Deep genotyping of the gene GmSNAP facilitates pyramiding resistance to cyst nematode in soybean

Yu Tin,Bo Liu,Xuehui Shi,Jochen C.Reif,Rongxi Gun,Ying-hui Li,*,Li-jun Qiu,*

aThe National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI)/Key Lab of Germplasm Utilization (MOA), Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,100081 Beijing,China

bDepartment of Breeding Research,Leibniz Institute of Plant Genetics and Crop Plant Research(IPK),Gatersleben, Germany

Keywords:SCN α-SNAP Allelic combination Marker-assisted selection(MAS)Haplotype analysis

ABSTRACT Soybean cyst nematode (SCN) is a highly destructive pathogen. The soybean host genome harbors at least two major genes for resistance (rhg1 and Rhg4), as well as a minor locus(SCN3-11). In the present study, a splicing site in GmSNAP11, the potential causal gene of SCN3-11, was identified by comparison of the GmSNAP11 cDNA sequences generated from resistant and susceptible soybean accessions. The sequence information was used to design a codominant CAPS marker, GmSNAP11-2565, which was used to genotype a panel of 209 soybean accessions varying with respect to SCN resistance. Analyses of the effect of the haplotypes formed by GmSNAP11-2565 and another large-effect (nonsynonymous)locus,GmSNAP11-2307,previously identified in GmSNAP11,revealed linkage disequilibrium(P ＜0.0001) between the two loci, suggesting that GmSNAP11-2565 could be used as a marker for GmSNAP11. GmSNAP11-2565 was accordingly used, along with established markers for GmSNAP18(rhg1)and GmSHMT(Rhg4),to characterize the panel accessions.The mean SCN female index of accessions carrying only the GmSNAP11 allele associated with resistance (20.3%) was higher than that associated with accessions carrying alleles for resistance at both GmSNAP11 and GmSNAP18 (12.4%), while the index for accessions carrying alleles for resistance at all of GmSNAP11, GmSNAP18, and GmSHMT was very low(1.9%).Selection on all three markers was effective for maintaining a high level of resistance to SCN race 3.

1. Introduction

Soybean (Glycine max [L.] Merr.) is a legume and oilseed crop accounting for over 60% of the oilseed produced worldwide and over 70% of vegetable protein consumed (http://soystats.com/). One of its most destructive pathogens is the soybean cyst nematode (SCN, Heterodera glycines) [1-3]. The pathogen has a distinct race structure, but by far the most prevalent race is race 3,especially in the USA and China[4,5].Although chemical control of SCN is possible,the deployment of genetic resistance, in conjunction with managed crop rotation, has emerged as the most effective management strategy [6,7].Because SCN resistance behaves as a quantitative trait, its assessment is time-consuming. For this reason, the development of genetic marker(s) linked to SCN resistance gene(s)greatly facilitates the breeding of resistant cultivars[8-10].

Genetic analysis of SCN resistance has revealed the presence in soybean germplasm of few major and several minor genes (https://www.soybase.org/) [11-13]. Two major loci conditioning resistance to SCN race 3 are known: rhg1 is located on chromosome 18 and Rhg4 on chromosome 8, and both of these genes have been isolated [14,15].Glyma.18G022500, one of three genes responsible for the rhg1-b resistance, is thought to encode an α-soluble Nethylmaleimide-sensitive factor attachment protein (SNAP)and is now referred to as GmSNAP18 [16]. Four DNA markers for GmSNAP18 targeting single-nucleotide polymorphisms(SNPs) have been developed and can differentiate between susceptible, moderately resistant, and highly resistant cultivars[3,17,18].The gene responsible for the Rhg4 resistance is Glyma.08G108900, which encodes a serine hydroxymethyl transferase and is referred to as GmSHMT [15]. Several DNA markers have been designed by targeting the two causal nucleotides in this gene [18,19]. GmSNAP18 and GmSHMT act additively. SCN3-11, a recently identified minor gene contributing to SCN race 3 resistance, lies in a region of chromosome 11 that shows pronounced similarity with the chromosome 18 region harboring GmSNAP18. The gene interacts with GmSNAP18 [20]. An association mapping analysis has suggested that a nonsynonymous substitution in Glyma.11G234500 (a version of the Glyma11g35820 sequence identified in soybean reference genome Glycine max Wm82.a2.v1) is the most likely causative polymorphism for SCN3-11, and the gene has accordingly been named GmSNAP11 [21].

