Abdulwhb S. Shibu, Bin Li*, Shengrui ZhngJunming Sun*

aThe National Engineering Laboratory for Crop Molecular Breeding/Ministry of Agriculture and Rural Affairs Key Laboratory of Soybean Biology(Beijing),Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

bDepartment of Agronomy,Bayero University,Kano,Nigeria

Keywords:Soybean cyst nematode (Heterodera glycines Ichinohe)Candidate gene Functional analysis Marker-assisted selection Resistance

A B S T R A C T 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 GmSNAP11. 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.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 893

2. The life cycle of soybean cyst nematode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 893

3. SCN population and race . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 893

4. Distribution of SCN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894

5. Management of SCN in soybean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894

6. Cytological and histological process of syncytium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894

7. Germplasm screening and sources of SCN resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895

8. Identification of SCN-resistance QTL and genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895

9. Functional gene analysis for SCN resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 898

10. Molecular mechanisms of SCN resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899

11. Strategies for soybean breeding for resistance to SCN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899

12. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900

Declaration of competing interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901

Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901

1. Introduction

Soybean(Glycine max L.Merrill)is a widely used legume crop accounting for about 70% of total protein meal and 28% of total vegetable oil consumption worldwide (www.soystats.com, 2019). It also serves as a source of fuel. The USA produces the highest percentage (34%) of the world’s soybeans annually, followed by Brazil (32%), Argentina(15%), and China (4%) (www.soystats.com, 2019). Soybean average yield in the USA increased annually by 22 kg ha?1from 1924(740 kg ha?1)to 2013(2959 kg ha?1)[1]and rose to 3500 kg ha?1in 2018 (www.soystats.com, 2019). Biotic and abiotic constraints are the two major challenges limiting soybean production. Management practices can prevent some abiotic stresses,but most,such as drought and floods,cannot be easily controlled [2]. Biotic stresses, meanwhile,have negative effects on the growth and development of soybean that often result in significant yield losses and cannot be easily controlled using management practices.The use of fertilizers and pesticides can reduce the incidence of biotic stresses; however, host plant resistance/tolerance is a more promising alternative for farmers.

The soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is an economically important pest of soybean causing estimated annual losses valued at billions of dollars worldwide [3]. The damage is often devastating because aboveground symptoms are not always visible and infestations are usually recognized at the later stage of infection when a significant proportion of the damage has already occurred and the nematode is difficult to eradicate;as a result,yields are significantly reduced due to inhibited root growth and reduced nodule formation [4,5]. The use of resistant varieties and crop rotation practices are the most effective strategies for controlling SCN[4].

This review assesses the trends and progress in the identification of nematode-resistance genes, available sources of resistance, and mechanisms underlying SCN resistance. Management strategies for SCN resistance and gene function analysis are also discussed.

2. The life cycle of soybean cyst nematode

The life cycle of SCN can be separated into four juvenile stages and one adult stage. Development from one stage to another usually begins in the egg and is interposed by moulting. SCN at the second-stage juvenile(J2)stage penetrates into the root of the host plant and migrates until it reaches the vascular cylinder. The J2 nematode initiates the formation of a permanent feeding cell (syncytium) and becomes sedentary[5]. The syncytium provides the nutrients for growth and development of SCN and is metabolically active as well as important for a complete life cycle. Sexual dimorphism usually becomes prominent in the third stage (J3) before the nematode moves to the fourth stage (J4). The adult females are visible on the host root surface because of their large size.The vermiform male migrates out of the root to fertilize the female before dying. The cyst is formed from dead females and protects the eggs in the soil for many years when there is no available host. In the presence of a favourable host, the eggs hatch and the life cycle continues [5]. The life cycle of SCN is usually completed in three to four weeks depending on nutrient availability, soil temperature, and location. Because SCN requires a living host to complete its life cycle, it is classified as a biotrophic pathogen.

