Genetic mapping of soybean cyst nematode race-3 resistance loci in the soybean PI 437.654

Resistance to the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is difficult to evaluate in soybean [Glycine max (L.) Merr.] breeding. PI 437.654 has resistance to more SCN race isolates than any other known soybean. We screened 298 F₆₇ recombinant-inbred lines from a cross between PI 437.654 and BSR101 for SCN race-3 resistance, genetically mapped 355 RFLP markers and the I locus, and tested these markers for association with resistance loci. The Rhg ₄ resistance locus was within 1 cM of the I locus on linkage group A. Two additional QTLs associated with SCN resistance were located within 3cM of markers on groups G and M. These two loci were not independent because 91 of 96 lines that had a resistant-parent marker type on group G also had a resistant-parent marker type on group M. Rhg ₄ and the QTL on G showed a significant interaction by together providing complete resistance to SCN race-3. Individually, the QTL on G had greater effect on resistance than did Rhg ₄, but neither locus alone provided a degree of resistance much different from the susceptible parent. The nearest markers to the mapped QTLs on groups A and G had allele frequencies from the resistant parent indicating 52 resistant lines in this population, a number not significantly different from the 55 resistant lines found. Therefore, no QTLs from PI 437.654 other than those mapped here are expected to be required for resistance to SCN race-3. All 50 lines that had the PI 437.654 marker type at the nearest marker to each of the QTLs on groups A and G were resistant to SCN race-3. We believe markers near to these QTLs can be used effectively to select for SCN race-3 resistance, thereby improving the ability to breed SCN-resistant soybean varieties.     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

Relationship of cyst nematode gene frequencies to soybean resistance

Summary Soybean (S, Glycine max (L.) Merr.) lines with relatively few cysts of soybean cyst nematode (CN, Heterodera glycines Ichinohe) populations are usually called CN-resistant. The phenotype of number of cysts per plant is of the CN-S (Cyst Nematode-Soybean) association and determined by the interactions of genes for avirulence-resistance. The acronym alins was proposed for these alleles for incompatibility, with xalin representing the interaction X of one microsymbiont malin with its host h-alin. These alins are dominant in the gene-for-gene model but may be mostly recessive with CN-S. Definitive genetic studies have been hindered by the heterogeneity of sexually reproducing CN populations and lack of the appropriate genetic models. Loegering's abstract interorganismal genetic model was modified so that one model represented all four possible interactions of dominant-recessive alins for an incompatible phenotype. This involved redefining the Boolean algebra symbol 1 to represent both the alins AND their frequencies. The model was used to derive the relationship: {ie893-01} where the expectation E of cysts (of any CN-S combination, as proportion of number of cysts on a check cultivar) is proportional to the product of CN genotypic frequencies expressed as functions of m-alin frequencies. Each m-alin is at a different locus, i.e., {ie893-02}. The number of terms multiplied for each CN-S is equal to the number of alins in the S line (or F₂ plant). There are too many unknowns in the equation to solve for any of them. The relationship does explain the continuous distributions of phenotypes that were nearly always observed. Basic genetic principles were used to concurrently derive the models and to obtain discontinuous distributions of numbers of cyst phenotypes in segregating generations due to one recessive alin in a CN-susceptible soybean line.Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 9739     

基于大豆胞囊线虫病抗性候选基因的SNP位点遗传变异分析

以我国363份栽培和野生大豆资源为材料, 对大豆胞囊线虫抗性候选基因(rhg1和Rhg4)的SNP位点(8个)进行遗传变异分析, 以期阐明野生和栽培大豆间遗传多样性及连锁不平衡水平差异。结果表明, 与野生大豆相比, 代表我国栽培大豆总体资源多样性的微核心种质及其补充材料的连锁不平衡水平较高(R²值为0.216)。在栽培大豆群体内, 基因内和基因间分别有100％和16.6％的SNP位点对连锁不平衡显著, 形成两个基因特异的连锁不平衡区间(Block)。在所有供试材料中共检测到单倍型46个, 野生大豆的单倍型数目(27)少于栽培大豆(31), 但单倍型多样性(0.916)稍高于栽培大豆(0.816)。单倍型大多数(67.4％)为群体所特有(31个), 其中15个为野生大豆特有单倍型。野生大豆的两个主要优势单倍型(Hap_10和Hap_11)在栽培大豆中的发生频率也明显下降, 推测野生大豆向栽培大豆进化过程中, 一方面形成了新的单倍型, 另一方面因为瓶颈效应部分单倍型的频率降低甚至消失。     

