大豆抗胞囊线虫病种质rhg1和Rhg4位点的单核苷酸多态性(SNPs)

本研究以57份中美大豆抗胞囊线虫病种质资源为实验材料,利用基于检测微珠的单碱基延伸方法,对与大豆胞囊线虫病(SCN)抗性基因rhg1和Rhg4紧密连锁的SNPs进行分析,目的是阐明我国大豆抗性种质在这两个位点的SNPs等位变异分布频率,为中国大豆种质抗SCN资源的利用奠定基础。分析结果表明,SNPs的抗性等位基因与中国大豆种质综合抗性的关系比不同生理小种的抗性关系更为密切。在rhg1和Rhg4位点,美国的9份抗性种质中,有7份抗性种质的SNPs均为纯合抗病基因型,而中国48份抗性种质中有32份。分别占鏊定总数的77．8％和66．7％,推测大豆抗SCN种质中,以rhg1和Rhg4这两个基因协同作用表现出的抗性可能占多数．但还存在其他的抗性机制。     

大豆的孢囊线虫抗性研究新进展

大豆孢囊线虫(Heterodera glycines Ichinohe,soybean cyst nematode,SCN)病害是大豆(Glycine max(L.)Merr.)生产上危害最严重的病害,每年造成巨大的经济损失.种植抗性大豆品种是防治SCN最经济、有效且对环境友好的措施.大豆对SCN的抗性受多基因位点控制.近年来,大豆的SCN抗性基因研究取得了突破性进展,几乎同时鉴定出了大豆的2个主要SCN抗性位点基因rhg1和Rhg4,并揭示了2种完全不同的植物抗病机制.rhg1采取的是一种由一段约31 kb长的基因组序列上的3个基因共同控制的多拷贝抗病机制,而Rhg4采取的是一种由丝氨酸羟甲基转移酶控制、可能由一碳代谢参与的抗病新机制.本文就近年来(2003年7月以来)在大豆的抗SCN位点的鉴定及新抗性种质资源挖掘、rhg1和Rhg4基因克隆与功能鉴定以及特异性分子标记开发与对SCN抗性的大豆资源品种的高通量筛选等研究方面取得的一些最新进展进行综述.     

大豆抗SCN3种质资源的创新

大豆孢囊线虫(SCN)是危害大豆的主要病害之一,它发生范围广、危害比较严重,培育抗病品种是目前最经济有效的控制措施.培育抗病品种首先需要筛选和鉴定抗源,得到优良抗源材料至关重要.为此,针对目前我国大豆抗SCN3种质资源存在的弱点问题进行创新研究.采用高抗大豆孢囊线虫病3号生理小种的抗源与当地优良品种进行杂交,对后代(F6)进行盆栽抗性筛选和田间丰产性能鉴定,从中鉴定出抗性强、综合农艺性状优良的创新种质资源,为今后抗线虫育种工作奠定基础,对加快育种进程、缩短育种年限具有重要作用.     

Chib1和PAL5基因在AM真菌Glomus fasciculatus诱导大豆抗线虫病害防御反应中的作用

本研究系统分析了大豆（品种：‘鲁豆4’）接种AM真菌Glomus fasciculatum和胞囊线虫（SCN,Heterodera glycines）4号生理小种后各处理菌根和线虫侵染率、几丁质酶和苯丙氨酸解氨酶（PAL）活性及几丁质酶基因Chib1和苯丙氨酸解氨酶基因PAL5转录物的动态变化。结果表明,接种SCN对AM真菌的侵染率没有产生显著影响,但先接种AM真菌后接种SCN的大豆根内线虫侵染率明显低于只接种SCN的处理。另外,先接种AM真菌后接种SCN的大豆根内几丁质酶和PAL活性显著提高,活性高峰出现在接种线虫后的第3天。值得注意的是,先接种AM真菌后接种SCN的大豆根内两种基因Chib1和PAL5转录物高峰也出现在接种SCN后的第3天,即AM真菌侵染率快速上升而SCN侵染率快速下降时期。所以Chib1和PAL5基因的表达可能是AM真菌诱导的抗大豆胞囊线虫病害防御反应的一种表现。因此推测Chib1和PAL5直接参与了AM真菌诱导大豆抗胞囊线虫病害的防御反应。     

