QTL mapping of qSCN3-1 for resistance to soybean cyst nematode in soybean line Zhongpin 03-5373

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is one of the most economically destructive pathogens.The soybean line Zhongpin03-5373(ZP),which combines resistance genes from several donors,is highly resistant to SCN race 3(SCN3).In our previous study,two QTL(rhg1 and GmSNAP11)were identified in a population of recombinant inbred lines derived from a cross between ZP and the susceptible parent Zhonghuang 13.The two QTL explained around one-third of the resistance,suggesting the presence of further QTL contributing to SCN resistance.In the present study,we used an improved version of the geneticmap comprising the previously applied 1062 molecular markers and 47 newly developed InDel(insertion-deletion)markers.The improved map revealed a novel locus contributing to SCN3 resistance:qSCN3-1,flanked by InDelmarker InDel1-7 and SNPmarker Map-0047,explained 4.55%of the phenotypic variance for resistance to SCN3 and was not involved in digenic epistatic interaction with rhg1 and GmSNAP11.Haplotypes of Map-0047_CAPS(a CAPS marker developed for Map-0047)and InDel1-7 were significantly associated with SCN3 resistance in a panel of 209 resistant and susceptible accessions.Using further allele-combination analysis for three functional markers representing three cloned resistance genes(rhg1,Rhg4,andGmSNAP11)and twomarkers flanking qSCN3-1,we found that adding the resistance allele of qSCN3-1 greatly increased soybean resistance to SCN,even in diverse genetic backgrounds.The qSCN3-1 locus will be useful for marker-assisted polygene pyramid breeding and should be targeted for the future identification of candidate genes.

Research Advances in Genetic Markers for Resistance to Soybean Cyst Nematodes(SCN)

Soybean cyst nematode(SCN), Heterodera glycine, is one of the important diseases affecting the yield of soybean(Glycine max) worldwide. Host-plant resistance is the most economically effective way to control SCN. Identification of DNA markers linked to SCN resistance genes is one of the important steps in the marker-assisted selection(MAS) for nematode resistance breeding. Quantitative trait loci(QTLs) controlled by mul-tiple resistance genes plays a powerful role in MAS-breeding program. In this paper, we reviewed genetic resistance of SCN, molecular markers of resistance gene and Genome-Wide Associated Study(GWAS) of QTLs linked to SCN resistance, explored the present problems of genetic resistance,and prospected the future of QTL analysis.

Identification of Endophytic Bacillus BHL3501 from Wild Soybean and Its Inhibitory Effect against Soybean Cyst Nematode (Heterodera glycines Ichinohe)

[ Objective] The paper was to identify endophytic bacillus BHL3501 from wild soybean and its inhibitory effect against soybean cyst nematode. [ Meth- od] Through morphological characteristic, physiological and biochemical characteristics and 16S rDNA analysis, a strain of endophytic bacterium BHL3501 with ne- maticidal activity isolated and screened from the root of wild soybean was identified, the effects of BHL3501 metabolite solutions with different dilution multiples on egg hatching and activity of 2rd instar juvenile of soybean cyst nematode were also studied. [ Result ] BHL3501 strain was preliminarily identified to be Bacillus sp.. The relative inhibitory rate of its original fermentation broth on egg hatching of soybean cyst nematode after 24 h was 98.3% ; the relative inhibitory rate under 10- time diluted solution treatment was 79.5%, which had significant difference with sterile water control. The corrected mortality rate of 24 instar juvenile was 93.1% after treated by original fermentation broth for 24 h, the treatments of all diluted solutions had significant difference with sterile water control. [ Conclusion ] BHL3501 metabolites had strong inhibitory effect against egg hatching of soybean cyst nematode, which also had strong toxic effect on its 2nd instar juvenile.

Effects of Abiotic Environmental Factors on Soybean Cyst Nematode

As a pest, in order to complete its life history and reproduces effectively, soybean cyst nematode （SCN） （Heterodera glycines Ichinche 1952） must adapt to various environments and conditions for long periods of evolution. The nematode is widely dispersed year after year. Controlling this pest requires understanding characters and adaptability of SCN. Effects of abiotic factors, such as temperature, soil humidity, agrotype, pH value, ions, plant exudates, agricultural chemical and cultivation systems on SCN, are reviewed in this paper. The results show that SCN is able to endure various environmental stresses, especially low temperature. Because of its special life history, cyst stage help SCN over winter, resistance of SCN to environmental stress is strong. A few studies have reported the mechanism of SCN environmental adaptability. We emphasized the importance of studying environmental adaptability of SCN, which would benefit the control of SCN by ecological means.

