Genetic analysis of net-like cracking in soybean seed coats

Cracking of seed coats in soybean (Glycine max (L.) Merr.) deteriorates the external appearance of seeds and reduces their commercial value. Two types of cracking have been reported that occur in some cultivars: Type I with irregular cracks and Type II with net-like cracks. This study was conducted to determine the genetic basis of net-like cracking. Genetic analysis was performed using F₁ plants produced by crossing Uzuramame, a Japanese landrace with black seed coats having net-like cracking and a Clark mutant with black seed coats, their F₂ population and F₃ lines. Degree of cracking in individual plants was calculated by averaging cracking index (no cracking: 0 to severe cracking: 4) of total or 100-seed samples (average cracking index, ACI). Uzuramame exhibited intense cracking, whereas the Clark mutant showed slight cracking. Intermediate degree of cracking in F₁ plants suggested incomplete dominance. ACI of F₂ plants was continuously distributed. Gene number involved was estimated to be 1.4 by Wright's method. All F₃ lines derived from F₂ plants with ACI more than 2.8 displayed severe cracking phenotypes. In contrast, F₃ lines derived from F₂ plants with ACI less than 2.8 segregated from low to high ACI (0.5 to 3.2). When F₂ plants were classified as slight (ACI<2.8) or severe (ACI>2.8) cracking, the frequency distribution of the F₂ plants fitted to a 3:1 ratio. Genotypes of SSR marker Satt264 that is closely linked to SoyPRP1 locus for proline-rich cell wall protein had a minor effect on ACI. Further, seed weight was positively associated with ACI (r =0.46^**). Our results suggest that net-like cracking is controlled primarily by a major gene, and SoyPRP1 and gene(s) contributing to seed weight may have minor effects on the intensity of cracking. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of IP and SCAR markers linked to the yellow seed color gene in Brassica juncea L.

Previous studies showed that the yellow seed color gene of a yellow mustard was located on the A09 chromosome. In this study, the sequences of the molecular markers linked to the yellow seed color gene were analyzed, the gene was primarily mapped to an interval of 23.304 to 29.402M. Twenty genes and eight markers’ sequences in this region were selected to design the IP and SCAR primers. These primers were used to screen a BC₈S₁ population consisting of 1256 individuals. As a result, five IP and five SCAR markers were successfully developed. IP4 and Y1 were located on either side of the yellow seed color gene at a distance of 0.1 and 0.3 cM, respectively. IP1, IP2 and IP3 derived from Bra036827, Bra036828, Bra036829 separately, co-segregated with the target gene. BLAST analysis indicated that the sequences of newly developed markers showed good collinearity with those of the A09 chromosome, and that the target gene might exist between 27.079 and 27.616M. In light of annotations of the genes in this region, only Bra036828 is associated with flavonoid biosynthesis. This gene has high similarity with the TRANSPARENT TESTA6 gene, Bra036828 was hence identified as being the gene possibly responsible for yellow seed color, in our research.     

Methods for evaluation of soybean chilling tolerance at the reproductive stage under artificial climatic conditions

Several evaluation methods for soybean chilling tolerance at the reproductive stage were examined under artificial climatic conditions. Comparisons were made on plants in control and treated plots using three cultivars differing in the level of chilling tolerance. In all methods, plants were grown at 22/17°C (day/night) until first flowering, and then transferred to growth chambers at 24/17°C for control and 15/15°C for chilling treatment, respectively. A method, in which plants were grown at 20/16°C after 4 weeks of the different temperature treatments, proved comparable to the conventional one, in which chilling tolerance at the flowering stage is evaluated using natural and artificial conditions. Another method, in which the plants in chilling treatment plots were grown at 15°C until maturity, also proved usable to evaluate genotypic differences in chilling tolerance independently of maturity time.     

Overexpression of a small heat‐shock‐protein gene enhances tolerance to abiotic stresses in rice

Rice (Oryza sativa L.) is one of the most important crops in the world which survives from various abiotic stresses in natural environments with specific stress‐involved genes expressed. Plant sHSPs (small heat‐shock proteins) were reported to respond to abiotic stresses. To improve the understanding of sHSPs in rice, we characterized heat‐shock‐protein gene OsHSP18.6 here. OsHSP18.6 could be induced by diverse stresses, such as drought, salt and cold, especially under heat. The gene was found expressed in root, stem, leaf, internode and spikelet. Overexpression of OsHSP18.6 results in increased thermotolerance and exhibits universal tolerance to stresses tested, including heat, drought, salt and cold. Lower levels of malondialdehyde (MDA) and greater activities of catalase (CAT) and superoxide dismutase (SOD) were observed in OsHSP18.6‐overexpression rice under heat and drought. OsHSP18.6‐overexpression lines indicated decreased sterile rates under hot weather without remarkable changes in most of other agronomic traits compared with wild‐type plants.     

