Combining focused identification of germplasm and core collection strategies to identify genebank accessions for central European soybean breeding

Environmental adaptation of crops is essential for reliable agricultural production and an important breeding objective. Genebanks provide genetic variation for the improvement of modern varieties, but the selection of suitable germplasm is frequently impeded by incomplete phenotypic data. We address this bottleneck by combining a Focused Identification of Germplasm Strategy (FIGS) with core collection methodology to select soybean (Glycine max) germplasm for Central European breeding from a collection of >17,000 accessions. By focussing on adaptation to high-latitude cold regions, we selected an “environmental precore” of 3,663 accessions using environmental data and compared the Donor opulation of Environments (DPE) in Asia and the Target Population of Environments (TPE) in Central Europe in the present and 2070. Using single nucleotide polymorphisms, we reduced the precore into two diverse core collections of 183 and 366 accessions to serve as diversity panels for evaluation in the TPE. Genetic differentiation between precore and non-precore accessions revealed genomic regions that control maturity, and novel candidate loci for environmental adaptation, demonstrating the potential of diversity panels for studying adaptation. Objective-driven core collections have the potential to increase germplasm utilization for abiotic adaptation by breeding for a rapidly changing climate, or de novo adaptation of crops to expand cultivation ranges.     

Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

Identification of <Emphasis Type="Italic">GmSALT3</Emphasis> haplotypes and development of molecular markers based on their diversity associated with salt tolerance in soybean

Soybean [Glycine max (L.) Merr.] is a major agricultural crop and generally known as a salt-sensitive crop. In a previous study, GmSALT3 was identified as a salt tolerance gene in soybean, and its nine haplotypes (H1 to H9) were reported in Chinese soybean accessions. In the present study, we aimed to identify new haplotypes of GmSALT3 in soybean accessions and to develop molecular markers for selection of salt-tolerant and -sensitive accessions. To do so, we examined genomic variations in the GmSALT3 coding region of 216 accessions of G. max and G. soja from Korea, China, and Japan. As a result, 40 different haplotypes, including three known haplotypes (H1, H2, and H5), were identified. Performing salt tolerance tests for the haplotypes, we were able to classify them into salt-tolerant (8) and salt-sensitive (32) categories. We also found that more variations in the haplotype composition of G. soja accessions exist than in that of G. max accessions. Quantitative expression analysis showed that almost all of the salt-tolerant haplotypes had much higher levels of GmSALT3 expression than the salt-sensitive haplotypes did. Finally, we developed molecular markers and applied them to screen salt tolerance of soybean accessions. The molecular markers performed well with an accuracy of 98.8% in identifying phenotypes of soybean accessions.     

Worldwide core collections of tea (Camellia sinensis) based on SSR markers

Tea (Camellia sinensis (L.) O. Kuntze) is the world’s most popular beverage crop. However, to date, no core collection has been selected from worldwide germplasm resources on the basis of genotype data. In this study, we analyzed 788 tea germplasm accessions using 23 simple sequence repeat (SSR) markers. Our population structure analysis divided the germplasms into a Japanese group and an exotic group. The latter could be divided into var. sinensis and var. assamica. The genetic diversity was higher in germplasms from China, Taiwan, India, and Sri Lanka than in those from other countries, and low in germplasms from Japan. Using the number of SSR alleles as a measure of genetic diversity, we developed a core collection consisting of 192 accessions and three subcore collections with 96, 48, and 24 accessions. Although the results might be affected by marker-selection bias, the core 192 collection adequately covered the range of variation of the 788 accessions in floral morphology, and the chemical composition of first-flush leaves. These collections will be powerful tools for breeding and genetic research in tea.     

Developing core collections to optimize the management and the exploitation of diversity of the coffee Coffea canephora

The management of diversity for conservation and breeding is of great importance for all plant species and is particularly true in perennial species, such as the coffee Coffea canephora. This species exhibits a large genetic and phenotypic diversity with six different diversity groups. Large field collections are available in the Ivory Coast, Uganda and other Asian, American and African countries but are very expensive and time consuming to establish and maintain in large areas. We propose to improve coffee germplasm management through the construction of genetic core collections derived from a set of 565 accessions that are characterized with 13 microsatellite markers. Core collections of 12, 24 and 48 accessions were defined using two methods aimed to maximize the allelic diversity (Maximization strategy) or genetic distance (Maximum-Length Sub-Tree method). A composite core collection of 77 accessions is proposed for both objectives of an optimal management of diversity and breeding. This core collection presents a gene diversity value of 0.8 and exhibits the totality of the major alleles (i.e., 184) that are present in the initial set. The seven proposed core collections constitute a valuable tool for diversity management and a foundation for breeding programs. The use of these collections for collection management in research centers and breeding perspectives for coffee improvement are discussed.     