Development of SNP markers and haplotype analysis of the candidate gene for rhg1, which confers resistance to soybean cyst nematode in soybean

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is one of the most destructive pests in the cultivation of soybean (Glycine max (L.) Merr.) worldwide. Markers based on the SCN resistance gene will enable efficient marker-assisted selection (MAS). We sequenced the candidate gene rhg1 in six resistant and two susceptible soybean genotypes and identified 37 SNPs (single nucleotide polymorphisms) among the sequences, of which 11 were in the coding region. Seven of these 11 SNPs led to changes in the amino acid sequence of the gene. The amino acid sequence we obtained differs from the previously published one by a stretch of 26–27 amino acids. Six codominant allele-specific SNP markers based on agarose gel detection were developed and tested in 70 genotypes, among which occurred only nine different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the six SNP loci in the 70 genotypes, which is consistent with intensive directional selection. A strong LD pattern was detected among five SNPs except 2868T > C. Two SNPs (689C > A and 757C > T) formed one haplotype (689C-757C) that was perfectly associated with SCN resistance. The new allele-specific PCR markers located in the alleged sequence of the rhg1 candidate gene, combined with the microsatellite marker BACR-Satt309, will significantly improve the efficiency of MAS during the development of SCN-resistant cultivars.     

Kinase-dead mutation: A novel strategy for improving soybean resistance to soybean cyst nematode Heterodera glycines

Protein kinases phosphorylate proteins for functional changes and are involved in nearly all cellular processes, thereby regulating almost all aspects of plant growth and development, and responses to biotic and abiotic stresses. We generated two independent co-expression networks of soybean genes using control and stress response gene expression data and identified 392 differentially highly interconnected kinase hub genes among the two networks. Of these 392 kinases, 90 genes were identified as “syncytium highly connected hubs”, potentially essential for activating kinase signalling pathways in the nematode feeding site. Overexpression of wild-type coding sequences of five syncytium highly connected kinase hub genes using transgenic soybean hairy roots enhanced plant susceptibility to soybean cyst nematode (SCN; Heterodera glycines) Hg Type 0 (race 3). In contrast, overexpression of kinase-dead variants of these five syncytium kinase hub genes significantly enhanced soybean resistance to SCN. Additionally, three of the five tested kinase hub genes enhanced soybean resistance to SCN Hg Type 1.2.5.7 (race 2), highlighting the potential of the kinase-dead approach to generate effective and durable resistance against a wide range of SCN Hg types. Subcellular localization analysis revealed that kinase-dead mutations do not alter protein cellular localization, confirming the structure–function of the kinase-inactive variants in producing loss-of-function phenotypes causing significant decrease in nematode susceptibility. Because many protein kinases are highly conserved and are involved in plant responses to various biotic and abiotic stresses, our approach of identifying kinase hub genes and their inactivation using kinase-dead mutation could be translated for biotic and abiotic stress tolerance.     

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC₃F_2:3 and F_2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11₇₉₅, OPAE-08₈₃₇, OPR-07₅₄₈ and OPY-07₂₀₃₀) were identified in the BC₃F_2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F_2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean. Received: 12 October 1999 / Accepted: 14 April 2000     

Genomic analysis of a region encompassing QRfs1 and QRfs2: genes that underlie soybean resistance to sudden death syndrome.

Candidate genes were identified for two loci, QRfs2 providing resistance to the leaf scorch called soybean (Glycine max (L.) Merr.) sudden death syndrome (SDS) and QRfs1 providing resistance to root infection by the causal pathogen Fusarium solani f.sp. glycines. The 7.5 +/- 0.5 cM region of chromosome 18 (linkage group G) was shown to encompass a cluster of resistance loci using recombination events from 4 near-isogenic line populations and 9 DNA markers. The DNA markers anchored 9 physical map contigs (7 are shown on the soybean Gbrowse, 2 are unpublished), 45 BAC end sequences (41 in Gbrowse), and contiguous DNA sequences of 315, 127, and 110 kbp. Gene density was high at 1 gene per 7 kbp only around the already sequenced regions. Three to 4 gene-rich islands were inferred to be distributed across the entire 7.5 cM or 3.5 Mbp showing that genes are clustered in the soybean genome. Candidate resistance genes were identified and a molecular basis for interactions among the disease resistance genes in the cluster inferred.     

