Elusive Diagnostic Markers for Russian Wheat Aphid Resistance in Bread Wheat: Deliberating and Reviewing the Status Quo.

Russian wheat aphid, Diuraphis noxia (Kurdjumov), is a severe pest of wheat, Triticum aestivum L., throughout the world. Resistant cultivars are viewed as the most economical and environmentally viable control available. Studies to identify molecular markers to facilitate resistance breeding started in the 1990s, and still continue. This paper reviews and discusses the literature pertaining to the D. noxia R-genes on chromosome 7D, and markers reported to be associated with them. Individual plants with known phenotypes from a panel of South African wheat accessions are used as examples. Despite significant inputs from various research groups over many years, diagnostic markers for resistance to D. noxia remain elusive. Factors that may have impeded critical investigation, thus blurring the accumulation of a coherent body of information applicable to Dn resistance, are discussed. This review calls for a more fastidious approach to the interpretation of results, especially considering the growing evidence pointing to the complex regulation of aphid resistance response pathways in plants. Appropriate reflection on prior studies, together with emerging knowledge regarding the complexity and specificity of the D. noxia-wheat resistance interaction, should enable scientists to address the challenges of protecting wheat against this pest in future.

1. Introduction

The Russian wheat aphid (RWA; Diuraphis noxia (Kurdjumov), (Homoptera: Aphididae)) has been known as a severe pest of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) since devastating losses were reported in the Crimea in 1901 [1], as quoted by [2]. This atypical grain aphid now appears throughout the world [3,4,5,6,7,8,9,10,11,12,13,14], following the 2016 report of its arrival in Australia [15]. Sometimes where D. noxia occurs, population levels remain below injurious levels. Damage is however regularly reported in areas characterized by medium-to-lower yield potentials, rain-fed conditions and sporadic droughts [16]. Several traits contribute to causing severe yield loss (60% or more) if this aphid is not controlled [17]. D. noxia infestation leads to dramatic chlorotic streaks on leaves [18], leaf-rolling, general stunting, and head-trapping [19] resulting in a sizeable loss of the photosynthetic area. Furthermore, D. noxia has a low developmental-threshold temperature [19], a vast host range throughout the grasses [20], and is protected from many generalist natural enemies by the rolled-leaf pseudo-gall it engenders [21]. Climate change and increased crop pest dispersal make finding tools for breeding resistant cultivars so as to control D. noxia more important than ever before.

In South Africa, D. noxia control was achieved using resistant cultivars, which formed the basis of an integrated pest management program [22]. Since 1992, >40 Dn-resistant (Dn after D. noxia) wheat cultivars have been released for cultivation. Research, started in 1978, successively focused on four South African D. noxia biotypes, namely RWASA1, RWASA2, RWASA3 and RWASA4, with an additional biotype, RWASA5, reported in 2019 [23]. These biotypes now occur concurrently in wheat-producing areas of the country [24,25,26,27]. Providentially, numerous sources of Dn resistance were rapidly identified following the incursions of D. noxia in both South Africa [28] and the United States [29]. On-going research [30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53] provided an ample number of accessions with genetic resistance to this pest. Numerous different mechanisms of resistance to D. noxia occur in these sources.

A 2016 review [54] concluded that, for aphid–plant interactions, multiple mechanisms could function at different stages of the interaction, and that these could differ for species pairs at different stages of co-evolution. Furthermore, the stealthy nature of aphid feeding in phloem makes these interactions highly distinct [54]. Within the known sources of Dn resistance, large variation occurs with respect to the mechanisms of resistance (antibiosis, antixenosis and tolerance [55]) that are expressed. Different plant metabolic processes of resistance were reviewed by [56]. Considerable evidence points to the role of phloem as a signaling network in addition to its primary role in the partitioning of photo-assimilates [57].

Dn resistance, which maintains chlorophyll functionality and thus yield under D. noxia infestation, was deployed in winter/facultative cultivars (Supplementary Figure S1 Map of South African wheat production regions) with considerable success and economic benefit [58]. Conventional back-cross-breeding, and the phenotypic screening of host plant resistance using bioassays with live aphids, was used to breed these cultivars [51]. Recently, marker-assisted selection (MAS) for Dn resistance breeding has been explored to facilitate gene/quantitative trait loci (QTL) stacking in order to achieve durable resistance to different pests/diseases or several biotypes of the same pest [59,60,61]. This could replace the phenotypic screening of plants [62,63] with a faster and higher throughput methodology.

Combining R-genes is no guarantee that resistance will be improved or more durable. There are arguments both for and against stacking aphid resistance genes in single accessions. For Acyrthosiphon kondoi Shinji (blue alfalfa aphid) resistance in Medicago truncatula, gene stacking enhanced aphid resistance with a complex interaction between genes in the pyramid [64,65]. The combination of R-genes Rag4 and Rag1b against Aphis glycines (soybean aphid), however, resulted in very susceptible progeny [66]. Importantly, the durability of successful gene stacks is not yet predictable. Naturally occurring R-gene groups named ‘hot spots’ occur where genes that confer resistance to aphids, other insects and pathogens occur together [67]. Some aphid R-genes/QTL identified to date appear to be pleiotropic [68,69], and others epistatic [65,70]. Much research is needed to fully understand how R-gene combinations function. An alternate option to stacking R-genes is the use of mosaic-planting of crop cultivars with different R-genes. This production practice challenges pests with a complex genetic environment, which has been shown to decrease pest fitness [71,72,73].

1.1. Chronicling Marker Development for D. noxia Resistance

1.1.1. Initial Studies

The search for Dn resistance markers in bread wheat began in South Africa and the USA in the early 1990s, using RAPD, RAPD-SCAR, PCR-RFLP and RFLP markers to explore donor landraces and near-isogenic lines [74,75,76,77,78]. As new marker technology developed, it was harnessed. By 2001, microsatellite/simple-sequence repeat (SSR) markers on chromosome 7D had been identified to tag Dn resistance genes. The gene Dn2 was sub-divided into three “types” based on band size heterogeneity [79], while SSRs with specific size bands were reported to mark Dn1, Dn2, Dn5(sic), Dnx, Dn8 and Dn9 [80]. Ambiguity ensued regarding the location and naming of Dn5 following this paper [80,81]. New SSR markers on chromosome 1D for Dn4 and Dn6 [60] followed shortly, while a Dn1 marker was confirmed [82], as cited by [83]. Dn4 markers Xgwm106 and Xgwm337, with estimated genetic distances of 7.4 and 12.9 cM [60], were confirmed in a second study with shorter linkage distances (5.9 and 9.2 cM, respectively) and slightly different band sizes using a different F2:3 population [84,85]. As with the 7D marker studies, variance between 1D marker studies caused confusion.

In 2005, the authors of [86] attempted to clarify the inconsistency in literature regarding the location and genetic relationships of the Dn resistance genes on 7D, namely Dn1, Dn2, Dn5, Dn6 and Dnx. This study also included five additional donor accessions with uncharacterized Dn-genes. It concluded that the majority of Dn-genes on 7D are located on the 7DS arm, and that the genes appear either allelic or are tightly linked to one another in a Dn-gene cluster. A smaller resistance cluster was confirmed on chromosome 1DS [86] with Dn4 [60] forming a part of this cluster. The position of Dn5, however, remained contested.

