Kunitz trypsin inhibitor polymorphism in the Korean wild soybean (Glycine soja Sieb. & Zucc.)

Kunitz trypsin inhibitor (KTi) in 1368 accessions of wild soybean (Glycine soja Sieb. & Zucc.), collected from three regions of Korea, was examined for allelic diversity and geographical distribution. Five electrophoretically distinguishable KTi forms were detected: three were common (Tia, Tib and Tia/Tib) and two were previously unreported (Tib^i7‐1 and Tibⁱ⁵). The Tia allele was predominant (93.49%). Alleles Tib, Tib^i7‐1 and Tibⁱ⁵ were detected with the frequencies of 3.47, 0.55 and 0.11%, respectively. The heterozygous form (Tia/Tib) was detected with the frequency of 2.26%. The nucleotide sequence of Tib^i7‐1 was identical to that of the Tib‐derived variant allele Tif, with the exception of three nucleotides: A→G at position +244, A→C at position +286 and G→C at position +601. The latter two were similar to Tia, suggesting that Tib^i7‐1 is an intermediate allele between Tia and Tib. The gene for Tibⁱ⁵ showed 100% similarity with the Japanese intermediate allele Tibⁱ⁵. This study demonstrates that Korean wild soybeans are remarkably rich source of new KTi alleles not reported before.     

Saponin polymorphism in the Korean wild soybean (Glycine soja Sieb. and Zucc.)

Seed saponin composition of 3025 wild soybean (Glycine soja Sieb. and Zucc.) accessions collected from nine regions of Korea was analysed by thin‐layer chromatography to determine its polymorphic variation and geographical distribution and find mutants in saponin components. The saponin composition of seed hypocotyls was primarily divided into seven phenotypes, designated as Aa, Ab, AaBc, AbBc, Aa+α, AaBc+α and AbBc+α. The predominant phenotypes were AaBc (55%), Aa (33%), AaBc+α (7.5%) and Aa+α (3.3%). The frequencies of Ab, AbBc and AbBc+α were very low (0.3‐0.5%). Codominant alleles Sg‐1^a and Sg‐1^b and dominant allele Sg‐4 occupied 98.6, 1.1 and 63.3%, respectively. Alleles Sg‐3 and Sg‐5 were found to be dominant in all the analysed accessions except the mutants. Three accessions were discovered as mutants via LC‐PDA/MS/MS. The accession CWS0115 did not produce saponin Aa and Ax, CWS2133 did not produce saponin Aa and Ab and CWS5095 did not produce any group A saponins. These newly determined mutants might be utilized in producing a new soybean variety with good taste as well as in biosynthetic studies.     

Genetic characterization of group A acetylsaponin‐deficient mutants from wild soybean (Glycine soja Sieb. and Zucc.)

Group A acetylsaponins are the main causative components for bitter and astringent tastes of soybean (Glycine max). In this study, we examined the genetic nature of the absence of group A acetylsaponins in 12 Korean wild soybean (Glycine soja) accessions. In all 12 accessions, the coding region (1431‐bp) of Sg‐1 locus was identical with Sg‐1^a, which adds the xylose sugar moiety at the terminal position of the C‐22 sugar chain of SS‐A, except one nucleotide (G→A change) at +948th position. This point mutation results in change of one amino acid from tryptophan (TGG) to stop codon (TGA). We observed that the mutated Sg‐1 was controlled by a single recessive gene (sg‐1^0‐a1). This gene was mapped between BARCSOYSSR_07_1561 and BARCSOYSSR_07_1598 on soybean chromosome 7. Our study demonstrated that the mutated Sg‐1 gene in Korean wild soybeans is genetically different from those identified in Japanese soybean cultivar ‘Kinusayaka’ and wild soybean JP‐36121. We believe that the new Sg‐1 mutants can also be utilized to produce a new soybean variety without bitter and astringent properties.     

Association mapping of yield-related traits and SSR markers in wild soybean (Glycine soja Sieb. and Zucc.)