Comparative Analysis of Gene Expression Profiling Between Resistant and Susceptible Varieties Infected With Soybean Cyst Nematode Race 4 in Glycine max

Soybean cyst nematode （SCN） is one of the most devastating pathogen for soybean. Therefore, identiifcation of resistant germplasm resources and resistant genes is needed to improve SCN resistance for soybean. Soybean varieties Huipizhiheidou and Wuzhaiheidou were distributed in China and exhibited broad spectrums of resistance to various SCN races. In this study, these two resistant varieties, combined with standard susceptible varieties （Lee and Essex）, were utilized to identify the differentially expressed transcripts after infection with SCN race 4 between resistant and susceptible reactions by using the Affymetrix Soybean Genome GeneChip. Comparative analyses indicated that 21 common genes changed signiifcantly in the resistant group, of which 16 increased and 5 decreased. However, 12 common genes changed signiifcantly in the susceptible group, of which 9 increased and 3 decreased. Additionally, 27 genes were found in common between resistant and susceptible reactions. The 21 signiifcantly changed genes in resistant reaction were associated with disease and defense, cell structure, transcription, metabolism, and signal transduction. The fold induction of 4 from the 21 genes was conifrmed by quantitative RT-PCR （qRT-PCR） analysis. Moreover, the gene ontology （GO） enrichment analyses demonstrated the serine family amino acid metabolic process and arginine metabolic process may play important roles in SCN resistance. This study provided a new insight on the genetic basis of soybean resistance to SCN race 4, and the identiifed resistant or resistant-related genes are potentially useful for SCN-resistance breeding in soybean.

Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode

The resistance of soybean(Glycine max(L.) Merr.) to soybean cyst nematode(SCN, Heterodera glycines Ichinohe), which is a devastating pathogen in soybean production and causes a large quantity of annual yield loss worldwide, can shift during the long-term interaction and domestication. It is vital to identify more new resistance genetic sources for identification of novel genes underlying resistance to SCN for management of this pathogen. In the present study, first, two ethane methylsulfonate-mutagenesis soybean M2 populations of PI 437654, which shows a broad resistance to almost all of SCN races, and Zhonghuang 13, which is a soybean cultivar in China conferring strong resistance to lodging, were developed. Many types of morphological phenotypes such as four-and five-leaflet leaves were observed from these two soybean M2 populations. Second, 13 mutants were identified and confirmed to exhibit alteration of resistance to SCN race 4 through the forward genetic screening of 400 mutants of the PI 437654 M2 population, the rate of mutants with alteration of SCNinfection phenotype is 3.25%. Third, these identified mutants were further verified not to show any changes in the genomic sequences of the three known SCN-resistant genes, GmSHMT08, GmSNAP18 and GmSANP11, compared to the wildtype soybean; and all of them were still resistant to SCN race 3 similar to the wild-type soybean. Taken together, we can conclude that the 13 mutants identified in the present study carry the mutations of the new gene(s) which contribute(s) to the resistance to SCN race 4 in PI 437654 and can be potentially used as the genetic soybean sources to further identify the novel SCN-resistant gene(s).

Sampling Survey and Identification of Races of Soybean Cyst Nematode (Heterodera glycines Ichinohe) in Huang-Huai Valleys

Soybean cyst nematode （SCN Heterodera glycines Ichinohe） is one of the most important nationwide soybean diseases in China. A total of 38 soil specimens or locations in the area was sampled and tested for SCN races during 2001-2003 for the inspection of race distribution in Huang-Huai Valleys. A map of race distribution was constructed according to the data from both the present study and the published reports cited. Three areas, namely, the area of southeast to Jinan in Shangdong Province; the area of northern Henan Province and its border region to south of Hebei Province; and the area of Luohe, Zhoukou of Henan Province and Fuyang of Anhui Province mainly infested with Race 1 were identified. Race 4 was predominant in Shanxi Province, Beijing and the adjacent area of Henan, Shandong, and Anhui provinces, and the delta of Huanghe River in Shandong Province. Race 2 was mainly found in Liaocheng, Dezhou of Shangdong Province and Shijiazhuang of Hebei Province, and Jiaozuo and Huojia of Henan Province. Race 7 was distributed in the west part of Jiaodong Peninsula of Shandong Province and Kaifeng, Huaxian, Wenxian of Henan Province. Race 5 was found and scattered in Hebei and Henan Province. Race 9 was found in Shangqiu of Henan Province, which was reported for the first time in China. It can be seen that Race 1 and Race 4 were the two predominant races in Huang-Huai Valleys, and that research should focus on developing resistant cultivars of these races. There might exist other races in an area with some predominant races. The race substitution in the past decade was not obviously found, therefore, the results should be meaningful to future breeding for resistance to SCN in Huang-Huai Valleys.