The objectives of the present study were to(1)characterize sequence variation at the GmSNAP11 locus, (2) use this sequence information to develop an informative molecular marker for the resistance, and (3) test the efficacy of markeraided selection based on simultaneous genotyping for resistance-associated alleles at GmSNAP18, GmSHMT, and GmSNAP11.

2. Materials and methods

2.1. Plant materials and SCN bioassay

Plant materials included two panels: a germplasm panel and a recombinant inbred line (RIL) panel. The germplasm panel comprised 209 soybean accessions (Table S1) [20]. Of these, three resistant (cv Zhongpin 03-5373 and landraces Peking and Huipizhiheidou (HPZHD)) and three susceptible(cvs Williams 82, Zhonghuang 13, and Lee) cultivars were selected for resequencing with the aim of developing new markers for the chromosome 11 locus determining SCN resistance. The RIL panel including 242 F11RILs derived from the cross Zhongpin 03-5373 × Zhonghuang 13 [20] was genotyped for constructing a localized linkage map around the site of the chromosome 11 locus, using markers developed in the present study in addition to previously developed markers known to map to the critical region.

The SCN bioassay was as described in Li et al. [20]. Briefly,plants were exposed to SCN race 3 infection under both field and greenhouse conditions at Heilongjiang Academy of Agricultural Sciences in 2011 and 2013, with four resistant accessions (Pickett, PI88788, Peking, and PI90763) and one susceptible one (Lee) used to identify the SCN race. The SCN resistance of the lines in the two panels were assayed in two environments (field and greenhouse) using a completely randomized design with three replications. The numbers of SCN females present on the roots were recorded 30 days after inoculation. The resistance level of each accession was evaluated using the female index FI, calculated as (the mean number of females on the accession) / (mean number of females on ZH13) × 100[20].

2.2. Sequencing and the identification of genetic variation

The similar genomic and mRNA sequences represented by copies of GmSNAP18 and GmSNAP11 in cv.Williams 82(Fig.S1)were downloaded from soybean reference genome Glycine max Wm82.a2.v1 (https://www.phytozome.net/) and aligned with the Seqman program implemented in DNAstar(https://www.dnastar.com/) and Multalin [22] (https://multalin.toulouse.inra.fr/multalin/). The alignment allowed two primer pairs(GmSNAP11 cDNA and GmSNAP18 cDNA) to be developed for specific amplification of the respective full-length cDNA sequences of GmSNAP11 and GmSNAP18 (Fig. S2, Table S2).Variation was sought between the two genomic DNA sequences from three SCN-resistant and three susceptible cultivars, extracted from leaf tissue using a rapid DNA extraction kit (MBI Fermentas, Vilnius, Lithuania). Total RNA was extracted from roots harvested from the same plants using a DP432 RNAprep pure Plant Kit (TianGen Biotech Co.Ltd., Beijing, China), after which it was reverse-transcribed using a KR106 FastQuant RT Kit (TianGen Biotech Co. Ltd.,Beijing,China).The resulting cDNAs were amplified using the GmSNAP11 cDNA and GmSNAP18 cDNA primer pairs and the amplicons were sequenced.