Suppressive mechanism of seed coat pigmentation in yellow soybean

In soybean seeds, numerous variations in colors and pigmentation patterns exist, most of which are observed in the seed coat. Patterns of seed coat pigmentation are determined by four alleles (I, iⁱ, i^k and i) of the classically defined I locus, which controls the spatial distribution of anthocyanins and proanthocyanidins in the seed coat. Most commercial soybean cultivars produce yellow seeds with yellow cotyledons and nonpigmented seed coats, which are important traits of high-quality seeds. Plants carrying the I or iⁱ allele show complete inhibition of pigmentation in the seed coat or pigmentation only in the hilum, respectively, resulting in a yellow seed phenotype. Classical genetic analyses of the I locus were performed in the 1920s and 1930s but, until recently, the molecular mechanism by which the I locus regulated seed coat pigmentation remained unclear. In this review, we provide an overview of the molecular suppressive mechanism of seed coat pigmentation in yellow soybean, with the main focus on the effect of the I allele. In addition, we discuss seed coat pigmentation phenomena in yellow soybean and their relationship to inhibition of I allele action.     

Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses

Summary Trehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 ^∘C and 35^∘C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops.     

Dynamic quantitative trait loci underlies isoflavone accumulation in soybean seed

Most of quantitative trait loci (QTL) underlying soybean seed isoflavone contents were derived from the harvest stage of plant development, which uncover the genetic effects that were expressed in earlier seed developmental stages. The aim of this study was to detect conditional QTL associated with isoflavone accumulation during the entire seed development. A total of 112 recombinant inbred lines developed from the cross between ‘Zhongdou 27’ (higher seed isoflavone content) and ‘Jiunong 20’ (lower seed isoflavone content) were used for the conditional QTL analysis of daidzein (DZ), genistein (GT), glycitein (GC) and total isoflavone (TI) accumulations through composite interval mapping with mixed genetic model. The results indicated that the number and type of QTL and their additive effects for individual and total isoflavone accumulations were different among R3 to R8 developmental stages. Three unconditional QTL and six conditional QTL for DZ, four unconditional QTL and five conditional QTL for GT, six unconditional QTL and five conditional QTL for GC, six unconditional QTL and seven conditional QTL for TI were identified at different developmental stages, respectively. Unconditional and conditional QTL that affect individual and total isoflavone accumulations exhibited multiple expression patterns, implying that some QTL are active for long period and others are transient. Two genomic regions, Satt144‐Satt569 in linkage group F (LG F; chromosome 13, chr 13) for DZ, GC, GT and TI accumulations and Satt540‐Sat_240 in LG M (chr 07) for TI and GC accumulations, were found to significantly affect individual and total isoflavone accumulations in multiple developmental stages, suggesting that the accumulation of soybean seed isoflavones is governed by time‐dependent gene expression.     

Studies on the flooding tolerance of soybean seed: varietal differences

Summary To evaluate seed tolerance of soybean seed to flooding, a screening method by soaking seeds at 25°C for 4 days has been established. Of 730 varieties tested, many from South America, India and Southeast Asia appeared highly tolerant to pre-germination flooding. Seed flooding tolerance correlated with seed coat color. Seeds with black colored coat exhibited high tolerance, and some varieties with black seed coat germinated well even after 10 days soaking at 25°C. All yellow seeds failed completely to germinate after 8 days of soaking.     

Weed tolerance in soybean: a direct selection system

Weed competition can severely reduce soybean (Glycine max (L.) Merr.) yields, particularly in organic systems. An efficient screening and breeding approach is needed to increase breeding progress for weed tolerance. This study sought to (i) establish a system for direct selection of competitive genotypes, (ii) evaluate genotypic differences in weed tolerance among six early‐maturing genotypes and (iii) assess the contribution of selected morphological traits to weed tolerance. A direct selection system providing two different levels of weed competition through all development stages of a soybean crop was developed, using mixtures of selected crop species as sown competitors. Two resulting mixtures induced intermediate (<30%) and strong (>50%) yield reduction, respectively. This selection system can be applied in screening and breeding programmes to facilitate breeding for weed tolerance. No significant difference in weed tolerance was detected between six soybean genotypes of maturity groups 000 to 00. Morphological traits that might influence competitive ability, for example light absorption, leaf area and lateral shoots, were assessed, and their potential for indirect selection for weed tolerance is discussed.     