Soybean cyst nematode-resistance: Gene identification and breeding strategies

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is the most economically damaging disease of soybean worldwide,and breeding host plant resistance is the most feasible option for SCN management.In this review,we summarise the progress made so far in identifying nematode-resistance genes,the currently available sources of resistance,possible mechanisms of SCN resistance and strategies for soybean breeding.To date,only two sources of SCN resistance have been widely used,from the accessions PI 88788 and Peking,which has resulted in a shift in SCN resistance and created a narrow genetic base for SCN resistance.These resistant germplasms for SCN are classified into two types according to their copy number variation in a 31-kb genomic region:PI 88788-type resistance requires high copy numbers of a rhg1 resistance allele(rhg1-b)and Peking-type resistance requires both low copy numbers of a different rhg1 resistance allele(rhg1-a)and a resistant allele at another locus,Rhg4.Resistance related to rhg1 primarily involves impairment of vesicle trafficking through disruption of soluble NSF-attachment protein receptor(SNARE)complexes.By contrast,resistance via Rhg4 involves disturbance of folate homeostasis at SCN feeding sites due to alteration of the enzymatic activity of serine hydroxymethyltransferase(SHMT).Other potential mechanisms,including plant defences mediated by salicylic acid(SA)and jasmonic acid(JA)signalling modulation,have also been suggested for SCN resistance.Indeed,genome-wide association studies(GWAS)have identified other candidate SCN resistance genes,such as Gm SNAP11.Although gene functional analysis in a transient expression system could increase the efficiency of candidate gene identification,information on novel genes and mechanisms for SCN resistance remains limited.Any beneficial candidate genes identified might,when fully exploited,be valuable for improving the efficiency of marker-assisted breeding and dissecting the molecular mechanisms underlying SCN resistance.

Breeding Soybeans for Resistance to Physiological Race 4 of Cyst Nematode

Soybean cyst nematode causes serious damage to soybean production. In 1991, we started breeding studies on the resistance of soybeans to the cyst nematode. We found that near the Beijing area the dominant race of the cyst nematode was race 4. We made more than 50 combinations of cross. The best combination was Dan 8 X PI 437654 which resulted in marked segregation in plant height, pod habit, resistance to cyst nematode and maturity. We obtained many new soybean lines highly resistant to the cyst nematode through the pedigree method of selection, enlarging the number of plants of good combinations, alternative breeding in the North and in the South, and identification at an early generation. We now have released three soybean cultivars, Zhonghuang 12, Zhonghuang 13 and Zhonghuang 17 with moderate resistance to the cyst nematode in Beijing, Anhui, Tianjin and Northern China. In addition, we obtained many lines which were highly resistant to the cyst nematode.

Molecular characterization and functional analysis of two new lysozyme genes from soybean cyst nematode(Heterodera glycines)

Soybean cyst nematode(SCN, Heterodera glycines(I.)) is one of the most important soil-borne pathogens for soybeans. In plant parasitic nematodes, including SCN, lysozyme plays important roles in the innate defense system. In this study, two new lysozyme genes(Hg-lys1 and Hg-lys2) from SCN were cloned and characterized. The in situ hybridization analyses indicated that the transcripts of both Hg-lys1 and Hg-lys2 accumulated in the intestine of SCN. The q RT-PCR analyses showed that both Hg-lys1 and Hg-lys2 were upregulated after SCN second stage juveniles(J2 s) were exposed to the Grampositive bacteria Bacillus thuringiensis, Bacillus subtilis or Staphylococcus aureus. Knockdown of the identified lysozyme genes by in vitro RNA interference caused a significant decrease in the survival rate of SCN. All of the obtained results indicate that lysozyme is very important in the defense system and survival of SCN.

A fragment of a 70-kDa Heterodera glycines heat shock protein(HgHSP70)interacts with soybean cyst nematode-resistant GmSHMT08

Soybean cyst nematode(SCN)Heterodera glycines is considered as the major constraint to soybean production.Gm SHMT08 at Rhg4 locus on chromosome 08,encoding a serine hydroxylmethyltransferase,is a major gene underlying resistance against H.glycines in Peking-type soybeans.However,the molecular mechanism underpinning this resistance is less well characterized,and whether Gm SHMT08 could interact with proteins in H.glycines remains unclear.In this study,yeast two-hybrid screening was conducted using Gm SHMT08 as a bait protein,and a fragment of a 70-kDa heat shock protein(Hg HSP70)was screened from H.glycines that exhibited interaction with Gm SHMT08.This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays.Our finding reveals Hg HSP70 could be applied as a potential candidate gene for further exploring the mechanism on Gm SHMT08-mediated resistance against SCN H.glycines.