Chib1和PAL5基因在AM真菌Glomus fasciculatus诱导大豆抗线虫病害防御反应中的作用

本研究系统分析了大豆(品种:‘鲁豆4’)接种AM真菌Glomusfasciculatum和胞囊线虫(SCN,Heteroderaglycines)4号生理小种后各处理菌根和线虫侵染率、几丁质酶和苯丙氨酸解氨酶(PAL)活性及几丁质酶基因Chib1和苯丙氨酸解氨酶基因PAL5转录物的动态变化。结果表明,接种SCN对AM真菌的侵染率没有产生显著影响,但先接种AM真菌后接种SCN的大豆根内线虫侵染率明显低于只接种SCN的处理。另外,先接种AM真菌后接种SCN的大豆根内几丁质酶和PAL活性显著提高,活性高峰出现在接种线虫后的第3天。值得注意的是,先接种AM真菌后接种SCN的大豆根内两种基因Chib1和PAL5转录物高峰也出现在接种SCN后的第3天,即AM真菌侵染率快速上升而SCN侵染率快速下降时期。所以Chib1和PAL5基因的表达可能是AM真菌诱导的抗大豆胞囊线虫病害防御反应的一种表现。因此推测Chib1和PAL5直接参与了AM真菌诱导大豆抗胞囊线虫病害的防御反应。     

AM真菌和胞囊线虫对大豆根内酶活性的影响

将‘鲁豆4号’大豆接种丛枝菌根(AM)真菌聚生球囊霉Glomus fasiculatum和大豆胞囊线虫(SCN)Heterodera glycines4号生理小种后,定期测定大豆根系中AM真菌及线虫侵染速率、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β—1,3葡聚糖酶及几丁质酶活性的动态变化。结果表明,接种AM真菌大豆根系中4种酶活性高于对照水平：先接种AM真菌后接种SCN处理根系中POD、PAL及几丁质酶的活性高于只接种SCN的处理,并且酶活性峰值出现的时间均早于或相当于后者。另外,PAL及几丁质酶活性出现高峰时期也正是AM真菌侵染率迅速升高及线虫侵染速率快速下降期。因此,AM真菌先激活了大豆的防御反应,然后使其对SCN的侵染产生快速反应,PAL及几丁质酶在AM真菌诱导的抗、耐线虫病害机制中起重要作用。值得注意的是,先接种AM真菌后接种SCN处理大豆根系中,β—1,3葡聚糖酶活性低于只接种AM真菌的处理。作者认为本试验条件下,该酶在大豆抗SCN病害中的作用表现不明显。     

AM真菌和胞囊线虫对大豆根内酶活性的影响*

将‘鲁豆4号’大豆接种丛枝菌根(AM)真菌聚生球囊霉Glomus fasiculatum和大豆胞囊线虫(SCN)Heterodera glycines 4号生理小种后, 定期测定大豆根系中AM真菌及线虫侵染速率、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3葡聚糖酶及几丁质酶活性的动态变化。结果表明, 接种AM真菌大豆根系中4种酶活性高于对照水平; 先接种AM真菌后接种SCN处理根系中POD、PAL及几丁质酶的活性高于只接种SCN的处理,并且酶活性峰值出现的时间均早于或相当于后者。另外,PAL及几丁质酶活性出现高峰时期也正是AM真菌侵染率迅速升高及线虫侵染速率快速下降期。因此,AM真菌先激活了大豆的防御反应,然后使其对SCN的侵染产生快速反应,PAL及几丁质酶在AM真菌诱导的抗、耐线虫病害机制中起重要作用。值得注意的是,先接种AM真菌后接种SCN处理大豆根系中,β-1,3葡聚糖酶活性低于只接种AM真菌的处理。作者认为本试验条件下,该酶在大豆抗SCN病害中的作用表现不明显。     