3. SCN population and race

The abilities of SCN populations to develop on resistant soybean varieties are diverse because of differences in generation time,fecundity, and extent of host damage [5]. The race description published in 1970, which was based on the ability of SCN to reproduce on four indicator lines(Pickett,Peking,PI 88788,and PI 90763), is still widely used for SCN classification [6]. When the number of nematodes produced on a particular line is equal to or greater than 10% of the number produced on cultivar Lee(susceptible to SCN), the race is designated “+”, whereas when the number is less than 10%, the race is designated “–” [7].However, the race scheme has faced various criticisms [8]. For one thing, according to that scheme, the virulence of a population on one differential line is linked to virulence on another, a situation that is impossible to test because of the obligate parasitic nature of SCN [5]. Another SCN classification scheme, the HG (Heterodera glycines) type classification scheme, was later developed to circumvent the challenges posed by the race scheme description. The HG type classification is based on the ability of SCN to reproduce on a larger and more diverse set of soybean indicator lines(seven), which also aids in management decisions for SCN[8]. The HG scheme allows the addition of new soybean lines and is flexible for different environments.

4. Distribution of SCN

Soybean cyst nematode is believed to have evolved in either China or Japan before spreading to other parts of the world[9].Riggs and Schmitt [7] reported 16 races of SCN, the first identified in 1954; a new race was also recently reported in the Shanxi province of China [10]. H. glycine is widespread in many countries, including the USA and China, where soybeans are grown on a large commercial scale. In the USA, Hg Type 0 or 7 (race 3) and Hg Type 1.2.5– (races 4 and 14) are primarily found in latitudes south and north of 37°N,respectively[11].In China,eight races of SCN are predominant(races 1, 2, 3, 4, 5, 6, 9, and 14) [9]. Hg Type 0 is the most common in the northeastern provinces of China (north of 41°N latitude), while Hg Type 1.2.5– is one of the two most common races in the Huang-Huai-Hai valley region (between 32°N and 41°N latitude) [9,12].

Until recently, race 4 was reported to be the most virulent race in China,but the new race 12 has recently been reported to be more virulent than race 4 [10]; however, so far it is not known to have spread broadly across China, having been reported only in Shanxi province.

5. Management of SCN in soybean

Soybean cyst nematode causes the most substantial yield reduction of any soybean disease or pest [13]. Damage by SCN to soybean plants is reported to have a positive association with the initial numbers of SCN present in the soil. Reducing the initial numbers and preventing the spread of SCN to noninfected areas are important aspects of its management that can minimize yield loss[13].Tillage and removal of root and soil debris from farm implements can also help reduce SCN numbers and its transfer to non-infested areas. Faghihi [14]reported that a one-year rotation with non-host crops(such as alfalfa [Medicago sativa], corn [Zea mays], sorghum [Sorghum bicolor], barley [Hordeum vulgare], and oat [Avena sativa]) in the absence of weeds could lead to a 55% decrease in SCN population.The use of nematicides can also control SCN but it is currently not feasible due to safety issues [13]. Biological control methods,when identified,can also be used to control or manage SCN. However, host plant resistance has always been the favoured choice for controlling pests and diseases because it is economically feasible and poses no threat to the environment. A summary of the integrated disease management(IDM)approaches that can be used for SCN control is presented in Fig.1.

6.Cytological and histological process of syncytium

Before SCN parasitism, root diffusate from soybean can cause increased hatching of SCN eggs. Moreover, the diffusate from susceptible varieties increases hatching more than that from resistant varieties [15]. Successful parasitism in soybean roots by SCN usually involves important physiological and morphological changes that result in the formation of a specialized feeding cell called a syncytium. The syncytium serves as the only source of nutrients during the life cycle of SCN,making it very important for the nematode's survival, and it has to be constantly replenished with nutrients that are required by the developing SCN[16].In susceptible plants,the syncytia develop into mature functional feeding sites sustained by the plants.This interaction occurs for weeks and, when successful, leads to failure in plant defence [17]. During the maturation of the syncytium into functional nurse cells, two developmental phases are observed. In the first or parasitism phase, SCN establishes the molecular circuitry to create a well-matched interaction with the plant cell; during this phase, the cytological features of syncytia for a susceptible or resistant process are similar. The second phase of syncytium maturation involves either a susceptible or resistant reaction, depending on the outcome of the plant’s defence[17].

