An Update of Recent Use of Aegilops Species in Wheat Breeding.

Aegilops species have significantly contributed to wheat breeding despite the difficulties involved in the handling of wild species, such as crossability and incompatibility. A number of biotic resistance genes have been identified and incorporated into wheat varieties from Aegilops species, and this genus is also contributing toward improvement of complex traits such as yield and abiotic tolerance for drought and heat. The D genome diploid species of Aegilops tauschii has been utilized most often in wheat breeding programs. Other Aegilops species are more difficult to utilize in the breeding because of lower meiotic recombination frequencies; generally they can be utilized only after extensive and time-consuming procedures in the form of translocation/introgression lines. After the emergence of Ug99 stem rust and wheat blast threats, Aegilops species gathered more attention as a form of new resistance sources. This article aims to update recent progress on Aegilops species, as well as to cover new topics around their use in wheat breeding.

Introduction

According to the latest revision of Aegilops L. taxonomy, (van Slageren system) which is used by most researchers (including this article), Aegilops consists of 23 species, having the D, S, U, C, N, and M genomes (van Slageren, 1994). Since the taxonomy has frequently changed (Kihara, 1954; Hammer, 1980; Witcombe, 1983; Kimber and Sears, 1987; van Slageren, 1994) this has led to some confusion about species names, and so a list of Aegilops species is provided below (Table 1). The biggest change from the previous taxonomy system is that Ae. mutica Boiss. has been removed and assigned a new species name: Ambylopyrum muticum (Boiss.) Eig. In the future, it will also be possible to make further modifications to reflect molecular findings (Edet et al., 2018).

Table 1

Taxonomy and genomic constitution of Aegilops species.

Genome∗	Taxonomy system
	van Slageren, 1994	Witcombe, 1983	Hammer, 1980	Kimber and Sears, 1987
D	Ae. tauschii	Ae. squarrosa	✓	Triticum tauschii
S	Ae. speltoides	✓	✓	T. speltoides
”	”	Ae. ligustica	✓	T. speltoides
Sb	Ae. bicornis	✓	✓	T. bicorne
Sl	Ae. longissima	✓	✓	T. longissimum
Ssh	Ae. sharonensis	✓	Ae. longissima	T. sharonense
Ss	Ae. searsii	✓	✓	T. searsii
C	Ae. caudata	✓	Ae. markgrafii	T. dichasians
M	Ae. comosa	✓	✓	T. comosum
N	Ae. uniaristata	✓	✓	T. uniaristatum
U	Ae. umbellulata	✓	✓	T. umbellulatum
CD	Ae. cylindrica	✓	Ae. cylindrica	T. cylindricum
DN	Ae. ventricosa	✓	✓	T. ventricosum
DM	Ae. crassa	✓	✓	T. crassum
DDM	”	✓	✓	T. crassum
DMS	Ae. vavilovii	✓	Ae. crassa	T. syriacum
DMU	Ae. juvenalis	✓	✓	T. juvenale
US	Ae. peregrina	✓	✓	T. peregrinum
US	Ae. kotschyi	✓	✓	T. kotschyi
UC	Ae. triuncialis	✓	✓	T. triunciale
UM	Ae. biuncialis	Ae. lorentii	Ae. lorentii	T. macrochaetum
UM	Ae. columnaris	✓	✓	T. columnare
UM	Ae. geniculata	Ae. ovata	✓	T. ovatum
UM	Ae. neglecta	Ae. triaristata	✓	T. neglectum
UMN	”	Ae. triaristata	✓	T. rectum
T	Amblyopyrum mutica	Ae. mutica	Ae. mutica	T. tripsacoides
	–	–	Ae. turcomanica	–