Wild soybean, the progenitor of cultivated soybean, is an important gene pool for ongoing soybean breeding efforts. To identify yield-enhancing quantitative trait locus (QTL) or gene from wild soybean, 113 wild soybeans accessions were phenotyped for five yield-related traits and genotyped with 85 simple sequence repeat (SSR) markers to conduct association mapping. A total of 892 alleles were detected for the 85 SSR markers, with an average 10.49 alleles; the corresponding PIC values ranged from 0.07 to 0.92, with an average 0.73. The genetic diversity of each SSR marker ranged from 0.07 to 0.93, with an average 0.75. A total of 18 SSR markers were identified for the five traits. Two SSR markers, sct_010 and satt316, which are associated with the yield per plant were stably expressed over two years at two experimental locations. Our results suggested that association mapping can be an effective approach for identifying QTL from wild soybean.     

Identification of a novel variant lacking group A soyasaponin in a Chinese wild soybean (Glycine soja Sieb. & Zucc.): implications for breeding significance

Group A saponins are the principal factors underlying the undesirable bitter and astringent tastes in soybean food products. Therefore, the genetic reduction or elimination of the undesirable tastes is of great significance in soybean taste breeding. Using thin‐layer chromatography and liquid chromatography–mass spectrometry, we identified a novel mutant lacking group A saponins in a wild soybean (Glycine soja Sieb. & Zucc.), reflecting the failed arabinose binding of arabinosyltransferase to the C‐22 hydroxyl group, consequently leading to a lack of group A saponins and the concomitance expression of two new components A‐αg and KA‐αg in seeds. The C‐3 glycosylated soyasapogenol A (SS‐A), A‐αg, is a steady precursor compound in biosynthesis of Aa and Ab types in the seeds of soybean plants and exhibits stable inheritance. In this study, we observed a critical step necessary for arabinose binding to the C‐22 hydroxyl group in the biosynthetic pathway of group A saponins and identified an important germplasm accession for the genetic improvement in the tastes of soybean milk and processed soybean foods.     

Genetic diversity and differentiation of Chinese wild soybean germplasm (G. soja Sieb. & Zucc.) in geographical scale revealed by SSR markers

Wild soybean (Glycine soja), as the progenitor of soybeans (G. max), is widely distributed in China and has been collected as a supplementary germplasm pool of soybeans. In this study, 375 wild soybean accessions from a set of genebank core collection were analysed for genetic diversity by using 42 simple sequence repeat primer pairs. The mean allele number per locus was 19.62. Ten‐percent unique alleles involving 35 or 83.33% loci differentiated among the geographical regions. The mean gene diversity (h) per locus was 0.89. A very low mean coefficient of gene differentiation (G_ST = 0.08) for geographical regions and a high mean within‐region gene diversity (H_S = 0.81) were observed, indicating that most genetic diversity existed within the regions. There was an obvious relationship between genetic distance and geographical distance. The results showed multiple centers of genetic diversity for Chinese wild soybean in North China, the Huanghe River Valley, and Central China as well as the Changjiang River Valley, implicating multiple site origins of soybeans within China.     

Development of a chromosome segment substitution line population with wild soybean (Glycine soja Sieb. et Zucc.) as donor parent

Chromosome segment substitution line (CSSL) population is potential in precisely detecting and pyramiding genes/QTL/segments due to the genetic background noise removed. To exploit and utilize the favorable wild alleles, a CSSL population with 151 lines (SojaCSSLP1) was generated using a wild soybean (Glycine soja Sieb. et Zucc.) N24852 as donor parent and the elite cultivar NN1138-2 as its genetic background. An improved CSSL construction strategy was used, i.e. continuous backcross after initial crossing followed with alternation of backcross and selfing combined with marker-assisted selection based on pedigree DNA pools and phenotypic differences among pedigrees. The SojaCSSLP1 with an average recovery ratio of 95.7?% of the NN1138-2 genome could cover the entire genome of wild soybean. Four wild alleles/segments for each of the two wild characteristics, longer plant height (PH) and more number of nodes on main stem (NN), in a total of six segments, were detected with additive effects all positive. Among them, Satt243 on Chr.10 and Sat_286 on Chr.19 associated with both PH and NN while Satt338 and SOYGPATR on Chr.4 and Satt314 neighboring with Satt192 on Chr.12 had the former and latter on each chromosome associated with PH and NN, respectively. That could explain the high positive correlation between the two traits (r?=?0.88). Compared with those in the literature, three QTL/segments for PH and one for NN were detected also among cultivated soybeans, indicating allele differentiation happened not only between wild and cultivated but also among cultivated soybeans. Therefore these QTL/segments might be the key ones to explain the domestication and evolution of soybean. In addition, SojaCSSLP1 should be also potential in studies for multiple wild traits due to its broad variation.     