Comparative Map-based Cloning for Genes Conferring Resistance to Soybean Cyst Nematode

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most dangerous disease to cause significant yield losses. Most recent estimates for SCN indicate losses of nearly 9 million metric tons worldwide in

RNA-seq analysis for soybean cyst nematode resistance of Glycine soja(wild soybean)

To explore genetic resource of wild soybean(Glycine soia. L), RNA-seq was used to investigate cyst nematode resistance of G. soja. Root transcriptome expressions were profiled at 9, 15 and 20 d post inoculation(DPI) in resistant and susceptible G. soja to SCN(soybean cyst nematode). A total of 1,594 differentially expressed genes(DEGs) were identified in roots infected by SCN compared with non-infected roots. In the resistant accession, 619, 65, and 8 DEGs were detected at 9, 15, and 20 DPI, respectively, while 327, 460 and 115 DEGs were detected at the same sampling point of susceptible accessions. DEGs were enriched in peroxidase gene sets which were involved in response to oxidative stress and oxidation reduction. Two gene families, ZIM transcription factor and WRKY transcription factor were enriched. WRKY transcription factor was only enriched in resistant accession. Moreover, gene expressions of 9 DEGs were validated by qRT-PCR. XLOC_023202, an unknown protein was up regulated more than 5 fold at 9 and 15 DPI in the resistant accession. These results provided an atlas of gene expressions of G. soja in response to SCN infection, and identified candidate DEGs for future research.

国家大豆区域试验品种对SMV和SCN的抗性分析

人工接种我国黄淮、长江流域及南方大豆产区流行SMV株系SC3、SC7和SC15、SC18及黄淮海流行SCN1号生理小种，对国内主要育种单位近年来选育的参加2010—2012国家大豆新品种区域试验的122个参试品种进行抗性鉴定和评价。结果表明：对SC3表现较好抗性（高抗、抗病）的品种32个，占接种该株系品种总数的32％，对SC7表现较好抗性的品种34个，占接种该株系品种总数的34％，同时对SC3和SC7表现较好抗性的品种22个，占接种2个株系品种总数的22％；对SC15表现较好抗性的品种3个，占接种该株系品种总数的13．6％，对SC18表现较好抗性的品种3个，占接种该株系品种总数的13．6％，同时对SC15和SC18表现较好抗性品种的2个，占接种2个株系品种总数的9．09％；近10年SMV抗性鉴定结果分析表明，我国抗SMV育种水平有所提高，新培育的中抗品种比率显著增加，感病品种比率下降。SCN抗性鉴定结果显示，仅沧豆11为高抗，高感品种34个，分别占参试品种总数的1．96％和66．67％，其余为中间型（中抗和中感）。近10年SCN抗性鉴定结果显示我国抗SCN育种水平没有明显改善。鉴定出鲁98011-14、沧豆11、开豆41、鲁97013-1兼抗SMV和SCN两种病害。

大豆抗SCN_3过程中总酚含量动态分析

大豆孢囊线虫 (SCN)是大豆生产上的毁灭性病害之一 ,土壤一经感染 ,则极难防治。利用植物本身的抗性 ,培育抗病品种是目前采用的最经济有效的控制措施。培育抗病品种首先要筛选和鉴定抗源材料。本研究采用抗感大豆孢囊线虫的野生、半野生、栽培大豆在接种和未接种大豆孢囊线虫 3号生理小种条件下总酚含量动态变化以揭示其抗SCN3的生化机制。初步探讨了在SCN3侵染过程中 ,抗源生化指标总酚的表达情况 ,整个侵染期动态变化以及其与抗性关系 ,目的在于揭示抗源品种抗病的内在规律。从而为抗SCN3资源的筛选和鉴定及抗SCN3育种提供理论依据 ,为研究抗SCN3的遗传提供参考。试验结果表明 ,总酚可以作为鉴定SCN3生化指标之一。