Chloride tolerance in soybean and perennial Glycine accessions

Diversity for chloride tolerance exists among accessions of perennial Glycine. Accessions whose tolerance thresholds exceed those of Glycine max cultivars may be useful germplasm resources. Soybean cultivars including ‘Jackson’ (sensitive) and ‘Lee’ (tolerant) and 12 accessions of perennial Glycine were evaluated for sodium chloride tolerance after 14 days in hydroponic culture at 0, 5, 10, and 15 g L^-1 NaCl. Sodium chloride had adverse effects on the growth of G. max cultivars and perennial Glycine accessions; however differential responses to salinity were observed among accessions. Considerably greater variation in sodium chloride tolerance existed among the perennial Glycine accessions than among the G. max cultivars. Sodium chloride tolerance thresholds ranged from 3.0 to 17.5 g L^-1 NaCl for the perennial accessions but only ranged from 5.2 to 8.0 g L^-1 for the cultivars, based on a Weibull model of leaf chlorosis. All G. max cultivars were severely injured or killed by NaCl at 10 g L^-1 and above. Five tolerant perennial Glycine accessions, G. argyrea 1626, G. clandestina 1388 and 1389, and G. microphylla 1143 and 1195, were significantly lower in leaf chlorosis score than any of the G. max cultivars at the 10 g L^-1 NaCl treatment. Two accessions, G. argyrea 1626 and G. clandestina 1389 were able to tolerate 15 g L^-1 NaCl with only moderate visual injury while all other accessions were severely injured or killed at this salt level. Variability for chloride tolerance observed among the perennial Glycine accessions has potential utility for developing enhanced salt tolerance in soybean. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and inheritance of a partially dominant gene for yellow seed colour in Brassica napus

A yellow‐seeded doubled haploid (DH) line no. 2127‐17, derived from a resynthesized Brassica napus L., was crossed with two black‐seeded Brassica cultivars ‘Quantum’ and ‘Sprint’ of spring type. The inheritance of seed colour was investigated in the F₂, and BC1 populations of the two crosses and also in the DH population derived from the F1 of the cross ‘Quantum’× no. 2127‐17. Seed colour analysis was performed with the colorimeter CR‐300 (Minolta, Japan) together with a visual classification system. The immediate F1 seeds of the reciprocals in the two crosses had the same colour as the self‐pollinated seeds of the respective black‐ and yellow‐seeded female parents, indicating the maternal control of seed colour. The F1 plants produced yellow‐brown seeds that were darker in colour than the seeds of no. 2127‐17, indicating the partial dominance of yellow seed over black. In the segregating BC1 progenies of the two crosses, the frequencies of the black‐ and yellow‐seeded plants fit well with a 1 : 1 ratio. In the cross with ‘Quantum’, the frequencies of yellow‐seeded and black‐seeded plants fit with a 13 : 3 ratio in the F₂ progeny, and with a 3 : 1 ratio in the DH progeny. However, a 49 : 15 segregation ratio was observed for the yellow‐seeded and black‐seeded plants in the F₂ progeny of the cross with ‘Sprint’. It was postulated from these results that seed colour was controlled by three pairs of genes. A dominant yellow‐seeded gene (Y) was identified in no. 2127‐17 that had epistatic effects on the two independent dominant black‐seeded genes (B and C), thereby inhibiting the biosynthesis of seed coat pigments.     