Monotelosomic 7DL plants carrying Dn5 on the telosome were developed, and both the 7DS and 7DL telosomes were confirmed using mapped microsatellite and endopeptidase markers to show unequivocally that Dn5 occurs on 7DL [87]. This 2006 study found an unknown Dn-gene, derived from the same donor as Dn5, i.e., PI 294994, on 7DS, substantiating the findings of a cluster on 7DS [86]. This Dn resistance gene on 7DS [80,87] has remained unnamed and is referred to in this paper as DnUnknown.

1.1.2. Diverse Approaches to Dn resistance Marker Identification

Argentinian studies from 1999 onward focused on the identification and mapping of antibiosis and antixenosis to D. noxia [68,88]. A 2004 study [69] reported markers Xpsr687 on 7DS and Xgwm437 on 7DL for antixenosis, Xpsr490 and Rc3 on 7DS, and Xgwm44, Xgwm437 and Xgwm121 on 7DL for antibiosis, with at least two QTL in the repulsion phase, one near the centromere (7DS or 7DL) and the other distal on 7DL for antibiosis. In 2005, loci Xgwm1293 and Xgwm1150 on 6AL were associated with antixenosis against a new biotype present in Argentina [89].

By 2007, the research focus for Dn resistance markers shifted to genes effective against multiple D. noxia biotypes. Resistance breaking biotypes had, by that time, occurred in both the USA [90] and South Africa [25]. Markers were developed for the Dn resistance genes Dn7 [91] and Dn2414 [92]. However, both genes are associated with the “sticky dough” trait from the donor 1RS:1BL wheat-rye translocation. This regrettably made them unsuitable for use in bread wheat breeding programs.

Efforts from 2010 thus focused on the bread wheat accession, CItr 2401 (PI 9781), as it is also resistant to multiple D. noxia biotypes. A study [93] of a doubled haploid population identified numerous QTL associated with the foliar area (Xpsp3103 on 4DS, and Xgdm3 on 5DS), chlorophyll content (Xgwm533 on 3BS and Xpsp3094 on 7AL) and number of expanded leaves (Xwmc264 on 3AS and XwPt8836 on 4DS). Pleiotropic effects between the 4DS QTL and Rht-D1 were noted, as were associations with orthologs of the markers [93]. Further scrutiny of CItr 2401 saw three papers [94,95,96] published, documenting the genetic basis of the Dn2401 resistance gene, which was mapped to 7DS. Four SSR markers, Xcfd68, Xbarc214, Xgwm473 [94,96] and Xcfd14 [96], and two single nucleotide polymorphisms (SNP), Xowm705 and Xowm711 [96], were identified closer to the Dn2401 gene region through focused genetic studies. A 2019 Dn2401 study [97] identified new SNP markers (Xowm713, Xowm714, Xowm715 and Xowm717) to delineate a 0.3 cM and 133.2 kb interval which contains six high-confidence resistance gene candidates. Again, several credible studies have stimulated new questions.

Genome-wide association studies (GWAS) were also conducted to identify loci/chromosome regions that control Dn resistance. In 2013, an ICARDA study using 134 diverse wheat accessions [98] identified marker wPt-733729 (7DS) associated with the leaf curling caused by D. noxia, as well as three markers, namely wPt-3018 (7DL), wPt-3291 (7DL) and wPt-665471 (7DS), associated with leaf chlorosis. In 2016, Australian research identified new QTL for Dn resistance that mapped to chromosome 7DS [99]. This study hypothesized that the active area on 7DS, close to the centromere, is controlled by several loci, each providing small additive effects. These loci are tightly linked, segregate together, and may be a single locus comprising multiple alleles associated with specific phenotypes. A novel model was proposed suggesting that the Dn-genes at the 7DS locus are possibly contained within a chromatin loop [99].

Sadly, the markers reported above have not been properly validated in multiple wheat backgrounds, and the questions raised regarding pleiotropic effects and marker orthologs were never answered. To illustrate the enigmatic literature, five well-studied SSR markers are listed together with the reported band size for each linked Dn-gene/QTL (Table 1). The applicable Dn resistance donor accession used in each study, or the accession from which the study material was developed, is provided with the reference to the relevant study. It is prudent to note that all the Dn-genes mentioned in Table 1 (Dn1, Dn2, Dn5, Dn6, Dn8, Dnx, DnUnknown, Dn2401 and Dn626580) are considered to occur on chromosome 7D near the centromere, but their exact position and how they interact with each other (i.e., alleles or part of an R-gene cluster) is still not yet entirely clear [49,60,80,86,87,95].

Table 1

Five D. noxia resistance-linked markers and reported fragment sizes/additive effects for different Dn-genes/QTL from specific donor accessions on chromosome 7D of bread wheat.

Marker	Fragment Size (bp)/QTL Additive Effect	D. noxiaR-Gene	Donor/(Test Accession(s))	Reference
Xgwm44	Four fragments between 80–182	None	(Chinese Spring, Thatcher) #	[60,86]
Xgwm44	185	None	(Chinese Spring)	[87]
Xgwm44	180	DnUnknown	PI 294994	[87]
Xgwm44	180	Dn6	PI 243781	[60]
Xgwm44	180	Dn6	PI 262660(sic)	[60]
Xgwm44	200	Dn6	PI 047545	[60]
Xgwm44	Instar duration −0.797 **Aphid fertility −3.940 **Longevity −13.457 ***	QTL	Doubled-Haploid Recombinant population of CS and 7D Synthetic	[69]
Xgwm111	Three fragments between 130–305	None	(Chinese Spring, Thatcher) #	[60]
Xgwm111	209	None	(Chinese Spring)	[87]
Xgwm111	200	Dn2	PI 262660	[80]
Xgwm111	200	Dn6	PI 243781	[60]
Xgwm111	210	Dn1	PI 137739	[87]
Xgwm111	210	Not yet named	PI 047545	[60]
Xgwm111	215	DnUnknown	PI 294994	[87]
Xgwm111	220	Dn5	PI 294994	[87]
Xgwm111	225	Dnx	PI 220127	[86]
Xgwm111	274	Dn2401	CItr 2401	[95]
Xgwm111	210, 240, 250	Dn1	PI 137739	[83]
Xgwm111	210, 240, 250	Dn5	PI 294994	[83]
Xgwm111	210, 240, 250	None	(Chinese Spring 7DS dt)	[83]
Xgwm437	112	None	Chinese Spring	[87]
Xgwm437	100 (Type III)	Dn2	PI 262660	[79]
Xgwm437	102 (Type II)	Dn2	PI 262660	[79]
Xgwm437	104 (Type I)	Dn2	PI 262660	[79]
Xgwm437	105	Dn5	PI 294994	[87]
Xgwm437	124	Dn626580	PI 626580	[49]
Xgwm437	Antixenosis +2.077 **Longevity −27.420 ***	QTL	Doubled-Haploid Recombinant population of CS and 7D Synthetic	[69]
Xgwm473	244	Dn626580	PI 626580	[49]
Xgwm473	244	Dn2401	CItr 2401	[95]
Xgwm635	100	Dn8	PI 294994	[87]

# Xgwm44182 and Xgwm111205 are considered characteristic or functional fragments. See [60] for discussion. **, ***: Significant at p = 0.01 and p = 0.001, respectively.