The Sg-6 saponins,new components in wild soybean (Glycine soja Sieb. and Zucc.): polymorphism,geographical distribution and inheritance

Hypocotyl saponin composition of 1,198 accessions of wild soybean (Glycine soja) collected from China, Korea, Japan and Russia Far East was analyzed by thin-layer chromatography to determine polymorphic variation and geographical distribution. Eight common distinguishable saponin phenotypes were identified: Aa, Ab, AaBc, AbBc, Aa+α, Ab+α, AaBc+α and AbBc+α. The latter four +α type were new. All eight types were identified in China. Type Ab+α was absent in Korea. Types Ab+α and AbBc+α, and Aa+α and Ab+α were not identified in Japan and Russia far east, respectively. Six new triterpene saponins were detected in +α type via LC-PDA/MS/MS analyses. They were, tentatively, designated as H-αg, H-αa, I-αg, I-αa, J-αg and J-αa. These saponins were inherited together by a single dominant allele. A gene symbol Sg-6 was assigned. Hence, the new saponins were collectively named as Sg-6 saponins. The frequency of Sg-6 allele was 17.6 % in Chinese, 10.0 % in Korean and 1.0 % in Japanese wild soybean. The wild soybeans having Sg-6 saponins can be utilized in soybean breeding programs as well as in saponin biosynthesis studies in soybean.     

Genetic characterization of wild barley populations (Hordeum vulgare ssp. spontaneum) from Kazakhstan based on genome wide SNP analysis

The wild ancestral form of barley, Hordeum vulgare ssp. spontaneum, is a valuable source for gene enrichment of cultivated barley. The purpose of this work was to study the area of distribution as well as the extent and structure of genetic variation of wild barley populations grown in Kazakhstan. It was found that distribution of wild barley populations in Kazakhstan is restricted to the most southern province. A genome wide single nucleotide polymorphism (SNP) analysis was performed in order to study the level of the genetic diversity in 96 accessions representing 14 wild barley populations from Kazakhstan and 25 accessions from the Middle East which is the center of diversity of this subspecies. The oligonucleotide pooled assay was used to genotype 384 SNPs distributed throughout the genome. In total 233 polymorphic SNPs were selected for further statistical analysis. The level of genetic diversity of wild barley populations from Kazakhstan was predictably narrower (He = 0.19 ± 0.01) in comparison with wild barley samples from the Middle East (He = 0.29 ± 0.01). The results suggested that H. vulgare ssp. spontaneum populations in Kazakhstan probably represent a recent spread of a limited number of plants from the primary distribution area and might be well adapted to winter low temperature.     

Genetic characterization of Malawian cowpea (Vigna unguiculata (L.) Walp) landraces: diversity and gene flow among accessions

Cowpea has been cultured from ancient timesin Africa and Asia and is now widespreadthroughout the tropics and subtropics.Cowpea accessions can be distinguishedphenotypically from one another by theirgrowth habit, time to maturity, yield, andseed size and colour. Data on geneticdiversity of farmer-developed accessionsare lacking. The main objective of thisstudy was to determine the pattern andextend of RAPD marker variation within andamong cowpea populations from differentagroecological zones and to determine thedegree of genetic relationships and geneflow among different landraces used bylocal farmers. Twenty of the 30 RAPDprimers tested allowed amplifications ofrandom polymorphic (RAPD) loci. Overall,80% of the scored loci were polymorphic. The degree of band sharing was used toevaluate genetic distance betweenaccessions and to construct a phylogenetictree. The genetic distance values amongaccessions varied between 0.09 to 0.57.Although some small clusters groupedaccessions of the same growth habits, ageneral lack of agreement betweenclustering and morphological features wasobserved. The analysis of molecularvariance revealed that within-region ortypes (among accessions) variationaccounted for 96% of the total molecularvariance. This high within-accessionvariability is being sustained by anuncontrolled gene flow among populations.     

