Assessing genetic diversity and geographical differentiation in a global collection of wild soybean (Glycine soja Sieb. et Zucc.) and assigning a mini-core collection.

Wild soybean (Glycine soja), the ancestor of the cultivated soybean (G. max), is a crucial resource for capturing the genetic diversity of soybean species. In this study, we used a set of 78 genome-wide microsatellite markers to analyse the genetic diversity and geographic differentiation patterns in a global collection of 2,050 G. soja accessions and a mini-core collection of G. max stored in two public seed banks. We observed a notable reduction in the genetic diversity of G. max compared with G. soja and identified a close phylogenetic relationship between G. max and a G. soja subpopulation located in central China. Furthermore, we revealed substantial genetic divergence between northern and southern subpopulations, accompanied by diminished genetic diversity in the northern subpopulations. Two clusters were discovered among the accessions from north-eastern China-one genetically close to those from South Korea and Southern Japan, and another close to those from Amur Oblast, Russia. Finally, 192 accessions were assigned to a mini-core collection of G. soja, retaining 73.8% of the alleles detected in the entire collection. This mini-core collection is accessible to those who need it, facilitating efficient evaluation and utilization of G. soja genetic resources in soybean breeding initiatives.

Mutant Tof11 alleles are highly accumulated in early planting-adaptable Japanese summer type soybeans.

To avoid crop failure because of climate change, soybean (Glycine max (L.) Merrill) cultivars adaptable to early planting are required in western Japan. Because current Japanese cultivars may not be adaptable, genetic resources with high early-planting adaptability, and their genetic information must be developed. In the present study, summer type (ST) soybeans developed for early planting were used as plant materials. We examined their phenological characteristics and short reproductive period as an indicator of early planting adaptability and performed genetic studies. Biparental quantitative trait loci (QTL) analysis of a representative ST cultivar revealed a principal QTL for the reproductive period duration on chromosome 11. The results of resequencing analysis suggested that circadian clock-related Tof11 (soybean orthologue of PRR3) is a candidate QTL. Additionally, all 25 early planting-adaptable germplasms evaluated in this study possessed mutant alleles in Tof11, whereas 15 conventional cultivars only had wild-type alleles. These results suggest that mutant alleles in Tof11 are important genetic factors in the high adaptability to early planting of these soybeans, and thus, these alleles were acquired and accumulated in the ST soybean population.

A Soybean Deletion Mutant That Moderates the Repression of Flowering by Cool Temperatures.

Ambient growing temperature and photoperiod are major environmental stimuli that summer annual crops use to adjust their reproductive phenology so as to maximize yield. Variation in flowering time among soybean (Glycine max) cultivars results mainly from allelic diversity at loci that control photoperiod sensitivity and FLOWERING LOCUS T (FT) orthologs. However, variation in the thermal regulation of flowering and its underlying mechanisms are poorly understood. In this study, we identified a novel mutant (ef1) that confers altered thermal regulation of flowering in response to cool ambient temperatures. Mapping analysis with simple sequence repeat (SSR) markers located the mutation in the upper part of chromosome 19, where no QTL for flowering has been previously reported. Fine-mapping and re-sequencing revealed that the mutation was caused by deletion of a 214 kbp genomic region that contains 11 annotated genes, including CONSTANS-LIKE 2b (COL2b), a soybean ortholog of Arabidopsis CONSTANS. Comparison of flowering times under different photo-thermal conditions revealed that early flowering in the mutant lines was most distinct under cool ambient temperatures. The expression of two FT orthologs, FT2a and FT5a, was dramatically downregulated by cool temperature, but the magnitude of the downregulation was lower in the mutant lines. Cool temperatures upregulated COL2b expression or delayed peak expression, particularly at the fourth trifoliate-leaf stage. Intriguingly, they also upregulated E1, a soybean-specific repressor of FT orthologs. Our results suggest that the ef1 mutation is involved in thermal regulation of flowering in response to cool ambient temperature, and the lack of COL2b in the mutant likely alleviates the repression of flowering by cool temperature. The ef1 mutant can be used as a novel gene resource in breeding soybean cultivars adapted to cool climate and in research to improve our understanding of thermal regulation of flowering in soybean.

Suppressed Methionine γ-Lyase Expression Causes Hyperaccumulation of S-Methylmethionine in Soybean Seeds.

Several soybean (Glycine max) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, we compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections of developing soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMM concentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met.

Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon.

KEY MESSAGE: A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465 bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon.