Inhibition of syncytium formation and growth is linked to plant resistance. Syncytium formation in both susceptible and resistant soybean includes the features of wall perforations,increased cytoplasm density, and increased abundance of the endoplasmic reticulum [18]. In susceptible plants, the syncytium undergoes a continuous development up to nematode maturity, which is usually accompanied by hypertrophy of nuclei,an increase in the rough endoplasmic reticulum,and the formation of wall ingrowths in early and late stages of infection[15]. Moreover, the syncytium cell in a susceptible variety of soybean is considerably larger than those in resistant varieties.In the resistant reaction, different mechanisms based on the syncytium have been proposed: necrosis of cells surrounding the immature juvenile SCN due to lack of syncytium formation[18], degeneration of syncytium [19], and formation of cell wall thickenings that seal off the syncytium[20].

In regard to their resistance reactions to SCN, soybeans are categorized into two main groups, the Peking and PI 88788 types, on the basis of their different histological responses to SCN infection. The Peking type includes the accessions Peking, PI 90763, PI 89772, PI 438489B, Pickett,Huipizhiheidou (ZDD2315), and Yuanboheidou (ZDD10261)[18,21,22], and the PI 88788 type includes typical genotypes such as PI 88788, PI 209332, and PI 548316 [22]. The effect of the resistant reaction on SCN development occurs much earlier in the Peking type than in the PI88788-type of resistance. The Peking-type resistant reaction causes mortality of SCN at the J2 stage [17–19,21,23], while in PI 88788-type resistance, SCN development can proceed to the J3 and J4 stages [21,24]. Thus, in PI 88788, syncytium function is not greatly affected in the early stage of resistance process.

Fig.1– Integrated disease management(IDM)strategies against soybean cyst nematode.

7. Germplasm screening and sources of SCN resistance

The major genetic groups of SCN are separated based on host compatibility described using the HG classification [8].The HG classification gives information on the standard indicator lines (SCN-resistant lines used in breeding for SCN resistance)on which the nematode population can reproduce.Thus, resistant cultivars derived from these indicator lines will harbour nematode populations that can reproduce on the indicator lines [25]. In some instances, resistance to a minimum of one SCN HG type has been reported for 158 soybean accessions across soybean germplasm collections(National Plant Germplasm System, NPGS, 2016; Crop: Soybean,Descriptors:Nematcyst_1,2,3,4,5,and 14,Observation:Resistant) [25]. Numerous resistant sources of germplasm(Table S1) have been identified from abundant soybean germplasm collections [26–28]. Notably, some germplasms,such as PI 437654,Huipizhiheidou,and Wuzhaiheidou,have a broad spectrum of resistance to SCN races. However, only a few of these have been used in soybean breeding because most of these collections carry undesirable traits that are difficult to improve through conventional breeding techniques[29].