2.3. Marker development

An alternative splicing site in GmSNAP11(GmSNAP11-2565,G/T) was identified by aligning the various gDNA and cDNA sequences. A CAPS primer pair (designated GmSNAP11-2565 CAPS_F/_R) was designed, using the dCAPS Finder 2.0 tool(https://helix.wustl.edu/dcaps/dcaps.html), to target this polymorphism. The primer sequences were 5′-CAACTTCTTGTGACTGGACAGCTTA and 5′-CTAGTGAATCAGCAAACAAAATAGT. Subsequently 20-μL PCRs contained 60 ng genomic DNA,1×PCR buffer,2 mmol L--1dNTP, 2 mmol L-1of each primer, and 1 U Taq polymerase(TransGen Biotech Co. Ltd., Beijing, China). The amplification regime comprised an initial denaturation step (94 °C/4 min),followed by 36 cycles of 94 °C/30 s, 56 °C/40 s and 72 °C/50 s,and was completed by a final extension step (72 °C/10 min).The amplicons were digested in a 10-μL reaction containing 5 μL of the PCR product, 0.2 μL 10 U μL-1Aci I (New England BioLabs (Beijing), Beijing, China), 1.5 μL buffer and 3.3 μL ddH2O incubated at 37 °C for 40 min. The digestion products were electrophoretically separated in 2.0% agarose gels and visualized by EtBr staining. In a previous study [23], a large number of small InDels were identified by comparison of the full genome sequences of Zhonghuang 13 and Zhongpin 03-5373. In the present study, one of these, a 3-nt InDel lying in the vicinity of Glyma.11G229500, was used to develop the Gm11I-1 marker (Table S2). The PCRs used to detect this marker were performed as above, and the amplification regime differed only in a change of the annealing temperature to 55 °C.The amplicons were electrophoretically separated in 6%polyacrylamide gels and visualized by silver staining.

2.4. Mapping GmSNAP11

A previous report [20] described the genotyping of the Zhongpin 03-5373 × Zhonghuang 13 RIL population with respect to 17 loci mapping in the vicinity of GmSNAP11.Those data were combined with the genotypic scores obtained from the GmSNAP11-2565_F/_R primer pair to perform a linkage mapping analysis. One-way analyses of variance(ANOVA) and Student’s t-tests were performed using SAS 9.3(http://www.sas.com/). A localized linkage map was constructed with QTL IciMapping 3.1 (https://www.isbreeding.net/) [24], which was also used to place the resistance locus,based on the composite interval mapping method[25].

2.5. Validation of the predictive power of markers for SCN resistance

In addition to the GmSNAP11-2565 CAPS marker, two established SCN resistance markers, the KASP (Kompetitive Allele-Specific PCR) marker rhg1-2, tagging GmSNAP18 [17],and the CAPS marker Rhg4-389, tagging GmSHMT [19], were used to genotype the panel of 209 soybean cultivars.

3. Results

3.1. Sequence variation in GmSNAP11

A phylogenetic analysis of SNAP paralogs suggested that the GmSNAP11 and GmSNAP18 sequences were closely related and shared a similar gene structure (Fig. S1). The length of the GmSNAP11 genomic sequence was 4763 nt, split into nine exons,and its 870-nt cDNA sequence was predicted to encode a 289-residue protein. The levels of similarity between the GmSNAP11 and GmSNAP18 genomic, mRNA, and peptide sequences were respectively 80.3%, 86.2%, and 99.0%. In order to discriminate between the paralogs, the GmSNAP11cDNA-F/-R primer pair, targeting the two UTRs(Fig. S2), was used to resequence the GmSNAP11 cDNA produced by the three SCN-resistant and the three susceptible cultivars. The alignment of the resulting six cDNAs spanned the full coding sequence (Fig. S3). Four genetic variants were identified, comprising three SNPs and one 17-nt InDel, and each of them distinguished the three resistant from the three susceptible cultivars (Fig. S3). The three SNPs comprised a synonymous T/C variant within the first exon (GmSNAP11-108), a nonsynonymous G/A in the sixth exon (GmSNAP11-2307)and a synonymous G/A in the seventh exon(GmSNAP11-2537).GmSNAP11-2307 is a synonym of Map-5149,the marker showing the strongest association with SCN race 3 resistance[20].The 17-nt InDel was spanned around the junction of the seventh exon and seventh intron, and was targeted by a primer pair with the aim of identifying sequence variation at this site.The alignment of the corresponding sequences from the six test accessions showed that the InDel,which extended from the beginning of the seventh intron(GmSNAP11 position 2565), induced the formation of an alternative splicing product, such that the GmSNAP11 transcript generated by SCN-resistant accessions included an extra 17-nt of sequence(Fig. S4). The three nucleotides (TAG) lying at positions 10-12 within the 17-nt sequence encoded a (premature) stop codon(Fig. 1). The implied alternative splicing site at GmSNAP11-2565 was predicted to result in the production of a truncated form of GmSNAP11 lacking the last 50 C-terminal residues(Fig.S5).