Other credible additional factors can be deduced from the literature in hindsight, and may shed light on significant aspects that could inadvertently have influenced this research field. The primary aim of this paper is thus to discuss the sometimes-contradictory literature pertaining to Dn resistance markers on chromosome 7D of wheat, and suggest plausible interpretations of the collective body of literature. Additional examples, obtained by testing some published SSR markers associated with Dn resistance on individual plants with known phenotypes from a panel of South African wheat accessions, will be presented. Prospective avenues for future research are alluded to, considering exciting current developments in the understanding of the complexities of the aphid–host plant resistance interaction.

2. Results

The mean phenotypic damage rating for the five example plants from each accession was used to rank them, from most resistant to least resistant to biotype RWASA2, and calculate the standard error of means, which is presented in Table 2 together with postulated potential genes in the accession.

Table 2

Rank of test entries using the t-distribution test (p = 0.05) of the mean damage rating (SEM) of each accession to biotype RWASA2.

Accessions Ranked from Most Resistant to Least Resistant	Mean RWASA2 Damage Rating (SEM) of Five Individual Example Plants of Each Accession	Postulated Potential Gene(s) in the Accession
PI 137739“S”	3.0 (0)	Dn1
CItr 2401	3.2 (0.5)	Dn2401
T06/16	3.2 (0.4)	Dn1, Dn5, Dn8, Dn9, DnUnknown
PI 586954	3.4 (0.5)	Dnx
PI 47545	3.8 (0.4)	Dn47545
PAN 3144	4.0 (0)	Gene not known
PI 626580	5.0 (1.1)	Dn626580
PI 586955	5.2 (1.9)	Dnx
T06/13	5.8 (2.7)	Dn5, Dn8, Dn9, DnUnknown
PI 243781	6.2 (2.6)	Dn6
PI 294994	6.8 (2.3)	Dn5, Dn8, Dn9, DnUnknown
T03/17	7.6 (2.2)	Dn1, Dn2
T05/02	7.8 (0.4)	Dn5, Dn8, Dn9, DnUnknown
PI 262660	8.0 (0.6)	Dn2
TugelaDn2	8.2 (0.4)	Dn2
Yumar	8.2 (0.7)	Dn4
BW991306	8.4 (0.8)	Dn2401, Dn5, Dn8, Dn9, DnUnknown
BW991405	8.4 (0.5)	Dn2401, Dn5, Dn8, Dn9, DnUnknown
PI 634775	8.5 (0.9)	Dn8
RIL-A50	8.6 (0.5)	Dn2401
Tugela-DN	8.8 (0.4)	Dn1
Betta-DN	9.0 (0)	Dn1
Gariep	9.0 (0)	Dn1
BettaDn2	9.0 (0)	Dn2
Hugenoot	9.0 (0)	Susceptible control
PI 634770	9.2 (0.4)	Dn9

2.1. Phenotyping

RWASA2, first reported as “Clone 2” [25], is virulent to Dn1, Dn2, dn3, Dn8 and Dn9 [27], while the genes Dn4, Dn5, Dn6, Dn7, Dnx and Dny remain effective against this biotype [27]. When considering the pedigrees of the accessions in the panel (see M&M), it is expected that several of them should be susceptible to RWASA2. This includes the susceptible control Hugenoot, as well as PI 137739“S” (Dn1), Betta-DN (Dn1), Gariep (Dn1), Tugela-DN (Dn1), PI 262660 (Dn2), BettaDn2, TugelaDn2, PI 634775 (Dn8), PI 634770 (Dn9) and T03/17 (Dn1, Dn2). The data confirm that, with the exception of PI 137739“S”, all the accessions one would expect to be susceptible to RWASA2 are indeed susceptible. There are, however, a number of accessions that are postulated to contain D. noxia resistance genes that should confer resistance to RWASA2, which are susceptible. It could thus be construed that T05/02 does not contain Dn5, Yumar does not contain Dn4, neither breeding-lines BW991306 nor BW991405 contain Dn2401 or Dn5, and RIL-A50 does not contain Dn2401. PI 137739“S”, a selection from the original landrace PI 137739, must then contain either an additional gene to the reported Dn1, or a different gene that confers RWASA2 resistance. In addition to PI 137739“S” (Dn137739”S”), accessions CItr 2401 (Dn2401), T06/16 (Dn5, Dn8, Dn9, DnUnknown), PI 58654 (Dnx), PI 47545 (Dn47545), PAN 3144 (gene not known) and PI 626580 (Dn626580) tested as being resistant to RWASA2, with PI 586955 (Dnx), T06/13 (Dn5, Dn8, Dn9, DnUnknown), PI 243781 (Dn6) and PI 294,994 (Dn5, Dn8, Dn9, DnUnknown) testing as being moderately resistant to this biotype.

Notably, multiple D. noxia biotypes [27,100] occur concurrently in wheat fields in South Africa, although the predominant biotype may vary from season to season and within particular geographic regions. This requires that genes with resistance to different biotypes be combined within a single accession order to make multiple-biotype resistant cultivars available to producers. Due to the variation in resistance reactions present in different plants of the same accession, is not possible to stack Dn resistance against different biotypes without diagnostic molecular markers. A single plant can only be accurately phenotyped with one biotype in each generation. For example, a robust molecular marker for any gene(s) resistant to RWASA1 but susceptible to RWASA2 would enable breeders to combine the RWASA1-effective gene(s) with RWASA2-effective gene(s), for the control of more than one biotype concurrently. Screening the germplasm with RWASA2 would identify plants with RWASA2-effective resistance, and RWASA1-effective resistance could be identified by selecting those RWASA2-resistant plants that also contain the marker. The reciprocate is not possible, as most genes effective against RWASA2 would mask the presence of genes effective against RWASA1 (Personal communication, data not shown VL Tolmay) if the plants were phenotyped with the RWASA1 biotype. This seeming anomaly could easily be explained if the particular Dn resistance in these accessions is complex in nature, and is contingent on the D. noxia biotype used to develop/select the accession, with other biotypes either recognizing the whole or only parts of the complex resistance.

2.2. Genotyping

Of the five markers tested on this panel of accessions, Xgwm473 and Xgwm635 did not reflect sufficient polymorphism, and the data are therefore not shown. Xgwm473 was reported to be linked to Dn resistance by two studies [49,95], with both groups describing a 244 bp fragment as the diagnostic band. However, the genes reportedly linked to this fragment were different, namely, Dn626580 [49] and Dn2401 [95]. Xgwm635 was reported to be linked to Dn8 from PI 294994 [80] with a 100 bp band. The three remaining markers, namely Xgwm44, Xgwm111 and Xgwm437, for which PIC values [101] were calculated from the panel data (Supplementary Table S1), will be discussed below in the order they occur on the wheat consensus map [102] of chromosome 7D. It is, however, prudent to note that markers Xgwm44 and Xgwm111 have multiple orthologs, as reported [86,103] (Supplementary Table S1), potentially compounding allelic interpretations.