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence‐related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full‐length cDNAs of GmSAMT1 from a SCN‐resistant soybean line and from a SCN‐susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli‐expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μm . To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN‐susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.     

大豆胞囊线虫的休眠

以前研究发现,辽宁地区大豆生长期间及收获期土壤中胞囊孵出的二龄幼虫量很少,推测线虫卵的休眠与大豆生长时期或季节相关。为明确该地区大豆胞囊线虫的休眠特点,2002-2003年采用田间随机多点取样、室内分离及模拟自然条件孵化等方法对大豆胞囊线虫的休眠进行深入研究。结果表明:在生长季节,感病品种辽豆10根围土壤中的白色雌虫、卵囊及褐色的胞囊均可孵出二龄幼虫,且孵化持续时间较长,第21d仍有幼虫孵出,白色雌虫及卵囊内的卵孵化率高于褐色胞囊;不同作物对其根围土壤中胞囊内卵的孵化影响不大,寄主作物大豆、非寄主作物玉米根围及休闲地土壤中的胞囊在条件适宜均可孵出二龄幼虫;季节对胞囊内卵的孵化有较大的影响,出苗期孵化率最高,收获期最低,2周时平均1个胞囊孵出幼虫分别为83.8和9.7条;胞囊皮对线虫卵的孵化有显著的影响。表明沈阳地区大豆胞囊线虫在正常和逆境条件下均有部分卵表现休眠。     

Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patellaris and Beta procumbens.

Summary In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n =18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.     

Targeted comparative genome analysis and qualitative mapping of a major partial-resistance gene to the soybean cyst nematode

A major partial-resistance locus to the soybean cyst nematode (Heterodera glycines Ichinohe; SCN) was identified on linkage group G of soybean [Glycine max (L.) Merr.] using restriction fragment length polymorphisms (RFLPs). This locus explained 51.4% (LOD=10.35) of the total phenotypic variation in disease response in soybean Plant Introduction (PI) 209332, 52.7% (LOD=15.58) in PI 90763, 40.0% (LOD=10.50) in PI 88788, and 28.1% (LOD=6.94) in Peking. Initially, the region around this major resistance locus was poorly populated with DNA markers. To increase marker density in this genomic region, first random, and later targeted, comparative mapping with RFLPs from mungbean [Vigna radiata (L.) R. Wilcz.] and common bean (Phaseolus vulgaris L.) was performed, eventually leading to one RFLP marker every 2.6 centimorgans (cM). Even with this marker density, the inability to resolve SCN disease response into discrete Mendelian categories posed a major limitation to mapping. Thus, qualitative scoring of SCN disease response was carried out in an F₅₆ recombinant inbred population derived from EvansxPI 209332 using a 30% disease index cut-off for resistance. Using the computer program JoinMap, an integrated map of the region of interest was created, placing the SCN resistance locus 4.6 cM from RFLP marker B53 and 2.8 cM from Bng30. This study demonstrates how a combination of molecularmapping strategies, including comparative genome analysis, join mapping, and qualitative scoring of a quantitative trait, potentially provide the necessary tools for high-resolution mapping around a quantitative-trait locus.     

A bacterial artificial chromosome library for soybean and identification of clones near a major cyst nematode resistance gene

 We constructed a bacterial artificial chromosome (BAC) library for soybean (Glycine max) consisting of approximately 30 000 clones with an average insert size of 120 kilobase pairs. The library was successfully screened with restriction fragment length polymorphism (RFLP) and microsatellite markers tightly linked to a major resistance gene for the cyst nematode, Heterodera glycines. Since many soybean RFLPs hybridize to duplicate loci, BACs homologous to duplicate RFLP loci were distinguished by digestion with the restriction enzyme originally used to map the RFLP, followed by a comparison of the hybridizing fragments. Linkage mapping of BAC clones identified with markers linked to the cyst nematode resistance gene demonstrated that these clones were located at the expected chromosomal positions and that there were no indications of chimeras within the genomic inserts. Received: 3 July 1997/Accepted: 26 August 1997     