AM真菌和胞囊线虫对大豆根内酶活性的影响

将‘鲁豆4号’大豆接种丛枝菌根(AM)真菌聚生球囊霉Glomus fasiculatum和大豆胞囊线虫(SCN)Heterodera glycines 4号生理小种后, 定期测定大豆根系中AM真菌及线虫侵染速率、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3葡聚糖酶及几丁质酶活性的动态变化。结果表明, 接种AM真菌大豆根系中4种酶活性高于对照水平; 先接种AM真菌后接种SCN处理根系中POD、PAL及几丁质酶的活性高于只接种SCN的处理,并且酶活性峰值出现的时间均早于或相当于后者。另外,PAL及几丁质酶活性出现高峰时期也正是AM真菌侵染率迅速升高及线虫侵染速率快速下降期。因此,AM真菌先激活了大豆的防御反应,然后使其对SCN的侵染产生快速反应,PAL及几丁质酶在AM真菌诱导的抗、耐线虫病害机制中起重要作用。值得注意的是,先接种AM真菌后接种SCN处理大豆根系中,β-1,3葡聚糖酶活性低于只接种AM真菌的处理。作者认为本试验条件下,该酶在大豆抗SCN病害中的作用表现不明显。     

大豆胞囊线虫抗性基因定位与克隆研究进展

大豆胞囊线虫（soybean cyst nematode,SCN）是大豆生产上一种危害严重的世界性害虫,能给大豆生产造成极大损失。大豆抗性品种选育是防治其措施中最经济、有效的方法。大豆SCN抗性的分子遗传学研究是开展大豆SCN抗性分子育种的理论基础,本文针对SCN抗性基因定位和克隆两个方面的研究现状进行了综述,并对当前研究中存在的问题及发展前景进行了讨论与展望。     

大豆胞囊线虫抗性基因定位与克隆研究进展

大豆胞囊线虫(soybean cyst nematode, SCN)是大豆生产上一种危害严重的世界性害虫, 能给大豆生产造成极大损失。大豆抗性品种选育是防治其措施中最经济、有效的方法。大豆SCN抗性的分子遗传学研究是开展大豆SCN抗性分子育种的理论基础, 本文针对SCN抗性基因定位和克隆两个方面的研究现状进行了综述, 并对当前研究中存在的问题及发展前景进行了讨论与展望。     

Rhg1 alleles from soybean PI 437654 and PI 88788 respond differentially to isolates of Heterodera glycines in the greenhouse

The production of resistant soybean [Glycine max (L.) Merr.] cultivars is the most effective means for controlling losses from soybean cyst nematode (SCN) (Heterodera glycines Ichinohe). The major resistance gene in most SCN resistance sources is rhg1, which has been mapped as a quantitative trait locus onto linkage group G. Our objective was to determine whether the SCN resistance sources PI 437654 and PI 88788 have different functional alleles at rhg1 based on resistance phenotypes. Populations segregating for resistance alleles at rhg1 from both PI 88788 and PI 437654 and at Rhg4, a second SCN resistance gene from PI 437654, were developed. These populations were screened for resistance to the H. glycines inbred isolates PA3 (HG type 7) and TN14 (HG type 1.2.5.7) in the greenhouse and evaluated with molecular markers linked to both rhg1 and Rhg4. Each isolate test was repeated, and the evaluations were done on a single-plant and a line-mean basis in Test 1, and solely on a single-plant basis in Test 2. Across two tests with the TN14 isolate, plants with the PI 437654 allele for a marker linked to rhg1 had significantly (P<0.0001) less SCN reproduction than plants carrying the PI 88788 allele. A marker linked to Rhg4, however, was not significantly associated with resistance to TN14. Across two tests with the PA3 isolate, alleles of rhg1 from both sources gave a resistant reaction, although plants homozygous for the PI 88788 allele had significantly (P<0.05) greater resistance than plants with the PI 437654 allele. The marker allele from PI 437654 linked to Rhg4 was significantly (P<0.0005) associated with greater resistance than the PI 88788 allele in both PA3 tests, and resistance was dominant. There was a significant interaction between alleles at rhg1 and Rhg4 in both PA3 tests. These results suggest that PI 437654 and PI 88788 each have a different functional SCN resistance allele at or close to rhg1. These allelic differences have implications that breeders should consider before incorporation into cultivars.     