SSR marker Xgwm44, located on 7DS [80,86,104], is reported to give a 180 bp band for resistance gene Dn6 from accessions PI 243781 [60,86] and PI 262660 (sic) [86], while resistance from accession PI 047545 was linked to a 200 bp fragment [86]. A 180 bp fragment was also reported for this marker for DnUnknown [87]. In this study, 12 haplotype combinations (Supplementary Table S1) of band sizes 0, 120, 130, 150, 175, 185, 190 and 200 bp were found in both individual resistant and susceptible plants.

Wheat microsatellite marker Xgwm111, on the short arm of chromosome 7D [86], has been associated with Dn resistance since the report [80] that it is tightly linked to Dn1, Dn2, Dn5(sic) and Dnx. The single band sizes reported for each of the genes in this study [80] were 210 bp [PI 137739], 200 bp [PI 262660], 220 bp [PI 294994] and 225 bp [PI220127], respectively. The resistance gene Dn5(sic) from PI 294994 identified by [80] is probably not the same as Dn5, named by and allocated to chromosome 7DL through a telosomic analysis [81]. A follow-up study [87] using Xgwm437 placed Dn5 on 7DL, as it was only amplified in 7DL monotelosomic plants. This corroborates the prior mapping [102,104] of Xgwm437 on 7DL. Furthermore, the landrace PI 294994 is known to contain several different resistance variants [105,106]. The most widely accepted explanation for the confusion regarding resistance genes from this landrace is that the resistant plants used in these and other studies [80,81,105,106], though all linked to landrace PI 294994, differ from each other because different single plants were selected for use. The biotype used for the phenotypic evaluation of the plant reaction could be an additional factor contributing variability to the results, as the contradictory genetic studies identifying Dn5 used different D. noxia biotypes, namely RWASA1 [81,87] and RWA1 [80], to evaluate for susceptible and resistant plants. Furthermore, the close proximity of the D. noxia R-genes to the centromere of chromosome 7D significantly affects recombination frequencies and further hinders clarity [87]. The literature reports Xgwm111 band sizes 200, 210, 215, 220, 225 and 274 bp associated with the phenotypic expression of Dn resistance (Table 1). In this study, more allelic variation was observed with band sizes 0, 130, 135, 150, 180, 190, 200, 210 and 220 bp recorded in 16 haplotype combinations (Supplementary Table S1) from both resistant and susceptible plants. None of the plants in this study gave a band size of 215 bp [87], 225 bp [80] or 274 bp [95], despite the donor accessions PI 294994 and CItr 2401, in addition to numerous accessions developed from these accessions, present in the test panel.

Three different ‘types’ of bands were found with marker Xgwm437, located on 7DL [79], that were associated with resistant plants derived from accession PI 262660 (Table 1). These fragments were reported as the ‘highest bands’, and the illustration provided in this manuscript clearly shows multiple bands obtained with this marker. These three ‘highest bands’ with band sizes 104 bp (Type I), 102 bp (Type II) and 100 bp (Type III) are very close in size to the 105 bp band reported for Dn5 [87] from PI 294994. A 105 bp fragment was also reported to be associated with Dn626580 [49]. In this study, each of the 11 haplotypes (Supplementary Table S1) contained a single band of either 0, 90, 95, 100, 105, 110, 115, 120, 125, 130 or 135 bp for this panel. These haplotypes appear to occur in specific combinations with the haplotypes associated with Xgwm111 and Xgwm44, alluding to the existence of a diverse resistance cluster or a block of allelic variants.

2.3. Correlation of Phenotype and Marker Results

To practically illustrate selection, using a combination of the phenotype resistance expression of one D. noxia biotype (in this case, RWASA2) and SSR markers for resistance to another biotype (for arguments sake, RWASA1), let us consider some examples (Table 3). Accessions derived from single R-gene-sources will be briefly discussed, before moving to those with potential combinations of R-genes from multiple sources.

Table 3

Accession (Sample name), D. noxia damage score (RWASA2) and marker haplotype for single plant examples screened with markers Xgwm44, Xgwm111 and Xgwm437.

Accession (Sample Name)	Single ExamplePlant RWASA2 Score	Xgwm44	Xgwm111	Xgwm437
Betta-DN_1	9	120; 190	135; 210	120
Betta-DN_2	9	120; 190	135; 210	120
Betta-DN_3	9	120; 190	135; 210	120
Betta-DN_4	9	120; 190	135; 210	120
Betta-DN_5	9	120; 200	135; 220	120
Gariep_1	9	120; 190	135; 210	115
Gariep_2	9	120; 190	135; 210	115
Gariep_3	9	120; 190	135; 210	115
Gariep_4	9	120; 190	135; 210	115
Tugela-DN (V4483)	9	120; 190	135; 210	115
Tugela-DN (V4484)	9	120; 190	135; 210	115
Tugela-DN (V4485)	9	120; 190	135; 210	115
Tugela-DN (V4486)	9	120; 190	135; 210	115
Tugela-DN (V4487)	8	120; 190	135; 210	115
BettaDn2 (V4493)	9	120; 150; 190	135; 210	100
BettaDn2 (V4494)	9	120; 150; 190	135; 210	100
BettaDn2 (V4495)	9	120; 150; 190	135; 210	100
BettaDn2 (V4496)	9	120; 150; 190	135; 210	100
BettaDn2 (V4497)	-	120; 150; 190	135; 210	100
TugelaDn2 (V4578)	9	Null	135; 220	115
TugelaDn2 (V4579)	8	120; 150; 190	135; 210	100
TugelaDn2 (V4580)	8	120; 150; 190	135; 210	100
TugelaDn2 (V4581)	8	120; 150; 190	135; 220	115
TugelaDn2 (V4582)	8	120; 150; 190	135; 220	115
T05/02 (V4553)	7	Null	135; 210	100
T05/02 (V4554)	8	120; 175	135; 200	100
T06/13 (V4543)	8	120; 185	135; 200	120
T06/13 (V4544)	8	120; 185	135; 200	120
T06/13 (V4546)	3	120; 175	135; 200	120
T06/13 (V4547)	2	120; 175	135; 200	120
T03/17 (V4548)	7	120; 175	130; 200	120
T03/17 (4549)	9	120; 175	130; 200	120
T03/17 (V4550)	7	120; 175	130; 200	120
T03/17 (V4551)	7	120; 175	130; 200	120
T03/17 (V4552)	8	120; 175	130; 200	120
T06/16 (V4538)	4	120; 190	130; 200	135
T06/16 (V4539)	3	120; 175	130; 200	135
T06/16 (V4540)	3	120; 175	130; 200	135
T06/16 (V4541)	3	120; 175	130; 200	135
T06/16 (V4542)	3	120; 175	130; 200	135
PAN 3144_1	4	120; 190	135; 200	120
PAN 3144_2	4	120; 190	135; 200	120
PAN 3144_3	4	120; 195	135; 210	120
PAN 3144_4	4	120; 190	135; 200	120
PAN 3144_5	4	120; 195	135; 210	120

Commercial cultivars Betta-DN, Gariep and Tugela-DN (all derived from PI 137739 and potentially containing Dn1) tested as being susceptible to RWASA2. They all contained the Xgwm44120;190 and Xgwm111135;210 haplotypes, while Betta-DN contained Xgwm437120, and both Gariep and Tugela-DN contained Xgwm437115 (Table 3). Advanced breeding-lines BettaDn2 and TugelaDn2 (derived from PI 262660 and potentially containing Dn2) were also susceptible to RWASA2. Within the 10 plants representing these two advanced breeding-lines, 9 plants contained the Xgwm44120;150;190 haplotype with 1 TugelaDn2 plant containing Xgwm44null. All five BettaDn2 plants as well as two of the TugelaDn2 plants contained haplotype Xgwm111135;210 as well as Xgwm437100. The remaining three TugelaDn2 plants contained Xgwm111135;220 and Xgwm437115. It is not unequivocally possible to confirm the presence of Dn1 or Dn2 based on these marker alleles, although the phenotypic data using RWASA2 is expected for plants containing these genes.