Soybean varieties with SCN resistance that are available for commercial purposes are derived primarily (90%) from three plant introduction (PI) accessions; PI 88788, PI 209332,and Peking [30]. Other resistant germplasms, including PI 437654, Huipizhiheidou, and Haerbinxiaoheidou, are also sources of resistance for some resistant soybean varieties[31]. Peking- and PI 88788-type sources of resistance are the most widely used against SCN. However, many breeding programs are primarily using PI 88788 as their major sources of SCN resistance.Joos et al.[32]reported that out of 336 SCNresistant entries evaluated,only 10 had resistance traits from sources other than PI 88788, and about 90% of SCN-resistant varieties in the central USA have PI 88788-type resistance[33].There has been a population shift in SCN resistance which led to new biotypes[34,35]and made the selection of new sources of SCN resistance difficult. Niblack et al. [36] observed that SCN could reproduce on PI 88788 in 70% of soil samples infested with SCN in Illinois, and similar population shifts have been reported in other soybean-producing regions[14,37]. Thus, PI 88788-type resistance has become less effective in reducing yield loss because SCN populations have adapted to overcome this type of resistance [38,39].Resistances from PI 437654 and Peking are effective, but combining them with increased yield and other agronomic traits is a major challenge[25].Thus,there is currently a great need to breed commercially viable varieties with resistance derived from different resistant sources.

8. Identification of SCN-resistance QTL and genes

Not all quantitative trait loci (QTL) mediating SCN resistance have been detected because of limited coverage of resistance sources, size of mapping population, and lack of adequate statistical techniques. However, the availability of soybean reference genome sequences [40] coupled with highthroughput single-nucleotide polymorphism (SNP) tools has provided insight for association mapping (AM), which has been used to study important soybean traits such as yield,disease resistance, and quality [41,42]. Breeding for SCN resistance in soybean will entail understanding the mechanism(s) underlying SCN resistance and mapping related QTL or genes. The first Rhg (resistance to H. glycines) locus was identified in the early 1960s [43], and since then, with the rapid developments in QTL mapping, many SCN-resistance loci have been mapped by utilizing different germplasms[44–65].Table 1 shows some of these QTL with their relatively high logarithm of the odds(LOD)values.Of these QTL,two loci found on chromosomes (Chr.) 18 and 8 (rhg1 and Rhg4,respectively) that are involved in resistance to SCN races 1 to 5 have been studied extensively [51,59,64,65]. The rhg1 locus has been consistently mapped to a subtelomeric region on Chr.18 in diverse soybean germplasms,including Peking[13].About 9%–28% of the total phenotypic variation observed in resistance to SCN HG types 2.5.7 (race 1) and 0 (race 3) is explained by Rhg4 genes from various resistant sources [38].Meksem et al. [51] reported that in the Forrest cultivar, rhg1 and Rhg4 together explain 98% of the observed phenotypic variance in resistance to SCN race 3.The resistance conferred by Rhg4 is primarily linked to SCN race 3 but also includes minor resistance to HG types 1.2.5.7 (race 2), 2.5.7, and 1.3.6.7(race 14). The Rhg4 and rhg1 loci are required to confer full resistance to some SCN races in cv. Peking and PI 437654[22,33,66,67].

In 2010, Vuong et al. [35] mapped two SCN-resistance QTL on Chrs. 10 and 18 in PI 567516C that are not related to rhg1 or Rhg4 loci. These QTL confer resistance against SCN races 1, 2, 3, and LY1. The QTL on Chr. 18 is physically distant from the rhg1 locus [35]. Furthermore, this is the first evidence of an SCN-resistance QTL on Chr. 10; all previously reported QTL for SCN resistance had been mapped to 15 of the soybean chromosomes, with none reported on Chrs. 2, 7,10,12, or 13 [7,38,50,57,68]. Kim et al.[13] also mapped two QTL on Chrs. 10 and 18 in PI 567305,which is highly resistant to multiple SCN HG types, similar to what was reported by Vuong et al.for 567516C[35].Thus,both PI 567516C and PI 567305 may harbour novel QTL that can mediate SCN resistance independently of the rhg1 and Rhg4 loci.