Genetic analysis of partial resistance to coffee leaf rust ( Hemileia vastatrix Berk & Br.) introgressed into the cultivated Coffea arabica L. from the diploid C. canephora species

The objective of this study was to identify polymorphic molecular markers associated with partial resistance to coffee leaf rust, Hemileia vastarix. A segregating F ₂ population derived from a cross between the susceptible Coffea arabica cv. Caturra and a C. canephora-introgressed Arabica line exhibiting high partial resistance was analyzed. Rust resistance measured as rust incidence (RI) and defoliation (DEF) was evaluated in field conditions in three consecutive years (2003–2005). During the 2003 season, which was characterized by favorable conditions for a rust epidemic, the F ₂ plants exhibited different levels of resistance ranging from very susceptible (50.1% for DEF and 49.5% for RI) to highly partial resistance (9.1% for DEF and 3.7% for RI). Molecular analysis enabled identification of seven polymorphic markers (5 AFLP and 2 SSR) exhibiting significant association with partial resistance. Coexistence of resistance homozygous alleles (RR) at codominant SSR loci was correlated with high resistance. This study is the first attempt to develop PCR-based sequence specific markers linked to partial rust resistance in coffee.     

Genetic and molecular analysis of the restoration of fertility ( Rf ) genes for Ogura male-sterility from a Japanese wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino)

Ogura male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. In this study, in order to gain better understanding of the variation and evolution of the restoration of the fertility (Rf) gene for Ogura male-sterile cytoplasm, the nucleotide sequence of the orf687 homologue in the Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino) was analyzed using an F₂ population made with a cross between a Japanese wild radish plant containing the Rf gene and ‘Uchiki-Gensuke’ (a maintainer of Ogura-male sterility). Segregation of male-fertile/-sterile plants in the F₂ generation suggested that another unidentified Rf gene unlinked to orf687 exists in the Japanese wild radish. The genotype of orf687 was determined for each F₂ plant by Southern hybridization with an orf687 gene probe, mismatch-specific endonuclease digestion of PCR products, and direct sequencing of a PCR product. Genotyping revealed that some fertility-restored plants are homozygotic for the ‘Uchiki-Gensuke’ type orf687 allele, supporting the idea that another gene different from orf687 also functions as an Rf gene for Ogura male-sterility. Protein analysis using an antibody raised against the Ogura-specific ORF138 protein suggests a mechanism of fertility restoration by the unidentified Rf similar to that by orf687. Sequence analysis of orf687 from a Japanese wild radish plant and ‘Uchiki-Gensuke’ revealed that both orf687 regions encode a mitochondrially-targeted protein consisting of 687 amino acids with 16 PPR motifs. Comparison of the deduced amino acid sequences with those of the known orf687 sequences from ‘Yuan hong’ and ‘Kosena’ containing Rf and recessive one (rf), respectively, showed that three unique amino acid replacements are present in ORF687 of the Japanese wild radish. Two of the three replacements, that from lysine to isoleucine at position 232 and from asparagine to asparate at position 240, confer negative charges to the protein. Since the Rf of ‘Yuan hong’ was reported to have a unique replacement that confers a negative charge to ORF687 (from asparagine to aspartate at position 170), it is proposed that the amino acid replacements conferring a negative charge to ORF687 are important for determining the status of the Rf/rf gene.     