Loci underlying resistance to Race 3 of soybean cyst nematode in Glycine soja plant introduction 468916

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is an important soybean [Glycine max (L.) Merr.] pest in the U.S. and throughout the world. Genetic resistance is the primary method for controlling SCN and there is a need to identify new resistance genes. Glycine soja Sieb. and Zucc. is the wild ancestor of domesticated soybean and is a potential source of new SCN resistance genes. The goal of this research was to map quantitative trait loci (QTLs) that provide resistance to SCN Race 3 from the G. soja plant introduction (PI) 468916. Fifty seven F₂-derived lines from a cross between the G. soja PI 468916 and the G. max experimental line A81-356022 were tested for resistance to an SCN population with a Race-3 phenotype. These lines were also genotyped with 1,004 genetic markers and resistance genes were mapped by composite interval mapping with the computer program QTL-Cartographer. In the F₂ population, three significant (LOD > 3.0) QTLs were detected that explained from 5% to 27% of the variation for Race-3 resistance. The two most significant QTLs identified in the F₂ population were tested in a population of 100 BC1F₂ plants developed by crossing A81-356022 to a line from the F₂ population that carried the two resistance QTLs from G. soja. In the backcross population, both Race-3 resistance QTLs were significant, which confirms the existence of these QTLs. The QTLs identified in this experiment map to positions where SCN resistance genes have not been previously identified, suggesting that these are novel genes that could be useful for diversifying the resistance genes currently used in cultivar development. Received: 7 August 2000 / Accepted: 4 December 2000     

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998     

Transfer of clover cyst nematode resistance from Trifolium nigrescens Viv. to T. repens L. by interspecific hybridisation

 Resistance to clover cyst nematode (Heterodera trifolii) has been successfully transferred from Trifolium nigrescens to T. repens by interspecific hybridisation. A sterile triploid hybrid (H-6909-5, 2n=3x=24) was initially produced with the aid of embryo culture. The hybrid was chromosome-doubled from axillary meristems by an in vitro colchicine method. Three chromosome-doubled plants were obtained, and these showed a marked increase in pollen stainability from 10% in 3x H-6909-5 to an average of 89% (range 88–91%) in 6x H-6909-5. T. nigrescens was a source of clover cyst nematode resistance. A mean of 23 (range 0–150) cysts per plant was recorded for T. nigrescens in comparison to a mean of 150 (range 50–240) cysts per plant for T. repens. The 3x and 6x interspecific hybrids were shown to be as resistant as the most resistant. T. nigrescens genotype and were significantly lower in cyst number per gram of root dry weight than the susceptible T. nigrescens and T. repens genotypes. Received: 11 July 1997 / Accepted: 15 July 1997     

Root responses to cereal cyst nematode (Heterodera avenae) in hosts with different resistance genes

The development of cereal cyst nematode (CCN; Heterodera avenae ) induced syncytia in the host roots of infected resistant bread wheat ( Triticum aestivum cv. AUS10894), diploid wheat ( Aegilops tauschii ), barley ( Hordeum vulgare cv. Chebec and cv. Galleon) and in the susceptible wheat cv. Meering and barley cv. Clipper were studied over a period of 13 d. The resistance to CCN in these cereal plants is conferred by the resistance genes Cre1 in the wheat cv. AUS10894, Cre3 in A. tauschii , Ha2 in barley cv. Chebec and Ha4 in barley cv. Galleon. Anatomical observations were made on the development of the syncytia in CCN-infected wheat and barley roots, which carry each of these four sources of resistance genes. Accelerated development of the syncytia in resistant plants, especially in the barley cultivars, was observed. The sites of syncytia development in susceptible wheat and barley were also closely associated with the vascular tissues in the stele, but less so in the resistant plants. The syncytia in the infected susceptible wheat and barley were also metabolically active at day 13. By contrast, the syncytia of resistant wheat plants carrying the Cre1 or Cre3 genes remained extensively vacuolated and less metabolically active. In barley plants with the Ha2 or Ha4 genes, the syncytia appeared non-functional and in early stages of degeneration by day 13 after inoculation.