Isolation and characterization of induced mutants in the gene associated with seed cadmium accumulation in soybean.

Food contamination by cadmium (Cd) is a serious threat to human health. Thus, it is imperative to prevent Cd accumulation in staple crops like soybean. The development of low Cd accumulating cultivars is an effective solution. To this end, it is essential to identify the gene(s) controlling seed Cd accumulation. Although Glyma.09G055600 (GmHMA3) seems to be associated with Cd accumulation in soybean, it has not been established if it is responsible for seed Cd accumulation. In the present study, the effect of GmHMA3 on seed Cd accumulation in soybean was validated using three independent GmHMA3 mutants isolated from an ethyl methanesulfonate-induced soybean mutant library. Each of mutant had an amino acid substitution in GmHMA3 and segregating progenies were developed by crossing the original cultivar with each of the three mutants. The relationship between these three mutations and seed Cd accumulation was investigated. While two of them significantly increased seed Cd accumulation corresponding to previous reports of a natural missense mutation in GmHMA3, the other slightly decreased seed Cd accumulation. Overall, these results indicate that GmHMA3 is responsible for seed Cd accumulation in soybean.

Effect of change from a determinate to a semi-determinate growth habit on the yield and lodging resistance of soybeans in the northeast region of Japan.

Although an indeterminate growth habit is attractive to develop high-yield soybean varieties with higher number of pods (Glycine max (L). Merr.), lodging in indeterminate varieties remains a problem in Japan. As the semi-determinate varieties have shorter main stem length than the indeterminate varieties, this trait can be useful to improve varieties with high yield and low lodging risk. We introduced the genes Dt1 and Dt2, which regulate stem growth habit, into three determinate varieties by backcrossing and evaluated the resulting effects on yield and lodging tendency under four different growing environments. The yield and lodging degree of the semi-determinate and indeterminate lines were higher and more severe than those of the determinate lines. Despite the lower overall lodging score, the semi-determinate lines had marginally lower overall yield than that of the indeterminate lines. However, the effect of introduction of semi-determinate traits on yield and lodging degree was different in the three backgrounds, with the yield of semi-determinate lines being the highest and the difference in lodging degree between the semi-determinate and determinate lines being under 1.0 in one background. Therefore, semi-determinate growth habit has potential to develop high yielding varieties with low lodging risk.

Erratum: Quantitative trait loci associated with short inter-node length in soybean.

[This corrects the article on p. 554 in vol. 68, PMID: 30697116.].

A QTL associated with high seed coat cracking rate of a leading Japanese soybean variety.

Seed coat cracking in soybeans [Glycine max (L). Merr.] leads to commercial and agronomic losses. The Japanese elite soybean cultivar 'Fukuyutaka' is often used as a parent for breeding, but its high rate of seed coat cracking is an obstacle to its further use in breeding programs. To establish a DNA marker-assisted selection system for seed coat cracking, genetic factors related to high rates of seed coat cracking were surveyed, and a quantitative trait locus (QTL) with a stable effect on seed coat cracking in both years of a two-year replication experiment was detected on chromosome 20. Comparison of a set of near-isogenic lines (NILs) around this locus verified that the presence of the 'Fukuyutaka' allele significantly increased seed coat cracking in the kernel. The locus is located in a genomic region spanning 3.2 Mb. Marker-assisted selection for the locus will improve the selection efficiency of 'Fukuyutaka'-derived breeding populations.

Mapping versatile QTL for soybean downy mildew resistance.

KEY MESSAGE: Three versatile QTL for soybean downy mildew resistance in Japan were detected using five RIL populations and confirmed using recombinant fixed pairs or a backcrossed line. Downy mildew reduces soybean seed quality and size. It is a problem in Japan, where 90% of soybean grown is used as food. In the USA, 33 downy mildew races have been reported, but race differentiation in Japan is unclear. To identify quantitative trait loci (QTL) for downy mildew resistance effective in the Kanto and Tohoku regions, we performed QTL analysis using five populations of recombinant inbred lines (RILs) originated from 'Natto-shoryu' × 'Tachinagaha' (NT), 'Natto-shoryu' × 'Suzumaru', 'Satonohohoemi' × 'Fukuibuki' (SF), 'Kinusayaka' × 'COL/Akita/2009/TARC/1,' and 'YR-82' × 'Harosoy' over a 4-year period (2014-2017). We evaluated spontaneously developed symptoms of the RILs and applied 112-233 polymorphic markers to each population. Out of 31 QTL detected, we found five on chromosome 3 in three populations and another five on chromosome 7 in three populations. Other QTL were detected in one population, nine of them in different years. In the NT population, two QTL were detected in a 3.0-Mb region on chromosome 7 and in an 8.1-Mb region on chromosome 18 by evaluating nine recombinant fixed pairs in both Kanto and Tohoku regions. In the SF population, a QTL on chromosome 8 was detected in both regions. This QTL was introduced into the 'Satonohohoemi' background by backcrossing, and its effect was confirmed in both regions. In summary, two QTL on chromosomes 7 and 18 from the NT population and one QTL on chromosome 8 from the SF population were confirmed to be effective in both Tohoku and Kanto regions.