Both T05/02 plants and two of the T16/03 plants (derived from PI 294994 using RWASA1) tested as being susceptible to RWASA2, suggesting that they do not contain Dn5, although they may well contain Dn8, Dn9 and DnUnknown, or any combination of the latter. The remaining two plants of T06/13 were resistant (a damage rating score of 6 or less) to RWASA2, and contained the Xgwm44120;175, Xgwm111135;200 and Xgwm437120 haplotypes. The susceptible plants contained different haplotypes, namely Xgwm44null, Xgwm111135;210 and Xgwm437100 (1 T05/03 plant); Xgwm44120;175, Xgwm111135;200 and Xgwm437100 (1 T05/03 plant) or Xgwm44120;185, Xgwm111135;200 and Xgwm437120 (2 T06/03 plants). Again, based on the above data, it is not possible to definitely confirm Dn5 present in the two RWASA2-resistant plants. These individual plants cannot be rescreened using RWASA1 or any other biotype, and there is no guarantee that their progeny or other seeds from the same mother plant will have the same haplotypes as these individual plants.

Advanced breeding-lines T03/17 (Dn1 and Dn2) and T06/16 (Dn1 and Dn5, Dn8, Dn9, DnUnknown) were purposefully developed to combine Dn-genes from multiple sources. The haplotypes of the five T03/17 plants are Xgwm44120;175, Xgwm111130;200 and Xgwm437120, and all are susceptible to RWASA2. Similar to the reasoning for accessions containing either Dn1 or Dn2, these results do not confirm the presence of either genes, nor whether the attempt to combine them was successful. According to the pedigree, advanced breeding-line T06/16 could potentially contain Dn1, Dn5, Dn8, Dn9 and DnUnknown, or any combination of these genes. All five plants of this accession tested as being resistant when screened with RWASA2, phenotypically substantiating the postulated presence of Dn5 as the only one of these genes reported to confer resistance to RWASA2. Haplotypes Xgwm44120;175, Xgwm111130;200 and Xgwm437135 occur in four plants with a higher level of resistance than the fifth, which contains marker haplotype Xgwm44120;190 instead of the 120; 175 bp band recorded for the other single plants. Again, the marker haplotypes do not correspond with the published information for Dn5 or DnUnknown, and it is not clear whether Dn1 is present in these plants at all. Had these plants been screened with RWASA1, the presence of Dn5 (which was substantiated in this case by the RWASA2 screening that took place) would have masked the presence of Dn1, as both genes confer resistance to RWASA1.

The phenotypic reaction of resistant commercial cultivar and the check accession of PAN 3144 (see M&M) shows that it contains gene(s) conferring resistance to RWASA1, RWASA2 and RWASA3. Biotype characterization studies [27,100] list Dn5, Dn6, Dn7 and Dnx as the only genes that confer resistance to all three of these biotypes. The haplotypes of the five resistant plants, namely Xgwm44120;190 or 195, Xgwm111135;200 or 210 and Xgwm437120, do not clearly indicate the presence of any of those genes in these plants.

Single plant data pertaining to the landrace R-donors and other accessions included in this study can be found in Supplementary Table S1. The five plants of landrace PI 137739”S” are uniform in terms of their genotype, as are those of PI 262660. In all likelihood, this is due to a specific, targeted selection in the case of PI 137739”S”, where a single plant with resistance to biotype RWASA3 = 5 was selected in January 2015. PI 262660 may inadvertently have become more uniform over many years of successive use and seed multiplication. Three haplotypes are present in the five plants of PI 294994, with only two plants, of the same haplotype, testing resistant to RWASA2. All four plants of landrace PI 047545 tested resistant to RWASA2, with the most resistant plant possessing a different haplotype to the others. Two haplotypes were also contained in the landrace PI 243781, with the single resistant plant different to the other susceptible ones, while in PI 626580 two haplotypes are present, but the most susceptible plant has the same haplotype as one of the most resistant. All three plants of CItr 2401 were resistant and contained the same haplotype. It would appear that the allelic diversity in landrace accessions may be dependent on whether the accession is still an amalgamation (bulked-up as collected) or whether a selection has been purified from it. Furthermore, allelic variation may be dependent on, or restricted by, which D. noxia biotype was used to characterize the accession or make the selection. The potential influence of the biotype used during the screening and selection would furthermore naturally affect/influence the robustness of the marker allele’s association with the trait. Generally, the haplotype data for the individual plants of single R-gene cultivars and advanced breeding-line accessions are sufficiently uniform to indicate that the accessions are true breeding. In the case of accessions developed to combine genes, the variation is somewhat greater.

3. Discussion

Many authors make an important distinction between markers useful for MAS and those that are not [103,107,108,109]. Generally, three critical requirements distinguish markers considered functional or diagnostic for MAS [107,110]. These are reliability, repeatability and robustness. The first requires flanking markers or tight (≤1 cm) linkage between the marker and the target gene/trait [108], as a larger distance can result in false selection [76,108]. The second is the validation of the marker–trait association across multiple genetic backgrounds [108,111], and the third is the suitability for large scale commercial application [108,109] versus the gain per unit time and cost [103]. The examples presented in this paper indicate that the markers tested did not meet the required level of reliability and repeatability across a panel of resistant and susceptible South African accessions. This is possibly due to the relatively large linkage distances and a lack of conclusive validation studies. In general, these examples show that multiple haplotypes exist in many of the test accessions, even when the phenotype is similar, and the accession is of an advanced enough generation to expect a true breeding response.