Genome-wide association studies (GWAS) have also been used to detect resistance loci. A study of 282 soybean accessions detected three resistance loci for SCN race 3 on chromosome 18, two of which corresponded to rhg1 and to another previously identified SCN-resistance locus, FGAM1,while the third was located at the other end of Chr. 18 [69].Vuong et al.[63]also reported eight novel QTL for resistance to SCN race 3. Zhang et al. [69] reported 13 significant SNPs for SCN resistance in seven different genomic regions. Three of these corresponded to previously mapped QTL including rhg1 and Rhg4,while the other 10 were novel.A study by Zhao et al.[70] revealed that 13 SNPs distributed on five chromosomes are associated with resistance to SCN race 1, four of which were novel loci.From these studies,more candidate genes for SCN resistance have been identified.Recently,12 SNPs located on Chrs. 7, 8, 10, and 18 were reported to be significantly associated with SCN resistance, among which three were located near the rhg1 locus [71]. Liu et al. [72] reported 27 mutations among 10 genes, and three of these genes overlapped between the two phenotypic mutants used in the study. These genes were reported to be possible candidate genes for SCN resistance[72].A summary of some associated loci is presented in Table 2.

Table 2–List of SNPs associated with resistance to soybean cyst nematode races 3 and 4.

The rhg1 copy number has been classified into high (> 6 repeats, e.g. PI 88788) and low (3 repeats, e.g. Peking) copy numbers [33]. Yu et al. [73] observed that for rhg1, both copy number and polymorphism type are important for SCN resistance. Among the resistance sources carrying rhg1,those with higher rhg1 copy numbers have greater resistance.Thus, the rhg1 locus may mediate SCN resistance by copy number variation(CNV)of multiple genes encoding an amino acid transporter(AAT),an α-soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (α-SNAP), and a WI12 protein (wound-inducible protein) [33,67]. Therefore, Kadam et al. [74] differentiated the CNV at rhg1 into resistant-high copy (PI 88788-type), resistant-low copy (Peking type), and susceptible-single copy (Williams 82) numbers. The Pekingtype resistance is suggested to be bigenic, comprising rhg1-a and Rhg4 [67], whereas PI 88788 requires only rhg1-b for resistance to SCN. Moreover, one gene in rhg1-a locus, the GmSNAP18 is fully required for Peking-type resistance [67].Patil et al. [75] further classified the rhg1-b locus into two categories, rhg1-b (similar to those in PI 88788-type lines) and rhg1-b1(similar to those in Cloud-type lines).

Serine hydroxymethyltransferase(SHMT),which catalyses the reversible conversion of serine and tetrahydrofolate to glycine and tetrahydrofolate, respectively, contributes to the resistance at the Rhg4 locus[76].The gene GmSHMT08 has two polymorphisms located in the first and second exons,389 G/C and 1165 A/T, which result in amino acid variations of arginine vs. proline and tyrosine vs. asparagine, respectively,and affect the kinetic properties of the enzyme [76]. Kandoth et al. [77] further suggested that GmSHMT08 has additional functions aside from its main enzymatic role in SCN resistance and interacts with the GmSNAP18 protein. Further evidence from Kandoth et al. [77] showed that even after many years of selection on the recombinant inbred line (RIL)EXF63 (rhg1Frhg4E), the SCN population did not change,whereas SCN selected on the RIL EXF67 (rhg1FRhg4F) shifted to HG type 1.3.6.7, suggesting a shift in virulence upon exposure to Peking-type resistance.

Table 3–List of candidate genes for resistance to soybean cyst nematode.

Nonsynonymous SNPs distributed in GmSNAP11,which is a paralogous gene of GmSNAP18, have been identified as novel alleles contributing to SCN resistance [33,78]. Whole-genome sequencing revealed that a truncated α-SNAP was encoded by GmSNAP on chromosome 11 and not GmSNAP on chromosome 18[79].Also,Li et al.[53]demonstrated that a nonsynonymous SNP(map-5149)closely associated with resistance to SCN race 3 mapped to GmSNAP11.Thus,GmSNAP11 is a novel gene that can also confer SCN resistance.

