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.     

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.     

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.     

Identification and validation of an over‐dominant QTL controlling soybean seed weight using populations derived from Glycine max × Glycine soja

Heterosis, or hybrid vigour, has been used to improve seed yield in several important crops for decades and it has potential applications in soybean. The discovery of over‐dominant quantitative trait loci (QTL) underlying yield‐related traits, such as seed weight, will facilitate hybrid soybean breeding via marker‐assisted selection. In this study, F₂ and F_2 : 3 populations derived from the crosses of ‘Jidou 12’ (Glycine max) × ‘ZYD2738’ (Glycine soja) and ‘Jidou 9’ (G. max) × ‘ZYD2738’ were used to identify over‐dominant QTL associated with seed weight. A total of seven QTL were identified. Among them, qSWT_13_1, mapped on chromosome 13 and linked with Satt114, showed an over‐dominant effect in two populations for two successive generations. This over‐dominant effect was further examined by six subpopulations derived from ‘Jidou12’ × ‘ZYD2738’. The seed weight for heterozygous individuals was 1.1‐ to 1.6‐fold higher than that of homozygous individuals among the six validation populations examined in different locations and years. Therefore, qSWT_13_1 may be a useful locus to improve the yield of hybrid soybean and to understand the molecular mechanism of heterosis in soybean.     

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.     

Genetic variation in South Korean natural populations of wild soybean (Glycine soja)

Summary Wild relatives are valuable genetic resources for crop improvement. Evaluating genetic variation in these species is not only important for their use in breeding programs, but will also provide information about evolution of crops. Seeds representing six natural populations were used to study the level of variation in the South Korean wild soybean. Electrophoretic assays of the seeds on horizontal slab gels were conducted to determine the genotypes of each natural plant at 35 loci in 17 isozymes and one protein. The results indicated a surprisingly high variation. The number of alleles at each locus was as high as four. Seventy two of the 94 reported alleles for the 35 loci were present in these populations. The average number of alleles per locus, 99% polymorphism and the expected heterozygosity in the total population were 2.1, 77.1% and 0.215, respectively. This amount of variation was not only higher than that reported for 857 soybean cultivars and wild soybean populations from other geographic regions, but also higher than the average for 123 self-fertilized plant species and 473 plant species of all mating systems. The high variation in the South Korean wild soybean as well as cultivated soybean indicated in this and other population genetic studies prompts us to propose that South Korea is one of the major soybean gene centers.     

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.     

Identification of quantitative trait loci controlling soybean seed weight in recombinant inbred lines derived from PI 483463 (Glycine soja) × ‘Hutcheson’ (G. max)

The objective of this study was to identify quantitative trait loci (QTLs) controlling 100‐seed weight in soybean using 188 recombinant inbred lines (RIL) derived from a cross of PI 483463 and ‘Hutcheson’. The parents and RILs were grown for 4 years (2010–2013), and mature, dry seeds were used for 100‐seed weight measurement. The variance components of genotype (a), environment (e) and a × e interactions for seed weight were highly significant. The QTL analysis identified 14 QTLs explaining 3.83–12.23% of the total phenotypic variation. One of the QTLs, qSW17‐2, was found to be the stable QTL, being identified in all the environments with high phenotypic variation as compared to the other QTLs. Of the 14 QTLs, 10 QTLs showed colocalization with the seed weight QTLs identified in earlier reports, and four QTLs, qSW5‐1, qSW14‐1, qSW15‐1 and qSW15‐2, found to be the novel QTLs. A two‐dimensional genome scan revealed 11 pairs of epistatic QTLs across 11 chromosomes. The QTLs identified in this study may be useful in genetic improvement of soybean seed weight.     