Identification and sequence characterisation of molecular markers polymorphic between male kiwifruit (Actinidiachinensis var. deliciosa (A. Chev.) A. Chev.) accessions exhibiting different flowering time

Fruit set in kiwifruit is strongly dependent on pollination, which is limited by the lack of efficient male pollen donors, among other factors. We searched for molecular markers that could be polymorphic in relation to flowering time in order to classify male kiwifruit plants to discard those that are not likely to perform as efficient pollen donors. Random amplified polymorphic DNA (RAPD) and modified amplified fragment length polymorphism (AFLP) markers were generated using 41 male kiwifruit plants in two flowering groups, early- and late-flowering males (with respect to the female cultivar ‘Hayward’). One RAPD and nine modified-AFLP markers polymorphic between male plants exhibiting different flowering time were identified, sequenced and analysed in databases. Unweighted pair group method with arithmetic average (UPGMA) clustering and multidimensional scaling showed that these markers could be used to classify the male plants into flowering groups. Analysis of molecular variance (AMOVA) agreed with this classification, showing that most of the genetic variation is found between flowering groups. Sequence analysis based on a database search revealed that the polymorphism PolM contains a 7-nucleotide long element involved in the repression of the phytochrome A gene, that Pol4 is a partial sequence of a phytochrome B gene, and that sequences Pol3, Pol5, Pol7, and Pol9 show high identity with ESTs from kiwifruit buds treated with hydrogen cyanamide. Clustering analysis supported the previous classification of males into flowering groups, making it feasible to predict male plants’ flowering times with respect to the cultivar ‘Hayward’ based upon these molecular markers.     

Screening of wild crucifers for resistance to mustard aphid, Lipaphis erysimi (Kaltenbach) and attempt at introgression of resistance gene(s) from Brassica fruticulosa to Brassica juncea

A diverse array of wild and weedy crucifers was screened under laboratory conditions for their resistance to Lipaphis erysimi (Kaltenbach) (Homoptera: Aphididae). Among these, Brassica fruticulosa and Brassica montana were found to be the most promising. The availability of a synthetic amphiploid, AD-4(B. fruticulosa × Brassica rapa var. brown sarson) as well as a set of Brassica juncea lines carrying genomic introgressions from B. fruticulosa allowed us to investigate B. fruticulosa resistance in greater detail. This assessment was carried out along with susceptible check B. rapa ssp. brown sarson cv. BSH-1 in a series of choice and no choice experiments. The mustard aphid showed maximum preference for feeding on BSH-1 while least preference was recorded for B. fruticulosa followed by AD-4 as evidenced by the number of aphids that settled on circular leaf bits of these genotypes 24 and 48 h after release in a choice experiment. Brassica fruticulosa exhibited strong antibiosis against mustard aphid in no choice experiment and all the released aphids died within 5–8 days of their release, while the maximum survival (76.7%) was recorded on BSH-1. The survival on AD-4 (40%) was significantly lower than that on BSH-1. Almost similar trend was observed with respect to other demographic parameters of L. erysimi viz. development time, fecundity and longevity. In the screen house studies, there was no seedling mortality in B. fruticulosa and AD-4 after 30 days of aphid release while 80% mortality was observed on BSH-1. Excellent variation for aphid resistance was recorded in B. juncea introgression lines, emphasizing heritable nature of fruticulosa resistance. The biochemical analysis suggested the possibility of high concentration of lectins to be associated with low aphid infestation in B. fruticulosa.     

Genetic diversity of European spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.) revealed by glutenin subunit variations at the Glu-1 and Glu-3 loci

Summary High and low molecular weight glutenin subunit (HMW-GS and LMW-GS) compositions of 270 European spelts, 15 Iranian spelts and 25 bread wheat cultivars were analyzed by one- and two-dimensional gel electrophoresis. The results revealed a total of 22 HMW-GS alleles (4 at Glu-A1, 11 at Glu-B1 and 7 at Glu-D1) and 32 allele combinations among the three Glu-1 loci. Two major genotypes of HMW-GS: 1, 13+16, 2+12 and 1, 6.1+22.1, 2+12 were found to occur in Central European spelt wheat cultivars and landraces at higher frequencies of 35 and 28%, respectively. The Glu-B1 locus displayed the greatest variation and genetic diversity index (H) was 0.69 whereas Glu-A1 and Glu-D1 showed H index values of 0.26 and 0.19, respectively. The dendrogram constructed by HMW and LMW glutenin subunit bands revealed that European spelts form a separated cluster from common wheat suggesting that spelt and common wheat form distinct groups. In addition, all 15 Iranian spelt land variety accessions differed from European spelts and possessed similar HMW-GS alleles to common wheat. Because of a wider polymorphism Central European spelt wheats are an important genetic reserviour for improving common wheat quality. Both authors contributed equally to this work     

