild perennial relatives

Successful crosses were obtained in 390 different genotype combinations between the soybean, Glycine max (L.) Merr., and wild perennial Glycine species. A total of 1534 Immature embryos were excised prior to abortion and cultured, and 1.4 percent of these germinated to produce shoots representing 18 genotype combinations. Sixteen F1 Interspecific hybrids encompassing 12 different combinations were subsequently grown to maturity: 7 G. max (2n = 40) × G. tomentella Hayata (2n = 78), 3 G. tomentella × G. max, and 2 G. canescens F. J. Herm. (2n = 40) × G. max. The F1 hybrids differed morphologically depending on the genotype combination, and showed variable degrees of anther development. Hybrids with wild Glycine as the female parent gave the most normal anther development. Expected chromosome numbers of 2n = 59 and 2n = 40 were confirmed for G. tomentella and G. canescens hybrids, respectively. The F1 hybrids exhibited low chromosome pairing and perturbed melosis in pollen mother cells and were sterile. Eight hybrid combinations were doubled with colchicine to give 2n = 118 and 2n = 80 chromosomes for G. tomentella and G. canescens hybrids, respectively. Doubled plants exhibited high levels of chromosome pairing as expected, but remained highly sterile because of abnormal microspore formation. Fertility was partially restored in one G. max × G. tomentella (2n = 118), which set mostly one-seeded, cleistogamous pods at a low rate. Chromosome numbers of selfed progeny were similar to the doubled parent. Variation in viability and fertility shown by different hybrid combinations within one interspecific cross indicate that genotype interactions play a large part in determining the ease with which hybrids can be made between wild perennials and the soybean, and will most likely influence which Glycine collections represent potential candidates for gene exchange with the soybean.

Intersubgeneric Crossability in the Genus Glycine Willd.

AbstractIn intersubgeneric crosses between wild perennial Glycine species of submenus Glycine and soybeans, Glycine max (L.) Merr., of subgenus Soja, all the putative hybrid pods aborted from 9 to 30 days after pollination (DAP). Spraying of 70 ppm gibberellic acid on hybridized gynoecia facilitated pod retention. Fluorescence microscopy revealed normal pollen germination and pollen tube growth in all the intersubgeneric crosses except in the G. cyrtoloba × G. max cross.

Adventitious roots inGlycine subg.Glycine (Leguminosae): Morphological and taxonomic indicators of the B genome

Adventitious roots were observed on 3 wild perennialGlycine species, in short-poddedG. clandestina, G. latifolia and mostG. tabacina (2x, 4x, 6x), while other species lacked adventitious roots. This suggests that the adventitious roots trait is associated with B genome species. Intra- and interspecific F1 hybrids reveal that adventitious roots apparently are inherited recessively. The presence of adventitious roots or short-poddedG. clandestina, coupled with infertility with longer-poddedG. clandestina, and enzymatic and leaflet morphology differences between the twoG. clandestina subgroups, supports segregation of the short-podded form as a separate taxon.

An intersubgeneric hybrid between Glycine tomentella Hayata and the soybean, G. max (L.) Merr.

The objective of the present paper is to provide information on the morphology and cytology of an intersub-generic hybrid (2n=59) between Glycine tomentellaHayata (2n=78) and G. max (L.) Merr. (2n=40) obtained through in vitro immature seed culture. The hybrid plant was slow in vegetative growth and twinning like the female parent but morphologically was intermediate between both parents for several traits. At metaphase I, the average chromosome associations and ranges for 25 cells were 44.0 I (37–51)+7.5 II (3–11). The plant was completely pollen and seed sterile. The present investigation suggests that wild perennial Glycine species can be exploited as either the male or female parent in wide hybridization programs with the soybean, G. max.