The effect of stem growth habit on single seed weight and seed uniformity in soybean (Glycine max (L.) Merrill).

The timing of flower formation and length of the seed-filling period of indeterminate growth soybean varieties vary more than those of determinate varieties (Glycine max (L.) Merrill). These variations have been hypothesized to affect single seed weight and its uniformity which determine the processing quality of soybean used in foods. We derived near isogenic lines (NILs) with different growth characteristics from an indeterminate line (donor parent) and three determinate lines with heavy seeds (recurrent parents), and evaluated the effects of growth habit on seed weight and its uniformity. Each NIL population consisting of five indeterminate and five determinate BC4F4 lines tested at two locations in two different years with two replications. Split-plot analysis of variance, with main-plot and sub-plot being cross combination and growth habit, respectively, showed that indeterminate varieties had slightly heavier seeds than determinate varieties and that there was no significant difference in uniformity of single seed weights. The effects of growth habit on seed uniformity was related to genetic background, but differences between the two growth characteristics were less than the differences among genetic background. This indicates that indeterminate growth habit did not much influence seed weight or its uniformity.

Identification and dissection of single seed weight QTLs by analysis of seed yield components in soybean.

Single seed weight (SSW), or seed size, is a seed yield components (SYC) in soybean, and it is suggested that the genetic factors regulating SSW are involved in the control of other SYCs. The quantitative trait loci (QTLs) for SSW and their effects on the other SYCs were investigated using a recombinant inbred line population derived from typical small- and large-seeded cultivars that were cultivated in two different environments. QTL analysis detected four environmentally stable QTLs for SSW, two of which coincided with the defined loci, qSw17-1 and Ln. The effects of the other loci, qSw12-1 and qSw13-1, were confirmed by analyzing residual heterozygous line progenies derived from the recombinant population. These four QTL regions were also involved in the control of an additional SYC, namely the large-seeded allele at each locus that reduced either the number of pods per plant or the number of ovules per pod. These results suggest the presence of at least two different regulatory mechanisms for SSW. Isolation of genes responsible for these QTLs provides an important tool in the understanding and utilization of SSW diversity for soybean breeding.

Quantitative trait loci associated with short inter-node length in soybean.

Manipulating the genetic control of plant height is essential in soybean breeding to increase yield through the enlargement of the plant size while preventing lodging. A Japanese soybean germplasm, Y2, has distinctively shorter inter-node lengths than those of recently developed Japanese cultivars and is expected to provide new variation to prevent lodging. A quantitative trait loci (QTL) analysis for plant height-related traits was conducted using F2 individuals derived from a cross between the elite Japanese cultivar Fukuyutaka and Y2. A major QTL for average inter-node length (AIL) and plant height was identified on chromosome 13 and named qSI13-1 (QTL for short inter-node on chromosome 13). The Y2 allele of qSI13-1 was partially dominant for plant height. qSI13-1 exhibited no effect on either days to flowering or number of main stem nodes. The AILs and plant heights of the near-isogenic lines containing the Y2 allele of qSI13-1 in the genetic background of Fukuyutaka were significantly less than those of Fukuyutaka. No significant differences between the near-isogenic lines and Fukuyutaka were observed for seed yield and flowering date, indicating that qSI13-1 will be useful in developing cultivars with short plant heights without having negative effects on yield potential and days to flowering.

Confirmation of the pleiotropic control of leaflet shape and number of seeds per pod by the Ln gene in induced soybean mutants.