One of the most important aspects of Aegilops is that it is closely related to bread wheat Triticum aestivum L. (AABBDD), which is one of the most important calorie sources for human nutrition. The D genome originated from the diploid species of Aegilops tauschii Coss. ( = Ae. squarrosa L.) (Kihara, 1946; McFadden and Sears, 1946), and the B genome was derived from a closely related species to Ae. speltoides Tausch (Riley et al., 1958; Sasanuma et al., 1996; Petersen et al., 2006; Kilian et al., 2007; Zhang W. et al., 2018) which has the S genome. Aegilops species are distributed from Europe to western China in a species-specific manner (van Slageren, 1994), adapted to many different climatic zones including drought/heat environments, different disease hot spots and nutrient-poor areas. It has been reported that Aegilops possesses useful traits for wheat breeding (For review to see; Kilian et al., 2011) including drought tolerance (Damania et al., 1992; Waines et al., 1993; Rekika et al., 1998; Monneveux et al., 2000; Farooq and Azam, 2001), heat tolerance (Waines, 1994), salinity (Colmer et al., 2006), aluminum toxicity tolerance (Miller et al., 1995) and resistance to several pests and diseases such as rust (Mihova, 1988; Anikster et al., 2005; Liu et al., 2010; Rouse et al., 2011; Vikas et al., 2014; Huang S. et al., 2018; Olivera et al., 2018), powdery mildew (Lutz et al., 1994; Buloichik et al., 2008), Hessian fly (El Bouhssini et al., 2008), cereal aphid (Holubec and Havlıckova, 1994) and barley yellow dwarf virus (BYDV) (Makkouk et al., 1994). In addition, the species can adapt to low phosphorous environments (Liu et al., 2015) and can contribute to higher iron and zinc content in wheat grain (Rawat et al., 2009).

In order to effectively exploit these useful traits in wheat, it is necessary to overcome extra difficulties with the introgression process, including a hybridization barriers, incompatibilities/hybrid abnormalities, sterility of F1s and, reduced meiotic chromosome pairings. Despite these obstacles, many Aegilops genes have been transferred to wheat and have been heavily utilized over the last 60 years (For review to see; Schneider et al., 2008; Kilian et al., 2011). Aegilops is also contributing to abate two recent threats to the global wheat production: Ug99 stem rust race derivatives and wheat blast (Magnaporthe oryzae Triticum). When Ug99 (original pathotype TTKSK) appeared in the early 2000s (Pretorius et al., 2000), more than 80% of wheat varieties did not have resistance against the race (Pretorius et al., 2000) and as such, wheat breeders sought resistance traits in Aegilops. When Wheat blast disease emerged in Bangladesh in 2016 (Ceresini et al., 2018), resistant wheat varieties were non-existent in the country, as well as neighboring India. Yet, a resistant variety was released within 2 years because of a resistance gene from Aegilops that was previously introgressed and ready for use (Cruz et al., 2016; Velu et al., 2018a; Mahmud, 2019).

In this paper, I will first review some difficulties relating to the use of Aegilops species (Supplementary Figure S1). Then, I will provide information on the contribution of Aegilops to wheat breeding in terms of identified genes in Aegilops, as well as some recent information on how Aegilops has contributed to the crisis prevention of Ug99 stem rust and wheat blast disease, which may change perspectives of Aegilops species as important sources for wheat breeding.

Hybridization Barriers Between Wheat and Aegilops Species and Crossability Genes

To utilize the genetic resources in the Aegilops genus, it is necessary to first produce hybrids between wheat and Aegilops species. Wheat can be either a female or male parent of the F1s, depending on species and specific cross combinations.