Two inter-related sources of ambiguity can be identified, which could account for the observed phenomena. Firstly, in terms of the host plant genetics, the dominant inheritance of most Dn resistance, often attributed to single genes [32,38,41,42,47,48,49,52,56,81,105,106,112,113,114,115], has underpinned many studies, despite evidence of a more complex genetic control of resistance [16,40,53,60,68,69,80,86,89,98,99,116]. ‘Downstream’ support for the last-mentioned hypothesis includes studies reporting multiple resistance mechanisms present in specific accessions [40,117,118,119], QTL associated with specific mechanisms [68,69,88,89,93,99], and studies reporting differential gene expression [120,121,122,123]. Additional compelling results include a 2009 paper [118] reporting multiple loci of genetic control within a single accession. Breeding-line KS94H871, containing Dnx from PI 220127, was shown to contain two loci encoding resistance to RWA1, but only one locus encoding resistance to RWA2. This pattern is echoed in the 2016 GWAS of a DH population (ECA Gregory x PI 94365) from Australia [99], with two loci (on 7D and 1D) encoding resistance to RWASA1 and RWASA2, while only the 1D locus encodes resistance to RWASA3. The re-evaluation and selection of resistant plants from ‘Plant Introduction’ accessions following the discovery of biotype USA2 in the United States [53] could point to mixed landrace accessions, as stated by the authors, but could alternately be explained by the “Dn-biotype-specific–R-gene(s)” concept shown in the aforementioned studies [99,118].

The second source of confusion could result from the D. noxia biotype used for the phenotypic evaluation of wheat accessions used in specific studies. This is true for the evaluation during the development of the test population and/or the evaluation of the phenotype which is used for trait-association analysis. In some instances, the biotype(s) used for the initial development of study-accessions and association mapping studies are not the same, while in some they are. The biotype is rarely specified per “Dn-marker–R-gene association”. Furthermore, Dn markers have rarely been validated using different/multiple biotypes to assess the specificity of the marker–trait association. Table 4 contains a summary of germplasms utilized in 7D marker studies, listing the D. noxia biotype(s) used to develop the accessions and evaluate the phenotype for genotypic association/marker development.

Table 4

Biotype(s) used for selection and/or development of wheat accessions, for the linkage analysis phenotyping and 7D marker alleles reported in the literature.

Accession(s) Selected or Developed with D. noxia Biotype	D. noxia Biotype Used to Phenotype for Linkage Analysis ₸	7D Markers Found (Reference)
PI 137739, PI 262660, PI 294994 selected with RWASA1 * and Betta-Dn1, Betta-Dn2, Betta-Dn9, Tugela-Dn1, Tugela-Dn2, Karee-Dn2, Karee-Dn8 developed with RWASA1 * [124]	RWA1 *	Xgwm111 200,210,220 [80]Xgwm635 100 [80]
Sando selection 4040 × PI 220127 F2:3 developed with RWA1 [80]	RWA1	Xgwm111 225 [86]
Carson x PI 262660 F2:3 developed with RWA1 [79]	RWA1	Xgwm437100, 102, 104 [79]
PI 372129, PI 243781, Thunderbird × PI 372129 (Dn4), Wichita × PI 372129 (Dn4), Wichita × PI 243781 (Dn6), and AL359 × PI 243781 (Dn6) developed with RWA1 [60]	RWA1	Xgwm44180 [60]Xgwm111200 [60]
F2 Betta-Dn1 †/Tugela-Dn2 †F2 Betta-Dn5 †/Tugela-Dn1 †F2 Karee-Dn5 †/Tugela-Dn2 †F2 PI 220127 (Dnx)/Tugela-Dn1 †F2 PI 220127 (Dnx)/Tugela-Dn2 †F2 PI 243781 (Dn6)/PI 137739(Dn1) #F2 PI 243781 (Dn6)/PI 372129(Dn4) #TC1 F1 Wichita//(Betta-Dn1 †/Tugela-Dn2 †) #TC1 F1 Wichita//(Karee-Dn5 †/Tugela-Dn2 †)TC1 F1 Wichita//(PI 243781 Dn6/PI 137739 Dn1) #	RWA1	Xgwm44180, 200 [86]Xgwm111210 [86]
NIL 92RL28, (PI 294994/5 * ‘Palmiet’) developed with RWASA1 *	RWASA1	Xgwm44180 [87]Xgwm111215 [87]Xgwm437105 [87]
PI626580 × Yuma F2:3 developed with RWA2 [49]	RWA2	Xbarc214237 [49]Xgwm437124 [49]Xgwm473244 [49]MS1251 [49]
‘Glupro’ × CItr2401 F2:3 and CItr2401 × ‘Glupro’ F2:3 developed with RWA2 [95]	RWA2	Xgwm111274 [95]Xgwm473244 [95]
Tugela−Dn2, Tugela−Dn5, Palmiet−Dn5, PI 137739 (=SA1684), PI 262660 (=SA2199), PI 294,994 (=SA463), Chinese Spring 7DS dt, Chinese Spring 7DL dt, Tugela, Tugela × Tugela-Dn1 F3:4 developed with RWASA1 [83]	RWASA1	Xgwm111 210, 240, 250 [83]
134 diverse wheat accessions selected with RWASY [98]	RWASY	wPt-733729 [98]wPt-665471 [98]wPt-3018 [98]wPt-3291 [98]
DH mapping population derived from EGA Gregory × PI94365 developed without phenotyping [99]	RWASA1RWASA2RWASA3RWASYRWATR	§ QTL_RWASA1_7D [99] QTL_RWASA2_7D [99] QTL_RWATR_rolling_7D[99]

₸ RWASA1 = Original South African biotype; RWASA2 = second South African Biotype; RWASA3 = third South African biotype; RWA1 = Original USA biotype; RWA2 = second USA biotype; RWASY = Original Syrian biotype; RWATR = Original Turkish biotype. † Initial identification and development of near-isogenic-lines with RWASA1 [124], further development with RWA1 [80,125]. # developed with RWA1. Inferred as original USA D. noxia biotype based on year of study. * Inferred as original South African D. noxia biotype based on year of study. § Author original designation modified as follows to reflect common RWA biotype nomenclature: ‘QTL_RWA SAB1_7D’ presented as ‘QTL_RWASA1_7D’; ‘QTL_RWA_SAB2_7D’ presented as ‘QTL_RWASA2_7D’; ‘QTL_RWA_Trolling_7D’ presented as ‘QTL_RWATR_rolling_7D’.

Inconsistencies with respect to fragment size between different studies, over many years in many wheat accessions, indicate that the marker alleles are not diagnostic. This may be potential Dn-gene allelic variation that has gone unresolved or un-noticed in the past. Nevertheless, the same markers are repeatedly found to be linked to Dn resistance on chromosome 7D. This indicates that genetic resistance to this pest is coded within those regions in some way. The current shortage of diagnostic markers for this trait should be addressed, taking account of the growing evidence for the complex regulation of resistance gene expression [126,127].

Across multiple crops, the complexity of aphid–plant interactions is being progressively revealed [71]. Despite multiple D. noxia biotype studies [128,129,130,131,132,133,134], many unknowns still have to be clarified. In general, “research shows that aphid virulence may be a complex adaptation involving a myriad of factors, including epigenetically controlled phenotypic plasticity and contributions from endosymbionts, the gut and saliva” [126]. Likewise, studies of plant defence against insects reveal that resistance gene expression and defence metabolism is influenced by both exogenous and endogenous environmental factors [71]. New evidence shows that plants utilize sophisticated mechanisms to modulate their response to stressors [135]. Embracing these unknowns within the current knowledge base [136], and engaging with them by using the ever-improving understanding of plant defence against insects, may lead to what has eluded us thus far. It is imperative that breeders are enabled with diagnostic markers with which to address the challenges posed by not only the insect pests, but also the changing climatic conditions which will undoubtedly influence pest distribution and the extent of damage they cause. The tools we need to breed D. noxia-resistant wheat will probably be based on a far better understanding of the specific D. noxia–host plant interactions. Two of the current developments in wheat to follow closely involve studies applying advanced molecular technologies to pinpoint D. noxia resistance genes [96,97] and to understand the regulation of the resistance response pathway [127,137,138]. Genetic characterization of the various donor sources of R-gene(s)/QTL, an understanding of the functional plant metabolism encoded by each genetic component, as well as a clear understanding of how these components interact with each other and the specific D. noxia biotype, will be essential to harnessing this plant-resistance to protect wheat in future.