新育成大豆品种对SMV和SCN的抗性评价

存接种我国大豆产区主要流行SMV株系SC-3及SC-7和SCN1号生理小种的条件下，对新育成的参加2004～2007年国家及江苏、北京、山东等省市大豆区试的品种分别进行了抗性评价。结果表明：在抗SMV鉴定的334个品种中，对SC-3抗性较好（高抗和抗病）的品种数有148个，占参试品种数的44．31％；对SC-7抗性较好的有71个，占参试品种数的21．26％。同时对2个株系抗性表现较好的有55个，占参试品种数的16．47％。这些抗性较好的品种既可用于大豆生产，也可作为抗源用于抗病品种选育和与抗性相关的研究。研究还显示，来自于西北和黄淮海大豆产区的参试品种一般抗性较好。抗SCN鉴定的193个大豆品种中，未发现高抗品种，中抗品种有25个，占12．92％。汾9877-10、邯601、蒙9793—1、沧豆九号等7个品种兼抗SMV和SCN2种病害。

大豆抗源抗SCN过程中POD酶动态分析

通过对大豆抗源抗孢囊线虫（ＳＣＮ）过程中过氧化物酶（ＰＯＤ）活性和酶谱的变化研究，探讨了大豆抗源在抗病过程中是通过提高体内ＰＯＤ酶活性来抵御ＳＣＮ的侵染，抗源品种ＰＯＤ活性和酶谱呈动态变化。在侵染高峰期（３０～３５ｄ），高抗抗原体内ＰＯＤ酶活性最强，酶谱着色较深；而感病品种在侵染高峰期ＰＯＤ酶活性降至最低值，且一些酶带受损以至消失。另外，证实了ＰＯＤ酶在抗病过程中参与木质素的合成。抗病过程中酶活性强弱及酶谱表达形式可作为大豆抗源抗性强弱的生理指标。

大豆抗感SCN基因的一个RAPD标记

对组合ＲＮｇ（抗）× ７７０５（感）的Ｐ１、Ｐ２、Ｆ１、Ｆ２、ＢＣ１Ｆ１和ＢＣ１Ｆ２分离群体进行抗病性鉴定，遗传分析的结果表明，大豆对ＳＣＮ１号小种的抗性由４对相互独立的主基因、包括一对显性基因和三对重叠的隐性基因决定。用 ＢＳＡ法筛选在五个 ＢＣ１Ｆ２分离家系内具有多态性的 ＲＡＰＤ标记，发现 ＲＡＰＤ标记 ＯＰＡ０１９１２００在亲本间及 ＢＣ１Ｆ２分离群体内具有多态性，只能在感亲、 ＢＣ１Ｆ２家系 １１和 ４３的感病单株中扩增出来，表明和这两个家系独有的感病基因有关。

大豆抗SCN机制及抗病相关基因研究进展

大豆胞囊线虫(soybean cyst nematode,SCN)引起的病害能给大豆生产造成极大损失。目前在SCN抗性机制研究方面的结果表明,大豆通过分泌一些小分子物质,如防御酶系、酚类代谢物质、植保素等来抑制SCN的发育,是大豆对SCN产生抗病性的一种抗性机制;在抗病相关基因研究方面,大豆中的rhg1和rhg4位点是SCN病最重要的抗性位点,SCN中的果胶酸裂解酶(pel)、抗甜菜线虫同系物GmHs1pro-1、效应蛋白30C02、受体激酶GmRLK18-1是SCN病的抗性的关键基因和蛋白。本文针对大豆抗SCN机制研究及抗病相关基因克隆研究进展进行综述,以期为SCN的抗病分子育种研究提供参考。

不同大豆品种与SCN 3号生理小种互作效应研究

在田间研究了不同大豆品种对大豆胞囊线虫(soybean cyst nem atode,SCN)的发育及繁殖的影响不同。结果表明,辽K89102为抗侵入型品种,Peking、PI90763、应县小黑豆、磨石豆和Franklin等的根内线虫从J2向J3或J3向J4发育过程中受到抑制,这些品种均有不同程度的抗线虫发育特性;不同品种对其根内SCN的雌雄分化有较大影响,感病品种根内SCN雌雄比远大于抗病品种,辽豆10根内线虫雌雄比例最高,为11.5∶1.0,Peking根内线虫雌雄比例最低,为0.1∶1.0;不同品种根围SCN最终群体量有较大差异,辽豆10根围土壤中胞囊量增加了331.5%,Peking和磨石豆的增幅最小,为16.7%。SCN侵染后,不同品种产生的组织病理学反应不同,抗病品种产生明显的组织病理学反应,表现在品种Peking和PI437654的根内线虫虫体附近根组织细胞有坏死现象,而感病品种辽豆10和开育10则没有坏死现象。