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     

Evolution and selection of Rhg1, a copy‐number variant nematode‐resistance locus

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2‐kb unit contains four genes. One allele of Rhg1, Rhg1‐b, is responsible for protecting most US soybean production from SCN. Whole‐genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2‐kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high‐density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non‐neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1.     

Genome-wide association study for soybean cyst nematode resistance in Chinese elite soybean cultivars

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a highly recalcitrant endoparasite of soybean roots, causing more yield loss than any other pest. To identify quantitative trait loci (QTL) controlling resistance to SCN (HG type 2.5.7, race 1), a genome-wide association study (GWAS) was performed. The association panel, consisting of 120 Chinese soybean cultivars, was genotyped with 7189 single nucleotide polymorphism (SNPs). A total of 6204 SNPs with minor allele frequency >0.05 were used to estimate linkage disequilibrium (LD) and population structure. The mean level of LD measured by r ² declined very rapidly to half its maximum value (0.51) at 220 kb. The overall population structure was approximately coincident with geographic origin. The GWAS results identified 13 SNPs in 7 different genomic regions significantly associated with SCN resistance. Of these, three SNPs were localized in previously mapped QTL intervals, including rhg1 and Rhg4. The GWAS results also detected 10 SNPs in 5 different genomic regions associated with SCN resistance. The identified loci explained an average of 95.5% of the phenotypic variance. The proportion of phenotypic variance was due to additive genetic variance of the validated SNPs. The present study identified multiple new loci and refined chromosomal regions of known loci associated with SCN resistance. The loci and trait-associated SNPs identified in this study can be used for developing soybean cultivars with durable resistance against SCN.     

An efficient method for measuring copy number variation applied to improvement of nematode resistance in soybean

Copy number variation (CNV) is implicated in important traits in multiple crop plants, but can be challenging to genotype using conventional methods. The Rhg1 locus of soybean, which confers resistance to soybean cyst nematode (SCN), is a CNV of multiple 31.2‐kb genomic units each containing four genes. Reliable, high‐throughput methods to quantify Rhg1 and other CNVs for selective breeding were developed. The CNV genotyping assay described here uses a homeologous gene copy within the paleopolyploid soybean genome to provide the internal control for a single‐tube TaqMan copy number assay. Using this assay, CNV in breeding populations can be tracked with high precision. We also show that extensive CNV exists within Fayette, a released, inbred SCN‐resistant soybean cultivar with a high copy number at Rhg1 derived from a single donor parent. Copy number at Rhg1 is therefore unstable within a released variety over a relatively small number of generations. Using this assay to select for individuals with altered copy number, plants were obtained with both increased copy number and increased SCN resistance relative to control plants. Thus, CNV genotyping technologies can be used as a new type of marker‐assisted selection to select for desirable traits in breeding populations, and to control for undesirable variation within cultivars.     

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

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

Resistance Genes in a 'Williams 82' x 'Hartwig' Soybean Cross to an Inbred Line of Heterodera glycines

The number of resistance genes in soybean to soybean cyst nematode (SCN) Heterodera glycines was estimated using progeny from a cross of ''Williams 82'' x ''Hartwig'' (derived from ''Forrest''³ x PI 437.654) screened with a fourth-generation inbred nematode line derived from a race 3 field population of SCN. Numbers of females developing on roots of inoculated seedlings were assigned to phenotype cells (resistant, susceptible, or segregating) using Ward''s minimum variance cluster analysis. The ratio obtained from screening 220 F₃ soybean families was not significantly different from a 1:8:7 (resistant:segregating:susceptible) ratio, suggesting a two-gene system for resistance. The ratio obtained from screening 183 F₂ plants was not significantly different from a 3:13 (resistant:susceptible) ratio, indicating both a dominant (Rhg) and a recessive (rhg) resistance gene.