Glycine max salicylic acid methyltransferase (GmSAMT)mediates the resistance of soybean to SCN. Lin et al. [80]showed that overexpression of GmSAMT1 in susceptible lines reduces the number of SCN,and it also affects the expression of genes responsible for salicylic acid (SA) biosynthesis and signal transduction. Other genes involved in SA biosynthesis and signalling also confer partial resistance on SCN. Mathew et al.[81]showed that overexpression of AtNPR1,AtTGA2,and AtPR-5 in soybean roots reduced cyst count to less than 50%of that in controls not overexpressing these genes.Youssef et al.[82]reported that ectopic expression of Arabidopsis phytoalexindeficient4 (AtPAD4) can broaden resistance to SCN. Therefore,the genes involved in SA biosynthesis and signalling play important roles in SCN resistance.

In addition,Yang et al.[83]characterized the soybean WRKY gene family and identified five WRKY genes (GmWRKY154,GmWRKY62, GmWRKY36, GmWRKY28, and GmWRKY5) conferring high SCN resistance (>70% reduction in cyst number) and four (GmWRKY52, GmWRKY53, GmWRKY86, and GmWRKY136)conferring moderate SCN resistance (40% to 60% reduction in cyst number). A list of some identified candidate genes with their associated proteins is presented in Table 3.

9. Functional gene analysis for SCN resistance

Reverse genetics strategies, such as T-DNA insertion mutagenesis [84], targeting-induced local lesions in genomes (TILLING)[85], RNA interference (RNAi) [86], and virus-induced gene silencing(VIGS)[87–89],have been developed in recent decades.Also,the soybean hairy root transient transformation system has been developed and used to study SCN resistance [33,67,76,90].The early work on the functional analysis of SCN-resistance genes was done by Cook et al. [33], who used gene silencing in soybean hairy roots to show that three genes in a 31-kb segment at rhg1-b contribute to SCN resistance. Overexpression of these genes together, but not individually, in soybean hairy roots significantly increased SCN resistance [33].Yang et al. [83]used transgenic hairy roots to show that the overexpression of WRKY genes could increase SCN resistance.

A DNA-based VIGS vector based on bean pod mottle virus(BPMV) has been developed [87,88] and used to detect genes for resistance to Asian soybean rust (Phakopsora pachyrhizi)[89]. Liu et al. [76] used mutation analysis, BPMV-VIGS, and transgenic complementation in soybean hairy roots to show that the Rhg4 locus gene GmSHMT08 confers SCN resistance.Kandoth et al. [77] showed that the GmSHMT08 protein has other functions aside from mediating in SCN resistance using mutagenesis.

These reverse genetics strategies, as well as the soybean hairy root transient expression system, have significantly increased the efficiency with which SCN-resistance candidate genes can be identified and will facilitate future understanding of the molecular mechanisms of SCN resistance.

10.Molecular mechanisms of SCN resistance

The molecular mechanisms of SCN resistance are complicated and still not fully understood. GWAS results have suggested that numerous disease-resistance proteins are associated with SCN resistance,including leucine-rich region(LRR)-containing proteins, zinc-finger domain proteins, protein kinases, cytochrome P450s, RING domain proteins, and members of the MYB and WRKY transcription activation families [56]. Comparative RNA sequencing analysis using wild soybean(Glycine soja)suggested that amounts of defencerelated differentially expressed genes (DEGs) are associated with SCN resistance; these included genes involved in pathogen recognition, the MAPK signalling cascade, Ca2+/calmodulin-mediated signalling, phytohormone-mediated signalling, WRKY-involved transcription regulation, cell wall remodelling, and various other defence-associated signalling processes [91]. Gene ontology (GO) enrichment analyses revealed that the serine and arginine metabolic processes may also be vital for SCN race 4 resistance [29]. To date, the rhg1-and Rhg4-mediated mechanisms have received the most attention due to their high contributions to SCN resistance,although other mechanisms have also been investigated.