In wheat × Aegilops crosses, crossability genes on the wheat side have been highlighted for their significant role on the success rate of obtaining F1 hybrids with Aegilops species (Figure 1). This is a key point considering it is very difficult to produce F1s using low crossable wheat parents. While East Asian wheat landraces generally have higher crossability success rates with Aegilops and other alien species (e.g., rye), European ones have lower rates of success (Zeven, 1987), presumably because European wheat has had greater chances to cross-pollinate with rye histroically. Even though crossability is a QTL trait and controlled by several genes (Alfares et al., 2009), two dominant genes Kr1 (5BL) and Kr2 (5AL) were two major genes (Lein, 1943) affecting pollen tube growth (Riley and Chapman, 1967). These two genes have effects across different species including Hordeum and Aegilops (Snape et al., 1979; Koba and Shimada, 1993). Kr1 has a stronger effect than Kr2, and dominant alleles (Kr1 and Kr2) have inhibition effects. Plants with Kr1Kr2 show less than 10% crossability, Kr1kr2 showed between 10 and 25% crossability, kr1Kr2 between 25 and 50% and plants with the kr1kr2 genotype more than 50% crossability (Lein, 1943). Additionally, crossability genes were also reported as Kr3 on 5D (homoeologous of Kr1 and Kr2) and Kr4 on 1A (Krolow, 1970; Zheng et al., 1992). More recently, SKr on 5BS was reported to have a stronger crossable effect than Kr1 (Tixier et al., 1998; Alfares et al., 2009; Mishina et al., 2009).

FIGURE 1

Effects of crossability on seed setting. (A) A high crossable durum line. (B) A low crossable durum line. The durum spikes show seed setting 2 weeks after pollination with Ae. tasuchii. The high crossable line (A) sets six grains, while the low crossable line (B) sets zero grains.

If it is too difficult to produce F1 hybrids in wheat × certain Aegilops species (wheat as females), pollination in the opposite cross direction (Aegilops as females) may be more successful. Dale et al. (2017) reported 0% seed setting in bread wheat × Ae. tauschii crosses (probably due to a crossability problem of the bread wheat parents), while it was 30% in Ae. tauschii × bread wheat. The seed-setting rate with Aegilops as female parents is variable across these species. Yuan et al. (2017) reported the rate was about 0.2% in Ae. speltoides × bread wheat, 2–9% in Ae. cylindrica, 12–15% in Ae. ovata and 22–47% in Ae. tauschii. It must be cautioned that the seed setting does not always mean success in obtaining F1 plants.

Endosperm and Embryo Development Deficiency and Embryo Rescue

Gill et al. (1981) observed endosperm abortion and embryo lethality or semi-lethality and seedling death in crosses between Ae. tauschii and three diploid Triticum species. While the reaction types were different in each three Triticum species, the same thing is common in Ae. tauschii × bread wheat crosses. Even though the initial seed-setting rate was a 47% (Yuan et al., 2017), the seedling formation rate dropped to 1%. Sehgal et al. (2011) reported that an average of 35% initial embryo formation ended in an average of 7% F1 plants.

The degree of endosperm development deficiency is cross-combination specific. However, high polyploidy Aegilops tend to set endosperm more when crossed with wheat, while diploid Aegilops species set less (data not shown). To overcome endosperm abortion, embryo rescue is necessary to recover hybrid seedlings. In this procedure, embryos are dissected from developing grains and transferred to an agar medium with nutrients such as sugar and salts for proper development (Miller et al., 1987). While some wheat lines such as Langdon (durum wheat) or various East Asian landrace lines tend to develop enough endosperm for the embryo to form seeds (Koba and Shimada, 1993), the amount of endosperm sometimes will be lower than normal “wheat × wheat crosses” (Figure 2). It is possible to skip embryo rescue if using these lines. The genetic background of forming unreduced gametes in wheat is not known yet.

FIGURE 2

Endosperm development deficiency in F1 grains. (A) normal selfed-seeds of durum wheat, (B) durum wheat cv. LANGDON × Ae. tauschii; (C) durum wheat cv. CIRNO × Ae. tauschii. The seed sizes are smaller in durum × Ae. tauschii. The panel (B) has some amount of endosperm, and the seeds can germinate. The panel (C) has no endosperm, and the seeds will not germinate. Embryo rescue is necessary on the right.