4. Materials and Methods

4.1. Plant Materials

The 26 accession panel (Table 5) used to provide single plant examples in this study is comprised predominantly of wheat cultivars and advanced breeding-lines from the Agricultural Research Council-Small Grain (ARC-SG) D. noxia pre-breeding program, South Africa [139]. Based on pedigree data and phenotypic evaluation with multiple biotypes, the accessions are postulated to potentially contain different Dn-genes (Table 5) or combinations thereof. The wheat cultivars Gariep, Yumar and PAN 3144 are considered differential checks, and their different RWASA-biotype responses are shown in Table 6 together with those of the susceptible (Hugenoot) and resistant (CItr 2401) controls.

Table 5

Study panel of wheat accessions, their pedigree, accession status, postulated D. noxia gene information, and customary mean resistance reaction (SEM) to RWASA1 and RWASA2.

Wheat Accession	Pedigree	Accession Status	D. noxia R-Gene(s) Potentially Present	Mean (SEM) RWASA1 Score *	Mean (SEM) RWASA2 Score *
Hugenoot	Betta//Flamink/Amigo	Cultivar, Susceptible check	None	9.3 (0.45)	9.0 (0.58)
PI 137739”S”	Not applicable	Selection from Dn1 D. noxia R-donor, Landrace ex. Iran	Dn1 and/or Dn137739”S”	5.1 (1.68)	4.5 (1.94)
Betta-DN	PI 137739/*4Betta(4)	Cultivar	Dn1	5.5 (1.74)	8.2 (1.09)
Gariep	PI 137739/*4 Molopo(20)	Cultivar, Differential check	Dn1	5.3 (0.55)	8.0 (1.01)
Tugela-DN	Tugela*4/PI 137739	Cultivar	Dn1	5.4 (1.34)	7.7 (0.98)
PI 262660	Not applicable	D. noxia R-donor, Landrace ex. Azerbaijan	Dn2	4.4 (0.54)	6.7 (2.20)
BettaDn2	Betta*4/PI 262660	Advanced breeding-line [SYN = PI 634769]	Dn2	5.3 (1.07)	-
TugelaDn2	Tugela*4/PI 262660	Advanced breeding-line [SYN = PI 634772]	Dn2	6.0 (1.18)	-
Yumar	Yuma/PI-372129//CO-850034/3/4*Yuma	Cultivar, Differential check	Dn4	5.9 (1.36)	7.6 (1.82)
PI 294994	Not applicable	D. noxia R-donor, Landrace ex. Bulgaria	Dn5, Dn8, Dn9, DnUnknown	4.0 (0.80)	4.1 (0.25)
T05/02	PI-294994/*4Molen	Advanced breeding-line	Dn5, Dn8, Dn9, DnUnknown	3.9 (1.21)	3.9 (0.69)
T06/13	Karee/4/PI-294994/*4Gamtoos/3/YD”S”/BON//Dove”S” #	Advanced breeding-line	Dn5, Dn8, Dn9, DnUnknown	3.9 (1.74)	3.7 (0.92)
PAN 3144	PANNAR ® Proprietary information	Cultivar, Differential check	Gene not known	4.1 (0.80)	3.5 (0.59)
PI 243781	Not applicable	D. noxia R-donor, Landrace ex. Iran	Dn6	3.1 (0.87)	5.5 (2.02)
PI 634775	Karee*6/PI 294994	Advanced breeding-line	Dn8	8.1 (1.92)	-
PI 634770	PI 294994/*4Betta	Advanced breeding-line	Dn9	5.6 (0.66)	-
PI 586954 [KS94WGRC29]	PI-220127/P5//TAM200/KS87H66	Advanced breeding-line	Dnx	4.4 (0.75)	4.1 (0.46)
PI 586,955 [KS94WGRC30]	PI-220127/P5//TAM200/KS87H66	Advanced breeding-line	Dnx	3.2 (1.05)	4.1 (1.42)
PI 047545	Not applicable	D. noxia R-donor, Landrace ex. Iran	Dn47545	3.2 (1.49)	3.7 (0.74)
PI 626580	Not applicable	D. noxia R-donor, Landrace ex. Iran	Dn626580	5.1 (1.35)	4.5 (1.31)
CItr 2401	Not applicable	D. noxia R-donor, Resistant check, Landrace ex. Tajikistan	Dn2401	3.6 (0.58)	4.0 (0.58)
RIL-A50	Kavkaz*5/CItr 2401	F6 recombinant inbred line	None	8.0 (1.65)	6.6 (1.76)
T03/17	SST333(ex.PI262660)//661L1–33/Tugela-DN(ex. PI 137739)	Advanced breeding-line	Dn1 + Dn2	4.4 (1.11)	5.1 (1.20)
T06/16	Gariep(ex.PI137739)/4/PI-294994/*4Gamtoos/3/YD”S”/BON//Dove”S”	Advanced breeding-line	Dn1 + Dn5, Dn8, Dn9, DnUnknown	4.1 (1.93)	3.3 (0.94)
BW991405	PI-294994/*4BTA//TMP/CI13523-STW646408/4/FKS*3/3/W66136//Mayo/WRR4255-49-5/5/CItr 2401/*4Kariega	Advanced breeding-line	Dn2401 + Dn5, Dn8, Dn9, DnUnknown	7.0 (1.49)	6.4 (1.80)
BW991308	PI-294994/4*Molen//CItr 2401/*4Kariega	Advanced breeding-line	Dn2401 + Dn5, Dn8, Dn9, DnUnknown	-	4.9 (2.16)

* Scores based on visual D. noxia damage to seedlings which is rated from 1 to 10 where 1 = Small isolated chlorotic spots, 2 = Small chlorotic spots, 3 = Chlorotic spots in rows, 4 = Chlorotic splotches, 5 = Mild chlorotic streaks, 6 = Prominent chlorotic streaks, 7 = Severe streaks, leaves fold conduplicate, 8 = Severe streaks, leaves roll convolute, 9 = Severe streaks, leaves roll tightly, and 10 = Plant dying [16]. Means collated from multiple prior evaluations with n ≥ 11 ≤ 40 (Supplementary Table S2). # Note 1: Gamtoos = Veery#3 [140,141,142] is a susceptible cultivar with the 1B/1R translocation released in South Africa in 1983. Multiple resistant accessions were developed from it by ARC-Small Grain Centre, Bethlehem, South Africa, namely Gamtoos-DN (Dn1) [143] GamtoosDn2 and GamtoosDn5 [144] and Stellenbosch University, Stellenbosch, RSA, ‘GamtoosDn7′ [142,143].