For the rhg1 locus,Cook et al.[78]suggested that the protein product of the resistance-associated gene GmSNAP18 may not function as classical α-SNAP, but instead may mediate resistance through a novel mechanism. Bayless et al. [92] have shown that the disruption of SNARE complexes and vesicle trafficking resulting from the significant accumulation of abnormal α-SNAP protein at the syncytium confers resistance.Moreover, an unusual NSF allele (rhg1-associated NSF on chromosome 07; NSFRAN07) was discovered only in rhg1+germplasm.The protective effect against rhg1 α-SNAP cytotoxicity from NSFRAN07coexpression in planta is much greater than that of the WT NSFCh07. An examination of segregation distortion between rhg1 and NSFRAN07across 855 soybean accessions and rhg1+progeny showed a 100%coinheritance of NSFRAN07and rhg1 alleles [93]. These results suggest that rhg1-mediated SCN resistance may be accomplished through impairment of SNARE complexes, and NSFRAN07enables the sustainability of nematode resistance in rhg1 cultivars.

For the Rhg4 locus, Liu et al. [76] suggested that the GmSHMT08 gene responsible for Rhg4 resistance has two genetic polymorphisms that alter the function of the enzyme and thereby lead to the resistant or susceptible reaction. The enzyme plays an important role in oneQcarbon folate metabolism, and its alteration leads to folate deficiency, which can trigger hypersensitive-response-like programmed cell death of developing syncytia and also of the nematode[76].

The SA and jasmonic acid (JA) signalling pathways may also be involved in SCN resistance. AtPAD4 encodes a lipaselike protein that acts in mediating SA signalling, and its ectopic expression in soybean roots can reduce the number of SCN cysts [82]. The overexpression of GmSAMT1 induces the expression of the genes Isochorismate synthase (ICS) and Nonexpressor of pathogenesis-related 1 (NPR1), which modulate the SA biosynthesis and signal transduction pathway before SCN infection [80]. The ectopic expression of some Arabidopsis genes (AtNPR1, AtTGA2, and AtPR-5) influencing SA synthesis, regulation, and signalling can also confer SCN resistance [81]. The JA synthesis and signalling pathway may also be involved in SCN resistance. The ectopic expression of AtAOS, AtAOC, and AtJAR1 in soybean provides some level of resistance to SCN [81]. Guo et al. [94] reported that overexpression of rhg1-GmAAT increases the transportation of glutamate from shoots to roots,resulting in the accumulation of free glutamate in the roots and upregulation of the JA pathway,which may contribute to SCN resistance.

In brief (Fig. 2), the main molecular mechanisms of SCN resistance in soybean can be summarised as(1)Disruption of SNARE complexes and vesicle trafficking(involving SNAP and NSF) by cytotoxic effects of the extensive accumulation of abnormal α-SNAP protein at the syncytium can mediate SCN resistance; (2) Disturbance of folate homeostasis, which may trigger hypersensitive-response-like programmed cell death of developing syncytia, causes the death of the nematode,thereby preventing its growth and reproduction; and (3)Modulation of SA and JA signalling triggers defence processes against SCN.

11. Strategies for soybean breeding for resistance to SCN

Based on the mechanisms of SCN resistance mentioned above, although there are several existing strategies for breeding crops for resistance, the multigenic and widespread nature of SCN will require more reliable and efficient methods to breed SCN-resistant soybean varieties.

A conventional approach is to screen large collections of soybean accessions and select lines that are resistant to SCN.These lines can be employed in traditional breeding programs as the donor parents in developing SCN-resistant varieties.Another approach is the marker-assisted selection (MAS),which is the most economical,fast,and reliable strategy,and whose efficiency and accuracy could be further enhanced by the development of high-throughput markers. Shi et al. [95]have reported three functional Kompetitive allele-specific PCR(KASP) marker assays (GSM381 and GSM383 at rhg1 and GSM191 at Rhg4) that can be used for high-throughput MAS for SCN resistance with high accuracy [95]. Tian et al. [96]developed cleaved amplified polymorphic sequences (CAPS)markers targeting GmSNAP11 with a high selection efficiency.The race structure of the SCN population requires the use of gene pyramiding in developing resistant soybean varieties.The use of linkage and association mapping will allow the identification of numerous markers linked to SCN resistance[53] and will provide a basis for developing simple and effective markers for SCN resistance breeding.