Overcoming Sterility of F1S and Unreduced Gametes

The genome of F1s between wheat and Aegilops in haploids causes sterility until doubling the chromosome numbers. One option is to conduct direct backcrossing of F1s with wheat as a pollen donor. Even though the rate of seed set is extremely low, it is possible to obtain BC1 plants (Cox et al., 1990; Fritz et al., 1995; Zemetra et al., 1998; Olson et al., 2013). The alternative is through chemical treatments such as colchicine (Blakeslee and Avery, 1937; Tang and Loo, 1940; Bennett and Smith, 1979) and N2O gas (Hansen et al., 1988). Some wheat lines such as Langdon produce unreduced gametes, which is a gamete with a 2n nucleus resulting from abnormal meiosis (Fukuda and Sakamoto, 1992; Cai et al., 2010) that leads to spontaneous amphidiploid formation. The formation of unreduced gametes have been reported in durum × Ae. tauschii, Ae. speltoides, Ae. longissima, Ae. umbellulata, Ae. comosa, Ae. ovata, ( = Ae. geniculata), Ae. ventricosa, Ae. crassa and Ae. triuncialis (Xu and Dong, 1992; Matsuoka and Nasuda, 2004; Tiwari et al., 2008; Fakhri et al., 2016). The rate of formation is different among Aegilops species and prevented by the presence of a shared homologous subgenomes (Fakhri et al., 2016). Additionally, it depends on the genotype of the Aegilops parents (Matsuoka and Nasuda, 2004; Fakhri et al., 2016).

Hybrid Necrosis/Weakness Abnormality

Hybrid necrosis, chlorosis and bushy plant formation is very common in “normal” wheat × wheat cross (Hermsen, 1963; Hermsen and Waninge, 1972; Pukhalskiy et al., 2000; Chu et al., 2006). The Ne1-Ne2 necrosis system is the best known hybrid necrosis system in wheat, which is caused when two complementary genes of Ne1 (5BL) and Ne2 (2BS) are found in the same plant (Tsunewaki, 1960; Nishikawa et al., 1974; Chu et al., 2006). However, this phenomenon is more frequent and complex in wheat × Aegilops crosses. Necrosis in T. turgidum × Ae. tauschii was first reported in the 1960s (Nishikawa, 1960, 1962a,b; Figure 3). Mizuno et al. (2010) did further analysis using a set of synthetic wheat lines that had one common durum wheat parent “Langdon” and different Ae. tauschii accessions. They found four different types of hybrid abnormality and responsible genes Net1 (7DS), Net2 (2DS), and Hybrid chlorosis1 (Hch1; 7DS) in Ae. tauschii (Mizuno et al., 2010, 2011; Nakano et al., 2015). The mode of action of these genes should be complementary with genes on the durum side, because the hybrid abnormalities take place only when Ae. tauschii is crossed with durum wheat. Hypersensitive response-like reactions were observed for Net1 necrosis, indicating that it is a kind of disease response reaction (Jeuken et al., 2009; Mizuno et al., 2010). Okada et al. (2017) also reported growth abortion and grass-clump dwarf phenotype in durum × Ae. umbellulata. They also showed a repressed expression of the shoot meristem maintenance-related and cell cycle-related genes in the plants with the grass-clump dwarf phenotype. To avoid a problem with hybrid seedling death, Dhaliwal et al. (1986) reported the suppression of Ne1-Ne2 necrosis at high temperatures. The author also confirmed that incubation at 28°C suppressed necrosis in F1s between emmer × Ae. tauschii (Supplementary Figure S2). However, the high temperature causes pollen sterility.

FIGURE 3

Hybrid necrosis observed in tetraploid wheat × Ae. tauschii. (A) F1 from Triticum turgidum ssp. durum cv. CIRNO C 2008 × Ae. tauschii WX (224); all plants of this group are growing normally. (B) F1 from Triticum turgidum ssp. dicoccum (PI 233433) × Ae. tauschii WX (224); all plants are showing a necrosis symptom of reddish leaf color.