Most soybean cultivars possess broad leaflets; however, a recessive allele on the Ln locus is known to cause the alteration of broad to narrow leaflets. The recessive allele ln has also been considered to increase the number of seeds per pod (NSP) and has the potential to improve yield. Recently, Gm-JAG1 (Glyma20g25000), a gene controlling Ln, has been shown to complement leaf shape and silique length in Arabidopsis mutants. However, whether Gm-JAG1 is responsible for those traits in soybean is not yet known. In this study, we investigated the pleiotropic effect of soybean Ln gene on leaflet shape and NSP by using two independent soybean Gm-jag1 mutants and four ln near isogenic lines (NILs). The leaflet shape was evaluated using a leaf image analysis software, SmartLeaf, which was customized from SmartGrain. The leaflets of both the Gm-jag1 mutants were longer and narrower than those of the wild-type plants. Interestingly, the image analysis results clarified that the perimeter of the mutant leaflets did not change, although their leaflet area decreased. Furthermore, one mutant line with narrow leaflets showed significantly higher NSP than that in the wild (or Ln) genotype, indicating that soybean Ln gene pleiotropically controls leaflet shape and NSP.

Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69.

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized ß-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5.

A soybean quantitative trait locus that promotes flowering under long days is identified as FT5a, a FLOWERING LOCUS T ortholog.

FLOWERING LOCUS T (FT) is an important floral integrator whose functions are conserved across plant species. In soybean, two orthologs, FT2a and FT5a, play a major role in initiating flowering. Their expression in response to different photoperiods is controlled by allelic combinations at the maturity loci E1 to E4, generating variation in flowering time among cultivars. We determined the molecular basis of a quantitative trait locus (QTL) for flowering time in linkage group J (Chromosome 16). Fine-mapping delimited the QTL to a genomic region of 107kb that harbors FT5a We detected 15 DNA polymorphisms between parents with the early-flowering (ef) and late-flowering (lf) alleles in the promoter region, an intron, and the 3' untranslated region of FT5a, although the FT5a coding regions were identical. Transcript abundance of FT5a was higher in near-isogenic lines for ef than in those for lf, suggesting that different transcriptional activities or mRNA stability caused the flowering time difference. Single-nucleotide polymorphism (SNP) calling from re-sequencing data for 439 cultivated and wild soybean accessions indicated that ef is a rare haplotype that is distinct from common haplotypes including lf The ef allele at FT5a may play an adaptive role at latitudes where early flowering is desirable.

Transfer of the Rsv3 locus from 'Harosoy' for resistance to soybean mosaic virus strains C and D in Japan.

Resistance to soybean mosaic virus (SMV) is imperative for soybean (Glycine max (L.) Merr.) production in the Tohoku region. Molecular markers for SMV resistance were previously reported for U.S. SMV strains, but they cannot be applied because of the differences in strain classification between Japan and the U.S. A U.S. variety 'Harosoy' has been used mainly as a donor of resistance to SMV strains C and D in a Japanese breeding program, resulting in resistant varieties such as 'Fukuibuki.' Because 'Harosoy' harbors the Rsv3 gene conferring resistance to the virulent SMV strain groups, G5 through G7, it appears that the Rsv3 gene confers resistance to strains C and D. In this study, we introduced resistance to the two strains from 'Fukuibuki' into a leading variety 'Ohsuzu' by recurrent backcrossing with marker-assisted selection. All lines selected with markers near Rsv3 showed resistance to the strains, suggesting that the Rsv3 locus is responsible for the resistance. Three years of trials showed that one of the breeding lines, 'Tohoku 169,' was equivalent to 'Ohsuzu' with respect to agricultural characteristics such as seed size, maturity date, and seed yield, except for the SMV resistance.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BACKGROUND: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. RESULTS: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. CONCLUSIONS: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

Seed yield and its components of indeterminate and determinate lines in recombinant inbred lines of soybean.

The present study was conducted to evaluate the benefits of indeterminate growth habit in breeding to improve yield potential of Japanese soybean varieties, which exclusively have determinate growth habit. Two populations of recombinant inbred lines (RILs) derived from crosses between determinate Japanese cultivars and indeterminate US cultivars were grown in Akita and Kyoto, and seed weight per plant (SW) and its components were compared between indeterminate and determinate RILs. The difference of SW between the two growth habits in RILs varied depending on maturation time. The SW of early indeterminate lines was significantly higher than that of early determinate ones in Akita, but not in Kyoto. Among yield components, the number of seeds per pod was constantly larger in indeterminate lines than that in determinate ones irrespective of maturation time. The number of seeds per plant and the number of pods per plant of the indeterminate lines were greater than those of the determinate lines in early maturation in Akita. These results suggest that the indeterminate growth habit is an advantageous characteristic in breeding for high yield of early maturing soybean varieties in the Tohoku region.

Molecular basis of a shattering resistance boosting global dissemination of soybean.

Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world's leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.