大豆根部水压胁迫耐逆指数遗传体系解析

大豆是重要的植物蛋白质和植物油脂来源,干旱是影响大豆产量的重要环境因子之一。为解析大豆耐旱性的遗传基础,本研究在PEG水压胁迫条件下,对由409个家系组成的巢式关联作图群体(具有1个共同亲本的2个重组自交系群体组成)进行叶片脯氨酸含量测定,通过限制性二阶段多位点全基因组关联分析(restrictivetwo-stagemultilocus genome-wide association study,RTM-GWAS),解析了大豆根部水压胁迫耐逆指数(root hydraulic stress tolerance index,RHSTI)的遗传体系。结果表明,在春季和夏季环境下,3个亲本蒙8260(共同亲本)、通山薄皮黄豆甲和正阳白毛平顶在RHSTI上均存在显著差异,其衍生群体RHSTI表型变异丰富,变幅分别为0.11~2.94和0.03~1.93,遗传力分别为97.7%和97.9%;2个环境联合分析发现,家系遗传力和家系与环境互作遗传力分别为37.9%和60.1%,说明群体RHSTI的变异受遗传和环境共同控制。通过RTM-GWAS方法,共检测到45个与RHSTI相关的QTL,分布在大豆18条染色体上,可以解释37.58%的表型变异,其中7个QTL的表型贡献率超过1%,为大贡献位点;这些QTL中,有34个位点与环境存在显著互作,可以解释12.50%的表型变异。结合PEG胁迫下大豆转录组数据,在定位区间500kb范围内共筛选到38个差异表达基因,可归为ABA响应因子、逆境响应因子、转录因子、发育因子、蛋白代谢因子、未知功能和其他等7类,其中逆境响应因子、转录因子和发育因子是最大的3类;其中位于主效位点的6个基因,与ABA响应因子、逆境响应因子、转录因子相关,应为主要候选基因。上述结果表明,大豆耐旱性是一个由多位点、多基因控制的复杂数量性状,且与环境存在互作,遗传基础复杂。研究结果为大豆耐旱性分子育种提供了依据。     

QTL mapping of phosphorus deficiency tolerance in soybean (Glycine max L. Merr.)

Phosphorus (P) deficiency is a major abiotic stress that limits plant growth and crop productivity throughout the world. In the present study, 184 recombinant inbred line (RIL) families developed from soybean varieties Kefeng No. 1 and Nanong 1138-2 were used to identify quantitative trait loci (QTL) associated with P deficiency tolerance. Seven traits of plant height (HT), weight of fresh shoot (FSW), weight of fresh root (FRW), weight of dry root (DRW), length of main root (RL), phosphorus content in leaf (LP), phosphorus content in root (RP), were used as parameters to assess the phosphorus deficiency tolerance. The QTL mapping for the seven traits was performed using the program WinQTLCart. Seven QTLs were detected and mapped on two linkage groups for three traits of weight of fresh shoot, phosphorus contents in leaf and in root. The QTLs that had LOD scores more than three were detected for all of the three traits above. Most of the QTLs explained more than 10% of the total variation. The two QTLs for phosphorus content in leaf explained more than 20% of the total variation, respectively. Five QTLs were mapped on linkage group F2, and two on linkage F1. It was suggested that the genes related to phosphorus deficiency tolerance located on linkage group F in soybean.Contributed equally to this work.     

Identification and characterization of a RAPD/SCAR marker linked to a resistance gene for soybean mosaic virus in soybean

Soybean line `ICGR95-5383' [Glycinemax (L.) Merr.] is a newly releasedgermplasm from China and is resistant (R)to soybean mosaic virus (SMV). ICGR95-5383was crossed to the susceptible (S)cultivars `HB1', `Tiefeng21', `Amsoy', and`Williams' to investigate the inheritanceof SMV resistance. The F<sub>1</sub> and F<sub>2</sub>plants were inoculated with SMV-3 (the mostvirulent) strain from Northeast China. Theresults showed that F<sub>1</sub> plants from thefour R × S crosses were necrotic (N) andall F<sub>2</sub> populations segregated in a3(R+N):1S ratio, indicating thatICGR95-5383 carries a single gene withincomplete dominance for resistance to SMV. In a bulked segregant analysis (BSA) of theF<sub>2</sub>population from ICGR95-5383 × HB1, a codominant RAPD marker,OPN11<sub>980/1070</sub>, was found to be linkedto the resistance gene in ICGR95-5383. The980-base pair (bp) fragment OPN11<sub>980</sub>was amplified in the R parent ICGR95-5383,R bulk, and resistant F<sub>2</sub> plants. Theother 1070-bp fragment OPN11<sub>1070</sub> wasamplified in the S parent HB1, S bulk, andsusceptible F<sub>2</sub>plants.OPN11<sub>980/1070</sub> was amplified in theF<sub>1</sub> plants and the necroticF<sub>2</sub> plants from the R×S cross.Segregation analysis of the RAPD marker inthe F<sub>2</sub> population revealed that themarker OPN11<sub>980/1070</sub> is closely linkedto the resistance gene with a map distanceof 3.03 cM. OPN11<sub>980/1070</sub> was clonedand sequenced, and specific PCR primerswere designed to convertOPN11<sub>980/1070</sub> into sequencecharacterized amplified region (SCAR) makerSCN11<sub>980/1070</sub>. SCAR analysis of theF<sub>2</sub> population confirmed thatOPN11<sub>980/1070</sub> and SCN11<sub>980/1070</sub> areat the same locus linked to the SMVresistance gene. The RAPD markerOPN11<sub>980</sub> was used as RFLP probefor southern hybridization to soybeangenomic DNA. Southern analysis showed thatsoybean genome contains low-copy sequenceof OPN11<sub>980</sub>. Using a recombinant inbredmapping population of `Kefeng No.1' (R) ×Nannong1138-2'(S), OPN11_980/1070 was mapped to thesoybean molecular linkage group (MLG) Fbetween the restriction fragment lengthpolymorphism (RFLP) markers B212 (0.7 cM) and K07 (6.7 cM) and 3.03 cM apart from theSMV resistance gene.     