3.2. The development of a CAPS marker based on the GmSNAP11-2565 polymorphism

The sequence spanning nucleotide 2565 produced an Aci I recognition site in the allele carrying G in the key position,but not in the allele carrying T (Fig. 2-A). The GmSNAP11-2565 CAPS primer was designed to ensure that the primer pair did not co-amplify the GmSNAP18 sequence. The pair generated an amplicon of length 283 bp. When applied to the six test cultivars (three SCN-resistant and three susceptible), the CAPS assay based on this primer pair produced a 283-bp amplicon from each template,but following AciI digestion,the profiles of the susceptible cultivars displayed two fragments(171 + 112 bp),while those of the resistant ones remained as a single 283 bp fragment(Fig.2-B).

3.3. Linkage analysis of the GmSNAP11-2565 CAPS marker

Of the 242 RILs, 32 were resistant to SCN race 3 and 210 were susceptible.The RIL population was genotyped using the GmSNAP11-2565 CAPS marker along with 17 established markers (16 SNPs and one SSR [20]) as well as the newly developed InDel marker Gm11I-1 (Table S2). The segregation data were used to construct a localized linkage map(Fig.3-A).The position of the locus was confined to a 16 kb region,flanked by GmSNAP11-2565 CAPS and Map-2071(LOD score of 4.8), and the locus explained 9.7% of the variation for SCN resistance (Fig. 3-B). Of 242 RILs, 110 carried the GmSNAP11-2565 T allele and 132 the G allele.The mean SCN female index of the T allele lines was 82.4%,significantly (P ＜0.0001)lower than that of the 132 G allele lines(113%)(Fig.3-C).

3.4. Validation of the GmSNAP11-2565 CAPS marker and analysis of haplotype in soybean germplasm

When the panel of 209 soybean accessions was genotyped with the GmSNAP11-2565 CAPS marker to evaluate the effect of GmSNAP11 on SCN resistance,the mean SCN female index of the 144 accessions carrying the T allele(5.5%)was found to be significantly (P ＜0.0001) lower than that of the 65 accessions carrying the G allele (61.3%) (Fig. 4-A).Assignment of HR types was 90.8% correct, that of MR types 55.2%, that of MS types 75.0%, and that of HS types 86.4%correct (Fig. 4-B).

Fig.1-Nucleotide polymorphism in the GmSNAP11 sequence between cultivars displaying variation for resistance to SCN.Two of the variants are synonymous, one is nonsynonymous,and one generates an alternative splicing site.

To identify the haplotypes around the fine-mapped 16 kb region in all 209 accessions and assess their association with SCN resistance, we first searched for annotated genes in this genomic region in the Glycine max Wm82.a2.v1 soybean genome (https://phytozome.jgi.doe.gov/), and identified three annotated genes: Glyma.11g234500, Glyma.11g234600,and Glyma.11g234700. We then identified large-effect SNPs and small InDels(1-5 bp)that led to amino acid substitutions or caused stop-codon loss or gain and frameshifts in these three annotated genes between our previously resequenced ZP03-5373 (resistant to SCN) and ZH13 (susceptible to SCN),the two parents of the RIL panel [26]. None of these largeeffect SNPs and InDels was observed in the genic exon regions in Glyma.11g234600 and Glyma.11g234700 between ZP03-5373 and ZH13, whereas two large-effect SNPs were detected in Glyma.11g234500, one GmSNAP11-2565 and the other GmSNAP11-2307 (Fig. 1). In our previous study, GmSNAP11-2307,named Map-5149,showed a significant association with the SCN resistance inferred from previous association mapping analyses and a KASP marker (GmSNAP11-5149) was developed[20,27].