Fig.2– An illustration of proposed mechanisms for resistance to soybean cyst nematode.NSF,N-ethylmaleimide-sensitive factor;SNAP,soluble N-ethylmaleimide-sensitive factor attachment protein;SNARE,soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SHMT,serine hydroxymethyltransferase;THF,tetrahydrofolate;MTHF,5,10-methylene tetrahydrofolate;PCD,programmed cell death;SA,salicylic acid;ICS,isochorismate;SID2,salicylic-acid-induction-deficient 2;NPR1,nonexpressor of pathogenesis-related 1;EDS1,enhanced disease susceptibility 1;PAD4,phytoalexin-deficient 4;JA,jasmonic acid;JAile,jasmonoyl-isoleucine; AOS,allene oxide synthase;AOC,allene oxide cyclase;OPR,12-oxophytodienoic acid reductase;JAR,jasmonic acid-amido synthetase.

Reverse genetic tools have facilitated the identification of SCN-resistance genes such as rhg1[33,76,93] and Rhg4[67,76].The identified genes will facilitate the development of resistant varieties through gene editing, allowing the improvement of SCN-susceptible lines that are desirable for other traits. Therefore, genome-editing tools such as zincfinger nucleases (ZFNs), transcription-activator-like effector nucleases(TALENs),and clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins (CRISPRCas9) deserve more attention from soybean breeders, given their ability to knock out or modify specific targeted sequences [97]. These techniques can be used to develop SCN-resistant varieties through targeted editing of rhg1,Rhg4,and other resistance genes, given that nucleotide transitions or deletions in GmSNAP18, GmSNAP11, and GmSHMT08 are known to significantly influence SCN resistance. Moreover,editing these genes, individually or together, will produce a series of soybean materials with different levels of SCN resistance, which could be useful in soybean breeding.

12.Conclusions

With the availability of soybean genome sequence information and efficient rapid techniques for gene function studies,previously unknown resistance genes (GmSNAP18 and GmSHMT08) for SCN have been identified in soybean. Also,new discoveries about the mechanisms of gene resistance are in progress.Apart from the previously identified rhg1 gene on chromosome 18, other genes, such as GmSNAP11 on chromosome 11, have been identified as being associated with SCN resistance. These identified candidate genes are important tools for improving marker-assisted breeding efficiency and understanding the molecular basis of SCN resistance. The phylogenetic information about SCN-resistance loci and gene/QTL-specific markers that have been developed will accelerate breeding programs targeting SCN-resistance. Gene functional analysis using techniques such as RNAi, VIGS, and mutagenesis will also increase the efficiency of candidate gene identification. The mechanisms underlying SCN resistance identified to date primarily involve disruption of SNARE complexes and vesicle trafficking, disturbance of folate homeostasis, and regulation of the biosynthesis and signalling of plant hormones such as SA and JA,suggesting possible avenues of approach for developing novel sources of SCN resistance. The combination of conventional breeding, MAS,and genomic editing tools is highly promising for future soybean breeding programs for SCN resistance.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.03.001.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This study was supported by the National Key Research and Development Program of China (2016YFD0100504,2016YFD0100201, and 2017YFD0101400), the National Natural Science Foundation of China(No.31301345 and No.31671716),the National Major Science and Technology Project of China(2016ZX08004-003),and the Agricultural Science and Technology Innovation Program of CAAS.

Author contributions

Abdulwahab S. Shaibu wrote the manuscript. Bin Li conceptualized and supervised this work,and edited the manuscript,Shengrui Zhang edited the manuscript. Junming Sun supervised and financed this work,and edited the manuscript.