Allele-specific hybridization markers for soybean

 Soybean (Glycine max) is one of the world’s most important crop plants due to extensive genetic improvements using traditional breeding approaches. Recently, marker-assisted selection has enhanced the ability of traditional breeding programs to improve soybeans. Most methods of assessing molecular markers involve electrophoretic techniques that constrain the ability to perform high-throughput analyses on breeding populations and germplasm. In order to develop a high-capacity system, we have developed allele-specific hybridization (ASH) markers for soybean. As one example, restriction fragment length polymorphism (RFLP) locus A519-1 (linkage group B) was converted into an ASH marker by (1) sequencing the pA519 cloned insert, (2) designing locus-specific PCR amplification primers, (3) comparative sequencing of A519-1 amplicons from important soybean ancestors, and (4) designing allele-specific oligonucleotide probes around single nucleotide polymorphisms (SNPs) among soybean genotypes. Two SNPs were identified within approximately 400 bp of the sequence. Allele-specific probes generated a 100-fold greater signal to target amplicons than to targets that differed by only a single nucleotide. The A519-1 ASH marker is shown to cosegregate with the A519-1 RFLP locus. In order to determine ASH usefulness, we genotyped 570 soybean lines from the Pioneer Hi-Bred soybean improvement using both A519-1 SNPs. Combined haplotype diversity (D) was 0.43 in this adapted germplasm set. These results demonstrate that ASH markers can allow for high-throughput screening of germplasm and breeding populations, greatly enhancing breeders’ capabilities to do marker-assisted selection. Received: 10 August 1998 / Accepted: 17 September 1998     

SoySNP618K array: A high-resolution single nucleotide polymorphism platform as a valuable genomic resource for soybean genetics and breeding

Innovations in genomics have enabled the development of low-cost, high-resolution, single nucleotide polymorphism (SNP) genotyping arrays that accelerate breeding progress and support basic research in crop science. Here, we developed and validated the SoySNP618K array (618,888 SNPs) for the important crop soybean. The SNPs were selected from whole-genome resequencing data containing 2,214 diverse soybean accessions; 29.34% of the SNPs mapped to genic regions representing 86.85% of the 56,044 annotated high-confidence genes. Identity-by-state analyses of 318 soybeans revealed 17 redundant accessions, highlighting the potential of the SoySNP618K array in supporting gene bank management. The patterns of population stratification and genomic regions enriched through domestication were highly consistent with previous findings based on resequencing data, suggesting that the ascertainment bias in the SoySNP618K array was largely compensated for. Genome-wide association mapping in combination with reported quantitative trait loci enabled fine-mapping of genes known to influence flowering time, E2 and GmPRR3b, and of a new candidate gene, GmVIP5. Moreover, genomic prediction of flowering and maturity time in 502 recombinant inbred lines was highly accurate (>0.65). Thus, the SoySNP618K array is a valuable genomic tool that can be used to address many questions in applied breeding, germplasm management, and basic crop research.     

Genetic Diversity of Soybean and the Establishment of a Core Collection Focused on Resistance to Soybean Cyst Nematode

Soybean cyst nematode （SCN; Heterodera glycines） Is one of the most Important pests affecting soybean production. The best method of control of SCN is through the development of resistant cultlvars. However, limited progress has been made in soybean breeding In China because most modern cultlvars have no resistance to SCN. The distribution and phenotype of 432 immune or highly resistant Chinese accessions were surveyed and a primary core collection was selected as a representative sample for further analyses. Using evenly distributed simple sequence repeat markers, five selection methods were applied to the primary core collection and the optimal method was chosen to establish a core collection, which consisted of 28 accessions. These encompassed 70.8% of the ailelic variation present in the overall resistant collection. The 28 accessions differed from the reference resistant accessions at the genomlc level, Indicating that Chinese resistant accessions are distinct from known resistant accessions. This applied core collection provides a rational framework for undertaking diversity surveys, using genetic variation for the investigation of complex traits and for the discovery of novel traits.     