An effective field screening method for flood tolerance in soybean

Flooding is an abiotic stress that causes considerable reductions in crop growth and yield worldwide. Soybean (Glycine max [L.] Merr.) cultivars are generally sensitive to flooding stress. The objective of this study was to develop an effective flooding tolerance screening method in the field. A total of 40 soybean genotypes were evaluated for flooding tolerance at V5 and R1 growth stages. At each stage, genotypes were exposed to different durations of flooding stress (3, 6, 9, 12 and 15 days). Plant foliar damage score (FDS) and plant survival rate (PSR) were used as the indicators of flooding tolerance. Soybeans were more sensitive to flooding at R1 growth stage than V5 growth stage. Length of flooding duration accounted for the variance of FDS and PSR. Soybean genotypes exposed to a 3‐day flooding in either V5 or R1 growth stage, did not show obvious foliar damage, while genotypes exposed to a 12‐ or 15‐day flooding showed significant foliar damage and plant death. The optimum flooding duration to screen for flooding tolerance in the field was determined to be 9 and 6 days for V5 and R1 growth stages, respectively, as distinguishable responses to flooding allowed genotypes to be classified as either being flooding tolerant or flooding sensitive. High correlation between FDS and PSR (.99, p < .0001) was observed. Similarly, FDS and PSR were highly correlated with grain yield (.95 and .95, p < .0001). The field screening method for flooding tolerance developed in our study will be favourable for selection of soybean flooding‐tolerant germplasm.     

Wide genetic variation in phenolic compound content of seed coats among black soybean cultivars

Black soybeans have been used as a food source and also in traditional medicine because their seed coats contain natural phenolic compounds such as proanthocyanidin and anthocyanin. The objective of this research is to reveal the genetic variation in the phenolic compound contents (PCCs) of seed coats in 227 black soybean cultivars, most of which were Japanese landraces and cultivars. Total phenolics were extracted from seed coats using an acidic acetone reagent and the proanthocyanidin content, monomeric anthocyanin content, total flavonoids content, total phenolics content, and radical scavenging activity were measured. The cultivars showed wide genetic variation in PCCs. Each of the contents was highly correlated with one another, and was closely associated with radical scavenging activity. PCCs were also moderately associated by flowering date but not associated by seed weight. Cultivars with purple flowers had a tendency to produce higher PCCs compared with cultivars with white flowers, suggesting that the W1 locus for flower color can affect phenolic compound composition and content. Our results suggest that developing black soybean cultivars with high functional phenolic compounds activity is feasible.     

Identification and validation of quantitative trait loci associated with seed yield in soybean

In soybean [Glycine max (L.) Merrill], the genetic analysis of seed yield is important to aid in the breeding of high-yielding cultivars. Seed yield is a complex trait, and the number of quantitative trait loci (QTL) involved in seed yield is high. The aims of this study were to identify QTL associated with seed yield and validate their effects on seed yield using near-isogenic lines. The QTL analysis was conducted using a recombinant inbred line population derived from a cross between Japanese cultivars ‘Toyoharuka’ and ‘Toyomusume’, and eight seed yield-associated QTL were identified. There were significant positive correlations between seed yield and the number of favorable alleles at QTL associated with seed yield in the recombinant inbred lines for three years. The effects of qSY8-1, a QTL promoting greater seed yield, was validated in the Toyoharuka background. In a two-year yield trial, the 100-seed weight and seed yield of Toyoharuka-NIL, the near-isogenic line having the Toyomusume allele at qSY8-1, were significantly greater than those of Toyoharuka (106% and 107%, respectively) without any change for days to flowering and maturity. Our results suggest that qSY8-1 was not associated with maturity genes, and contributed to the 100-seed weight.