Four haplotypes (Hap1, Hap2, Hap3, and Hap4) were observed in 209 soybean accessions (Fig. 4-C). Hap2 and Hap4 were the main haplotypes, with proportions of 68.4%and 29.2%respectively.Hap1 and Hap3 were rare haplotypes,with proportions of only 0.5% and 1.9% respectively. A chisquare test showed a significant (P ＜0.0001) correlation between haplotype and SCN resistance. Among the 143 soybean accessions carrying Hap2, 133 showed resistance(HR/MR, 117/16), for a resistance identification efficiency of 93%. Among the 61 soybean accessions carrying Hap4, 24 showed resistance (HR/MR, 12/12), so that its resistance identification efficiency was only 39.3%. These findings suggested that Hap2 was the major resistant haplotype, and Hap3 the major susceptible haplotype.

3.5. Multiple marker-assisted selection for GmSNAP18,GmSHMT, and GmSNAP11

Fig.2-The GmSNAP11-2565 CAPS marker.(A)The sequences flanking the targeted G/T polymorphism(shown in red).The Aci I recognition site is indicated by underlining.(B)Gel separation of Aci I-digested amplicons generated from a genomic DNA template of six soybean cultivars(Zhongpin 03-5373[ZP03-5373], Huipizhiheidou[HPZHD]and Peking:resistant to SCN,and Zhonghuang 13[ZH13],Lee and Williams 82:susceptible to SCN).

Fig.3-The effect of GmSNAP11 on SCN resistance,derived from a genetic analysis of a RIL population developed from the cross Zhongpin 03-5373 × Zhonghuang 13.(A)A localized linkage map of chromosome 11 constructed using the two newly developed markers GmSNAP11-2565 CAPS and Gm11I-1 and 17 established markers.(B)A QTL plot showing the positive effect of GmSNAP11 on resistance to SCN race 3.(C)The presence of the T allele at GmSNAP11-2565 CAPS has a suppressive effect on the SCN female index.

Given that GmSNAP11-2307 and GmSNAP11-2565 showed linkage disequilibrium (P ＜0.0001, r2= 0.89, D′ = 0.98),GmSNAP11-2565 was selected to represent GmSNAP11. The rhg1-2 KASP marker (sixth exon of GmSNAP18), which was able to distinguish low-copy types from those carrying either a single copy or a high copy number of rhg1 [17], was applied to the germplasm panel,as was the Rhg4-389 CAPS marker for GmSHMT [19]. The relationships of the eleven allelic combinations with SCN race 3 resistance are shown in Fig. 5-A and were highly significant (P ＜0.0001) (Fig. 5-B). The mean SCN female index of accessions carrying the resistance allele of only GmSNAP11 was 20.3%, while that of accessions carrying the resistance alleles of both GmSNAP11 and GmSNAP18 was 12.4%. Accessions carrying the resistance alleles of all three genes showed the lowest mean SCN female index(1.9%).The predictability of the HR phenotype based on the allelic combination GmSNAP18 (G), GmSHMT (G), and GmSNAP11 (T)was 93.6%,while the combination GmSNAP18(G),GmSHMT(C),and GmSNAP11(T)was 88.9%predictive of MR.

4. Discussion

Soybean is a paleopolyploid that has experienced two wholegenome duplication events,the first occurring around 59 million years ago and the second around 13 million years ago[28]. The SNAP gene family comprises at least five members:GmSNAP02 (Glyma.02G260400), GmSNAP09 (Glyma.09G279400),GmSNAP11 (Glyma.11G234500), GmSNAP14 (Glyma.14G054900),and GmSNAP18 (Glyma.18G022500) [21]. The product of GmSNAP18 (syn. rhg1) involved in membrane fusion during vesicular trafficking and conducted a highly effective resistance against SCN on the host [16,29,30]. Functional allelic variation at GmSNAP18 is thought to be based on variation in the number of gene copies present,with the level of resistance increasing with the number of copies present [14,31]. In contrast, the GmSNAP11 resistance is associated with the production of a truncated gene product [21]. The truncation,which produces a protein lacking the final 50 C-terminal residues,is induced by an alternative splicing site at position 2565. Thus, the GmSNAP11-mediated SCN resistance differs mechanistically from that mediated by GmSNAP18. The increased SCN resistance in the presence of both GmSNAP11 and GmSNAP18 is likely due to the additional quantity of truncated protein [21,32]. The other known major SCN resistance gene GmSHMT encodes a serine hydroxymethyl transferase [15].