The genetic diversity of annual wild soybeans grown in China

Annual wild soybeans (Glycine soja), the ancestors of cultivated soybeans (G. max), are important sources of major genes for resistance to pests, diseases and environmental stresses. The study of their genetic diversity is invaluable for efficient utilization, conservation and management of germplasm collections. In this paper, the number of accessions, the variation of traits, the genetic diversity indexes (Shannon index) and the coefficient of variation were employed to study the geographical distribution of accessions, genetic diversity of characters and genetic diversity centers of annual wild soybean by statistical analysis of the database from the National Germplasm Evaluation Program of China. Most annual wild soybeans are distributed in Northeast China, and the number of accessions decreases from the Northeast to other directions in China. The genetic diversity indexes (Shannon index) were 0.49, 0.74, 0.02, 0.55, 1.45, 2.41, 1.27 and 1.89 for flower color, sootiness of seed coat, cotyledon color, pubescence color, hilum color, leaf shape, stem type and seed color, respectively. Coefficients of variation were 7.1%, 28.7%, 76.43% and 18.2% for protein content, oil content, 100-seed weight and days to maturity, respectively. Three genetic diversity centers, the Northeast, the Yellow River Valley and the Southeast Coasts of China, are proposed based on the geographical distribution of the number of accessions, genetic diversity and the multivariate variation coefficient. Based on these results and Vavilov’s theory of crop origination, two opposing possible models for the formation of the three centers are proposed, either these centers are independent of each other and the annual wild soybeans in these centers originated separately, or the Northeast center was the primary center for annual wild soybeans in China, while the Yellow River Valley center was derived from this primary center and served as the origin for the Southeast Coast center. Received: 25 June 2000 / Accepted: 18 October 2000     

Genome Size in Chinese Soybean Accessions--Stable or Variable?

Variation in genome size up to 1.12-fold has been recently reportedin 90 Chinese accessions ofGlycine max (soybean). Generallysuch results have to be viewed with reservation if rigorousinternal standardization and control tests for the repeatabilityof the results have not been done. Therefore, we reinvestigatedten accessions (five allegedly ranking high and five low) forgenome size using propidium iodide flow cytometry and Feulgendensitometry. Using flow cytometry, the maximum difference betweenaccessions was 1.018-fold (non-significant); the differencebetween the means of the high-ranking and low-ranking groupwas 1.002-fold (non-significant). With Feulgen densitometry,the maximum difference between accessions was 1.034-fold (non-significant).The present data suggest genome size constancy, in terms ofcytometric evidence, for the Chinese soybean accessions in question.Likewise, no reasonable evidence was obtained for a differencebetween Chinese and American soybeans. Copyright 1999 Annalsof Botany Company Glycine max, Chinese soybeans, U.S. soybeans, genome size variation, propidium iodide flow cytometry, Feulgen densitometry.     

8. Molecular biology and genetic conservation programmes

The techniques of molecular biology will become a standard part of germplasm conservation and exploitation. They are being used to gather information very rapidly about chromosome structure and genetic variation within the major crop species. Genetic maps with hundreds of DNA sequence markers covering the whole genome have already been created for some crops, such as maize, soybean, wheat and potato. Genetic variation is being revealed by the combined use of restriction endonucleases, fractionation of DNA fragments by electrophoresis and investigation of the size of specific allelic fragments. This kind of approach offers new opportunities to assess the extent of genetic variation among accessions in germplasm collections, thereby helping to decide which accessions are essentially duplicates and which should be maintained in a core collection. I recommend that germplasm banks will in the future also contain diagnostic DNA markers for characterizing and screening germplasm.
When material from germplasm banks is used in crop plant breeding programmes to transfer specific traits into the crop, the availability of a complete set of molecular markers covering the entire genome makes it straightforward to discover which segments have been transferred and which are essential to maintain, so as to preserve the introduced trait.
Germplasm banks are obviously a source of new genetic variation for the molecular geneticist as well as the plant breeder. The isolation of specific alleles determining self-incompatibility from Brassica oleracea accessions for subsequent introduction into oil seed rape is described as an example.     

The genetic diversity of cultivated soybean grown in China

Cultivated soybean (Glycine max) is an economically important crop that is grown for its oil and protein products. A better knowledge of its genetic diversity will be valuable for the utilization, conservation, and management of germplasm collections. Using the database of the National Germplasm Evaluation Program of China (NGEPC), we studied the geographical distribution of accessions, the genetic diversity of 15 qualitative and quantitative characters, and the genetic diversity centers of cultivated soybean in China using variation in these 15 traits and genetic diversity indexes (Shannon index). Cultivated soybean is widely distributed throughout China. As an indication of its distribution, a line can be roughly drawn from the Daxinganling mountains in northeastern China to the Qingzang plateaus in southwestern China based on the abundance of accessions and locations of the collections. Of the 22,637 known accessions in China, the 20,570 collected over a vast area between latitudes 18° and 53°N and longitudes 80° and 136°E were used in this study. The Shannon indexes of various morphological traits were calculated. Cultivated soybean accessions were found to exhibit a higher genetic diversity in the area between 34°–41°N and 110°–115°E. On the basis of the geographical distribution of a number of accessions, and their genetic diversity, one genetic diversity center—downstream of the Yellow River Valley—is proposed. Based on these results and on Vavilovs theory on crop origins, one possible diversity center was proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00122-003-1503-xCommunicated by C. Möllers     

Allele-specific marker development and selection efficiencies for both flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes in soybean subgenus soja

Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3′-hydroxylase (F3′H; EC1.14.3.21) and flavonoid 3′,5′-hydroxylase (F3′5′H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3′H and the W1 locus co-segregated with a gene encoding F3′5′H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3′H and F3′5′H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3′H and one of three alleles for GmF3′5′H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3′H and F3′5′H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3′H allele explained 92.2 % of the variation in tawny and two gmf3′h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3′5′H alleles and one gmF3′5′h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3′h-b and gmf3′5′h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.     