The critical N dilution curve for linseed (Linum usitatissimum L.) is different from other C3 species

The objective was to determine the critical N dilution curve of linseed, which is the minimal total N concentration in shoots necessary to produce the maximal shoot dry matter, and to explain possible differences with other C3 species. One main experiment was carried out in 1998/1999 on winter linseed with four levels of fertilizer N. Two plant densities were also studied, the recommended one (600 seeds m⁻²) and the minimum for canopy closure (150 seeds m⁻²), in order to investigate the stability with plant density of the critical N dilution curve. Shoot dry weights (W_S) and shoot N contents expressed in percentage (N_S) were measured for the determination of the critical dilution curve, along with organ N percentages and relative weights. The results of four other experiments were used to validate the critical N dilution curve. Three of these four trials were conducted on winter linseed (one in 1996/1997 and two in 1997/1998) with five levels of fertilizer N, and one on spring linseed in 1999 with six levels of fertilizer N.

The critical N dilution curve of linseed was different from those of other C3 species. The curve was steeper, indicating a greater decrease in the critical shoot N concentration (N_SC) as the critical shoot dry weight (W_SC) increased. This linseed curve determined with the data of the main experiment was relevant when compared to the data of the four other experiments. Organ weight ratios and N concentration of organs were investigated in a fertilizer N treatment resulting in N_S close to the critical N values, N_SC. In this treatment, the decrease in N_S was the result of both a decrease in the N percentage of all organs and a decrease in the leaf weight ratio. The difference between linseed and other C3 species was mainly due to an acceleration of the dilution of N when leaf emission stopped and the flower bud emission began. At this stage of development, the leaf weight ratio of linseed was less than that of wheat, resulting in lower N_S. For a given W_S, no significant differences in N_S, organ N percentages nor organ weight ratios were observed between the two plant densities. This indicates that the difference between linseed and other C3 species could not result from very high plant densities in linseed. Hence, it is concluded that the linseed N accumulation in shoot is different from other C3 species.     

Transfer of architectural traits from perennial Helianthus mollis Lam. to sunflower (H. annuus L.) and localisation of introgression

Common sunflower (i.e., Helianthus annuus) is occasionally crossed with other Helianthus species to gain favourable agronomic traits. An interspecific cross between H. annuus and Helianthus mollis was used to examine events that occurred during the five first generations. First generation hybrid plants were verified using GISH, and F₁ hybrid (#17) was back-crossed to common sunflower to obtain BC₁ (#4) and BC₂ (#21). Some BC₂ progeny possessed new traits, i.e., compact (Compact plant architecture is due to short inter-node length, short petiole length and short leaf lamina length) architecture without and with corrugated leaves, which were not observed in the parents. These traits were selected for and fixed after the BC₂-S₃ (#18) to produce a H. mollis introgression line (namely HM374). Although the size of the population was small, we tentatively construct a genetic map using data from the BC₂ generation. Two linkage groups were identified carrying H. mollis specific AFLP markers that were associated with compact plant architecture, and one unlinked marker was shown to explain variation for the corrugated leaves. Introgression line HM374 was used to test the inheritance of compact plant architecture in a cross with an elite high oleic line, OL75HO. Microsatellite (SSR) markers were used to genotype F₁ and F₂ offspring’s. Our data suggested that the line HM374 was still heterozygous for introgression after eight generations. HM374 displays new architecture traits (i.e. compact architecture with or without corrugated leaves) and carries H. mollis fragments anchored on LG1 and LG11 of sunflower public map.