Fig.4-Effect of GmSNAP11 on SCN resistance,derived from a comparison based on a panel of 209 soybean accessions.(A)The mean SCN female index was higher in accessions carrying the G allele of the GmSNAP11-2565 CAPS amplicon than in those carrying the T allele.(B)Distribution of GmSNAP11-2565 CAPS alleles according to the strength of SCN resistance(HR,highly resistant;MR,moderately resistant,MS:moderately susceptible;HS,highly susceptible).(C)Boxplot representation of the effect on the SCN female index of the four haplotypes(Hap).

Combining the deployment of genetic resistance with crop rotation has emerged as the optimal means of mitigating losses to soybean production caused by SCN.Where multiple independent genes for resistance are present in the crop gene pool, marker-assisted selection offers possibly the only effective means of stacking two or more genes in a cultivar,an operation aimed at increasing both the strength and the durability of the resistance. Genes conferring resistance to SCN have been shown repeatedly to interact in an additive manner [33-37]. The two known major genes for resistance are GmSNAP18 (syn. rhg1) and GmSHMT (syn. Rhg4), which together explain most of the resistance displayed by cultivar Forrest. The more recently discovered chromosome 11 locus SCN3-11(here referred to as GmSNAP11)interacts with rhg1 to provide a higher level of resistance than does either gene present on its own [20]. The isolation of both GmSNAP18 and GmSHMT has allowed the development of perfect markers for both that are highly informative with respect to SCN resistance [17,19]. In the present study, a CAPS marker was developed for GmSNAP11. When it was used in conjunction with the markers for GmSNAP18 and GmSHMT to associate genotype with SCN reaction among a set of 209 soybean accessions,the mean SCN female index was 20.3%for the set of accessions carrying only GmSNAP11, falling to 12.4% for those carrying both GmSNAP18 and GmSNAP11 and to just 1.9%when all three genes were present.Of the 130 accessions expressing a high level of resistance, ＞84% carried the resistance-associated allele of GmSNAP18 (referred to as the Peking type resistance), while the other 16% were resistant,owing to the presence of either a high number of GmSNAP18 gene copies(referred to as the PI88788 type resistance)or the as-yet unknown resistance genes. The genotyping exercise demonstrated the potential of marker-aided selection for resistance, especially in programs based on Peking type germplasm. The pyramiding of the three resistant alleles represents a powerful means of selecting for strong resistance to SCN race 3 across a highly heterogeneous set of genetic backgrounds, and thus should be effective for transferring gene-stacked resistance from one background to another.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.04.003.

Acknowledgments

Fig.5-Variation in the SCN female index as influenced by the alleles present at GmSNAP18,GmSHMT,and GmSNAP11,derived from a comparison based on a panel of 209 soybean accessions.(A)The upper part of the panel shows the distribution of the SCN female index and the lower part illustrates the distribution of the alternative alleles at GmSNAP18(rgh1-2),GmSHMT(Rhg4-389), and GmSNAP11(GmSNAP11-2565).The yellow color denotes a resistant reaction(HR,MR)and the green color a susceptible reaction(MS,HS).Gray lines represent heterozygous accessions. (B)A boxplot representation of the effect on the SCN female index of the four major allelic combinations.

This research was financed by National Key R&D Program for Crop Breeding (2016YFD0100602, 2016YFD0100201), the Agricultural Science and Technology Innovation Program (ASTIP)of the Chinese Academy of Agricultural Sciences, National Science and Technology Platform. The authors thank the China National Gene Bank (https://www.nationalgenebank.org/)for seed of the Chinese accessions.