Diversity of γ- glutamyl peptides and oligosaccharides,the “kokumi” taste enhancers,in seeds from soybean mini core collections

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the “kokumi” taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the “kokumi” taste components in soybean by performing a genetic analysis.     

Population Structure and Domestication Revealed by High-Depth Resequencing of Korean Cultivated and Wild Soybean Genomes

Despite the importance of soybean as a major crop, genome-wide variation and evolution of cultivated soybeans are largely unknown. Here, we catalogued genome variation in an annual soybean population by high-depth resequencing of 10 cultivated and 6 wild accessions and obtained 3.87 million high-quality single-nucleotide polymorphisms (SNPs) after excluding the sites with missing data in any accession. Nuclear genome phylogeny supported a single origin for the cultivated soybeans. We identified 10-fold longer linkage disequilibrium (LD) in the wild soybean relative to wild maize and rice. Despite the small population size, the long LD and large SNP data allowed us to identify 206 candidate domestication regions with significantly lower diversity in the cultivated, but not in the wild, soybeans. Some of the genes in these candidate regions were associated with soybean homologues of canonical domestication genes. However, several examples, which are likely specific to soybean or eudicot crop plants, were also observed. Consequently, the variation data identified in this study should be valuable for breeding and for identifying agronomically important genes in soybeans. However, the long LD of wild soybeans may hinder pinpointing causal gene(s) in the candidate regions.     

Genome-wide signatures of the geographic expansion and breeding of soybean

Soybean is a leguminous crop that provides oil and protein. Exploring the genomic signatures of soybean evolution is crucial for breeding varieties with improved adaptability to environmental extremes. We analyzed the genome sequences of 2,214 soybeans and proposed a soybean evolutionary route, i.e., the expansion of annual wild soybean (Glycine soja Sieb. & Zucc.) from southern China and its domestication in central China, followed by the expansion and local breeding selection of its landraces (G. max (L.) Merr.). We observed that the genetic introgression in soybean landraces was mostly derived from sympatric rather than allopatric wild populations during the geographic expansion. Soybean expansion and breeding were accompanied by the positive selection of flowering time genes, including GmSPA3c. Our study sheds light on the evolutionary history of soybean and provides valuable genetic resources for its future breeding.

     

Non-toxic and efficient DNA extractions for soybean leaf and seed chips for high-throughput and large-scale genotyping

In applied soybean (Glycine max L.) breeding programs, marker-assisted selection has become a necessity to select value-added quantitative trait loci. The goal of this work was to improve marker-assisted selection workflow by developing a reliable, inexpensive, high-throughput DNA extraction protocol for soybean seed and leaf samples that does not generate hazardous waste. The DNA extraction protocol developed allows for the leverage of robust SNP genotyping platforms such as the Simple Probe Assay and KASPar v4.0 SNP Genotyping System to genotype thousands of seeds or leaves non-destructively in a single day with a 95 % success rate. This methodology makes it possible to run up to 150 SNP markers on the DNA extracted from a single seed chip or leaf sample.     

Genetic divergence between North American ancestral soybean lines and introductions with resistance to soybean cyst nematode revealed by chloroplast haplotype

Domesticated soybean [Glycine max (L.) Merr.] is a major crop with an established ancestral relationship to wild soybean (Glycine soja Sieb. & Zucc.) native to Asia. Soybean genetic diversity can be assessed at different levels by identification of polymorphic alleles at genetic loci, in either the plastid or nuclear genomes. The objective of this study was to evaluate genetic diversity based on chloroplast haplotypes for soybean genotypes present in the USDA germplasm resource collection. Shared chloroplast haplotypes represent broad groups of genetic relatedness. Previous work categorized three-quarters of the cultivated soybeans from Asia into a single haplotype group. Our results confirmed the close relationship of North American soybean ancestors and G. max plant introductions previously identified as representing potential sources of soybean genetic variation with the finding that these genotypes belonged to a single chloroplast haplotype group. Genetic diversity was identified in soybean genotypes determined to have a high density of single nucleotide polymorphisms and in a screen of accessions with resistance to soybean cyst nematode. Characterization of soybean plant introduction lines into chloroplast haplotype group may be an important initial step in evaluating the appropriate use of particular soybean genotypes.