Table 6

Susceptible, differential and resistant checks used in the study, the D. noxia R-genes they reportedly carry and reactions to four South African D. noxia biotypes (Adapted from [145]). A typical damage rating score of 1–3 is considered highly resistant (HR); 4, 5 is resistant (R); 6, 7 is moderately resistant (MR) and 8–10 is susceptible (S).

Differential Checks	D. noxia R-Gene	RWASA1	RWASA2	RWASA3	RWASA4
Hugenoot	None	S	S	S	S
Gariep	Dn1	MR	S	S	S
Yumar	Dn4	MR	MR	S	S
PAN 3144	Gene not known	R	R	R	S
CItr 2401	Dn2401	R	R	R	R

RWASA2 was chosen to phenotype the individual plants for marker validation. It is sufficiently damaging to allow discrimination, and all checks (Table 6) give consistent responses to it, while with other resistance breaking biotypes (RWASA3, RWASA4), a measure of segregation is known to occur. The reaction of accessions to the original South African biotype (Supplementary Table S2) was considered the baseline reaction of each accession.

4.2. Phenotypic Screening and Tissue Collection from Single Example Plants

A 21-day seedling assay [16] was performed to phenotype the test plants. In total, 15 individual seeds of each accession were planted in Professional Potting Mix® (Cultera, Muldersdrift, South Africa, www.cultera.co.za). Five cones per accession containing three seeds were arranged in a randomized complete block design within two 98-cone trays and then watered with KynoPop™ (Kynoch, Sandton, South Africa, www.kynoch.co.za) seedling fertilizer. Seven days post-planting, fresh leaf tissue material for DNA extraction purposes was harvested from a single plant per cone for each accession, and the other plants that germinated within that cone were uprooted and discarded. Every accession was left with five individual plants that were then each infested with c. five individuals of apterous mixed instars of D. noxia biotype RWASA2. The RWASA2 biotype used in this study was obtained from a colony maintained at ARC-SG. The individual plants were scored 21 days post-infestation using a damage rating scale of 1–10, where 1 = Highly resistant and 10 = Dead [15].

4.3. DNA Isolation and Polymerase Chain Reaction (PCR)

The fresh leaf material, harvested from five individual plants of each test accession, was individually homogenized within 750 µL of extraction buffer for 1 min at 30 r/s with the Qiagen TissueLyser II. A modified cetyltrimethylammonium bromide (CTAB) DNA extraction protocol [146] was used to isolate genomic DNA, which was then treated with 2 µL RNase-A enzyme (Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa). A Nanodrop 2000 Spectrophotometer (Thermo Scientific (Pty) Ltd., Waltham, MA, USA) was used to determine the quality, purity and concentration of each sample at the absorbance ratio of 260/280 nm. DNA samples were diluted with 1x TE (Tris-EDTA) buffer to 50 ng µL−1 final concentration and stored at 4 °C before progressing to downstream PCR applications. Five SSR marker primer pairs for D. noxia resistance, which occur on chromosome 7D, vis. Xgwm44 [60,80], Xgwm111 [60,80], Xgwm437 [79], Xgwm473 [49] and Xgwm635 [80], were synthesized by Integrated DNA Technologies (Integrated DNA Technologies, Inc. Coralville, Iowa, USA, www.IDTDNA.com) and were provided by Whitehead Scientific PTY (Ltd) Cape Town, South Africa (www.whitesci.co.za). PCR reaction conditions recommended for the KAPA 2X Ready Mix PCR Kit (KAPA Biosystems, Cape Town, South Africa, www.kapabiosystems.com) were applied. Each PCR reaction consisted of 10 µL (1x) KAPATaq 2X Ready Mix, 0.5 µL (10 µM) per SSR primer and the remaining volume (5.0 µL) of DNAse-free water. PCR was performed with a profile comprising initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation involving 95 °C for 30 s, annealing at a specific temperature for individual marker for 30 s, and extension at 72 °C for 30 s. Thereafter, a final extension step of 5 min at 72 °C was performed.

Relevant SSR marker-specific PCR amplicons were separated on 3.0–3.5% (w/v) high resolution agarose gel (Certified Low Range Ultra Agarose, Bio-Rad Laboratories, Inc. Hercules, CA, USA) stained with GelStar™ Nucleic Acid Gel stain (Lonza, Morristown, NJ, USA). Fragment separation was performed in an electrophoresis chamber containing 1x Tris-borate-EDTA (TBE) buffer and run at 100–125 V for 1–4 h. The SSR product sizes were determined according to 100 bp and/or 20 bp DNA ladders (Lonza SimplyLoad R, Lonza, Morristown, NJ, USA). A digital photograph was taken of the gel under UV light exposure with the Bio-Rad Molecular Imager Gel DocTM XR Instrument. Observed SSR marker alleles were sized, recorded and analyzed per cultivar both visually and with Bio-Rad image LabTM gel analysis software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data for each single example plant (damage rating score and successful marker analysis) are tabled in Supplementary Table S2. The mean phenotypic damage rating for the five single plants from each accession was used to rank the accessions from most resistant to least resistant, and calculate the standard error of means presented in Table 3.

5. Conclusions

In recent years, the spread of agricultural crop pests has become broader [147,148]. Prediction models [149] estimate that by the middle of this century, many important crop-producing countries will be fully saturated with pests. These authors [149] further state that, in spite of the quarantine and phytosanitary measures that are designed to prevent pest spread, natural dispersal and trade eventually result in invasions of crop pest species into previously pest-free areas. The global redistribution of species is not limited to pests that spread to previously pest-free areas. Virulent biotypes of pests can similarly spread, causing the resurgence of a pest in an area where it was formerly controlled. Climate change will undoubtedly influence the distribution and pest status of D. noxia. A clear understanding of the genetic control of Dn resistance, together with robust diagnostic markers, will be important in addressing challenges posed by this aphid in a timely manner.

The landrace origins and proximity of Dn resistance gene(s) to the centromere of 7D have been put forward as possible explanations for the difficulties encountered in the search for diagnostic markers for this trait to date. However, following thorough deliberation, it appears that additionally, two inadvertent faults may have blurred the accumulation of a coherent body of information applicable to Dn resistance. The “single dominant gene” assessment, initially accepted as the model of genetic control for resistance to this pest, has paradoxically permeated and simplified the underlying assumptions of many studies. This may have hindered critical investigation, despite multiple studies contending that Dn resistance is controlled by closely linked genes, multiple alleles at the same locus, or QTL influenced by the genetic background they occur in. Reconsideration of inadvertent assumptions or omissions, with appropriate reflection on the D. noxia biotype used to generate the data, may help better understand previous studies and plan future ones. This review calls for a more fastidious approach to the interpretation of results. Should it hold true that the genetic control of D. noxia resistance is more complex than originally thought, it could follow that a D. noxia biotype-specific R-gene/allele/QTL interaction, or possibly even a D. noxia biotype-specific resistance–response pathway interaction, may be at play. This, together with the potential pleiotropic and epistatic effects of genes involved in Dn resistance, should be investigated in future studies.