Pyramiding three rust‐resistance genes confers a high level of resistance in soybean (Glycine max)

Pyramiding Asian soybean rust (ASR) resistance (Rpp) genes in a single genotype has been shown to increase ASR resistance in soybean. However, it remains unclear which combinations of Rpp genes are superior. Therefore, here, we developed six new Rpp‐pyramided lines carrying different combinations of Rpp genes and evaluated their resistance against 13 Bangladeshi rust (Phakopsora pachyrhizi) isolates (BdRPs) alongside seven previously developed Rpp‐pyramided lines. We found that lines carrying one, two and three Rpp genes had high ASR resistance without sporulation in 13.8%, 35.2% and 73.1% of the assays, respectively. Among the new lines that were developed, those with Rpp3 + Rpp4 and Rpp3 + Rpp4 + Rpp5 had high levels of ASR resistance, while the line containing Rpp2 + Rpp4 + Rpp5 showed immunity phenotype at two weeks after inoculation by the BdRP‐22 infection. Thus, pyramiding larger numbers of Rpp genes confers soybean with a higher level of resistance to ASR pathogens and can produce an immunity phenotype at two weeks after inoculation.     

Breeding and characterization of soybean Triple Null; a stack of recessive alleles of Kunitz Trypsin Inhibitor,Soybean Agglutinin,and P34 allergen nulls

Soybean (Glycine max) seeds contain bioactive proteins with antinutritional and immunological properties that affect metabolism and assimilation of nutrients. The presence of antinutritional proteins requires soybeans to be heat‐treated resulting in input energy costs. Nulls for bioactive seed proteins have been previously isolated from the USDA soybean collection, including Kunitz trypsin inhibitor (TI), soybean agglutinin (LE) and immunodominant soybean allergen P34 protein. Each of these nulls has the potential to partially address the concerns of soybean feed/food consumption. A stack of recessive nulls of TI, LE and P34 was created in a cv ‘Williams 82’ background termed ‘Triple Null’. Triple Null has a slight reduction of total protein compared with ‘Williams 82’ corresponding to aggregate contribution of TI, LE and P34 in the seed proteome. Triple Null's proteome analysis revealed P34 and TI nulls are frame‐shift mutants able to accumulate small amounts of authentic P34 and TI proteins. Triple Null has possible application as a conventional feed/food source and for immunotherapy to mitigate soybean allergenic response.     

Genetic characterization and gene flow in different geographical-distance neighbouring natural populations of wild soybean (Glycinesoja Sieb. & Zucc.) and implications for protection from GM soybeans

To design appropriate strategies for ex situ and in situ conservation of wild soybean and safeguard the biosafety of the wild soybean gene pool when genetically modified soybeans are grown, it is important to understand its genetic characteristics, and to quantify gene flow and kinship within and between neighboring populations. We analyzed 9 pairs of neighboring populations of wild soybeans using 20 pairs of nuclear SSR markers. Results showed that Chinese wild soybean natural populations had outcrossing rates of 0–3.5% and that most populations contained many kinship families. The kinship families could be attributed to the accumulation of outcrossed offspring within populations during the history of population colonization. Wild soybean is very sensitive to environmental selection, which results in genetic differentiation of populations, and the emergence of specific alleles. We used an index τ to explain why genetic differences would exist between the pairwise populations; the interpopulation genetic differentiation chiefly consisted in the differences of allele frequencies over the genome. We found long-distance dispersal (1.5 km) of wild soybean seeds in a land ecosystem. There was close correlation between genetic and geographical distance among natural populations of Chinese wild soybean. Within a distance of 50 km, there was greater gene flow when the distance between populations was shorter. These findings have implications for ex situ and in situ conservation in an ecogeographical region, and also for protection of the gene pool from contamination by GM soybeans in wild soybean species.     

Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

Characterization of broad‐spectrum resistance to Soybean mosaic virus in soybean [Glycine max (L.) Merr.] cultivar ‘RN‐9’

Soybean mosaic virus (SMV) can cause serious yield losses in soybean. Soybean cultivar ‘RN‐9’ is resistant to 15 of 21 SMV strains. To well‐characterize this invaluable broad‐spectrum SMV‐resistance, populations (F₁, F₂ and F_2:3) derived from resistant (R) × susceptible (S) and R × R crosses were tested for SMV‐SC18 resistance. Genetic analysis revealed that SC18 resistance in ‘RN‐9’ plus two elite SMV‐resistant genotypes (‘Qihuang No.1’ and ‘Kefeng No.1’) are controlled by independently single dominant genes. Linkage analysis showed that the resistance of ‘RN‐9’ to SMV strains SC10, SC14, SC15 and SC18 is controlled by more than one gene(s). Moreover, Rsc10‐r and Rsc18‐r were both positioned between the two simple sequence repeats markers Satt286 and Satt277, while Rsc14‐r was fine‐mapped in 136.8‐kb genomic region containing sixteen genes, flanked by BARCSOYSSR_06_0786 and BARCSOYSSR_06_0790 at genetic distances of 3.79 and 4.14 cM, respectively. Allelic sequence comparison showed that Cytochrome P450‐encoding genes (Glyma.06g176000 and Glyma.06g176100) likely confer the resistance to SC14 in ‘RN‐9’. Our results would facilitate the breeding of broad‐spectrum and durable SMV resistance in soybeans.     

Development of AS‐PCR marker based on a key mutation confirmed by resequencing of Wx‐mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding

Soft rice with low amylose content (AC) ranging by 5–15% is a unique type with special eating and appearance quality and has become popular in the rice market. We resequenced the Wx‐mp, a key allele from Milky Princess, a Japanese low AC variety, and found that the +473 mutation in exon 4 is the key mutation in both Wx‐mp and its ancestor allele, Wx‐mq from Milky Queen. Based on this functional mutation, an allele‐specific PCR (AS‐PCR) marker was developed and proven in a breeding population derived from a cross between a Chinese late variety Nan Keng 46 (Wx‐mp/Wx‐mp) and an early line Ning 63121(Wx‐b/Wx‐b). Based on the marker‐aided selection by our newly developed AS‐PCR marker for Wx‐mp and the known ST10 marker for Stvb‐i, a total of 12 Wx‐mp homozygotes were selected from 198 F₂ progenies, and four of them were immune to rice stripe virus (RSV) with averagely 11.3 days earlier heading than Nan Keng 46 without significant change in grain yield.     

Identification and marker‐assisted introgression of QTL conferring resistance to bacterial leaf blight in cowpea (Vigna unguiculata (L.) Walp.)

Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is widespread in major cowpea [Vigna unguiculata (L.) Walp.] growing regions of the world. Considering the resource poor nature of cowpea farmers, development and introduction of cultivars resistant to the disease is the best option. Identification of DNA markers and marker‐assisted selection will increase precision of breeding for resistance to diseases like bacterial leaf blight. Hence, an attempt was made to detect QTL for resistance to BLB using 194 F_2 : 3 progeny derived from the cross ‘C‐152’ (susceptible parent) × ‘V‐16’ (resistant parent). These progeny were screened for resistance to bacterial blight by the leaf inoculation method. Platykurtic distribution of per cent disease index scores indicated quantitative inheritance of resistance to bacterial leaf blight. A genetic map with 96 markers (79 SSR and 17 CISP) constructed from the 194 F₂ individuals was used to perform QTL analysis. Out of three major QTL identified, one was on LG 8 (qtlblb‐1) and two on LG 11 (qtlblb‐2 and qtlblb‐3). The PCR product generated by the primer VuMt337 encoded for RIN2‐like mRNA that positively regulate RPM1‐ and RPS2‐dependent hypersensitive response. The QTL qtlblb‐1 explained 30.58% phenotypic variation followed by qtlblb‐2 and qtlblb‐3 with 10.77% and 10.63%, respectively. The major QTL region on LG 8 was introgressed from cultivar V‐16 into the bacterial leaf blight susceptible variety C‐152 through marker‐assisted backcrossing (MABC).     

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

Shotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.