Gametocidal Genes

A group of gametocidal genes (Gc), sometimes considered as selfish genes, is another type of obstacle in which the genes cause chromosome breakages in gametes without Gc (Endo and Tsunewaki, 1975; Maan, 1975; Endo and Katayama, 1978). This happens when a plant becomes heterozygous in Gc —half of the gametes will have Gc and the other half will have no Gc. Gametes without Gc show reduce fitness, which is to the advantage of gametes with Gc for the transmission to the next generation (For review, see Tsujimoto, 2005; Endo, 2007; Niranjana, 2017). Gc genes have been identified in accessions of certain species that have C, S, Sl, or M genomes and mostly confined to three different homoeologous groups: 2, 3, and 4 (Endo, 2007). The identified genes include chromosome 3C of Ae. markgrafii ( = Ae. caudata) and Ae. triuncialis (Endo and Tsunewaki, 1975), 2C of Ae cylindrica (Endo, 1979), 2Sl and 4Sl of Ae. longissima, 2Ssh and 4Ssh of Ae. sharonensis (Maan, 1975; Endo, 1985), 2S and 6S of Ae. speltoides (Tsujimoto and Tsunewaki, 1984, 1988; Kota and Dvorak, 1988) and 4M of Ae. geniculata (Kynast et al., 2000). The effects of Gc genes are variable; some cause lethality to gametes, while others are mild, allowing incorporation of the gamete into progenies. King et al. (2018) reported the presence of a 2S chromosome segment in all of the developed wheat-Ae. speltoides introgression lines due to the gametocidal effect. When researchers use these species, it is better to keep in mind that extra difficulties may arise from Gc genes. The suppression of Gc genes was reported in Norin 26, which inhibits Ae. triuncialis Gc3-C1 action and is designated as Igc1 (Tsujimoto and Tsunewaki, 1985). The presence of additional suppressor genes can also be predicted because the effect of a Gc gene is different in various wheat backgrounds. The Gc of chromosome 3C is usually lethal but when found in “Chinese Spring” background, it is mild. In addition, Friebe et al. (2003) produced a mutant of the Ae. sharonensis Gc2 gene (designated as Gc2mut) which has a suppression effect on Gc2, which will be useful to reduce problems of Gc genes in wheat breeding scheme.

The Use of Ae. tauschii For Wheat Breeding

Aegilops tauschii is the easiest species in this genus to utilize in wheat breeding, because there is little to no inhibition to meiotic chromosome pairing with the D genome chromosomes of bread wheat. According to several sources, bread wheat originated about 10,000 years ago (Wang et al., 2013; Matsuoka and Takumi, 2017), which is relatively recent and not long enough for genomic differentiation. Furthermore, Ae. tauschii contrasts with diploid A genome ancestors. Luo et al. (2000) reported about a 1/6 recombination-rate reduction between Triticum monococcum 5A and bread wheat 5A chromosomes when compared to the recombination rate between two T. monococcum 5A homologous chromosomes. Even though the A genome of bread wheat and that of the diploid ancestor can form perfect bivalents during meiosis in the F1s of AAB (Gill et al., 1988), there are likely to be significant differences in base sequences and chromosome structures (such as inversion, translocations, deletion/duplications, or heterochromatin structures) after the tetraploid wheat formation – i.e., 100,000–500,000 years ago (Huang et al., 2002).

The spontaneous formation of bread wheat in nature was a rare event during which only a very limited number of Ae. tauschii plants were involved, based on molecular data and field observations (Dvorak et al., 1998; Matsuoka, 2011; Wang et al., 2013). The genetic diversity of Ae. tauschii is far greater in comparison to bread wheat’s D genome diversity (Dvorak et al., 1998; Wang et al., 2013). Matsuoka et al. (2013) proposed sub-dividing Ae. tauschii into three groups, TauL1, L2, and L3, and found that bread wheat is close to TauL2 but distinct from TuL1. Even though it is not obvious as in the case of T. monococcum, crosses of bread wheat with Ae. tauschii accessions of TauL1 may show a reduction in chromosome recombination rates of the A-genome chromosomes.

Figure 4 represents two ways to utilize Ae. tauschii in wheat breeding, either through direct crossing or indirect crossing (synthetic wheat). With indirect crossing, tetraploid wheat (AABB) will be crossed with Ae. tauschii (DD) to produce an F1 (ABD), and subsequently this F1 will have its chromosome number doubled naturally or artificially to produce so-called synthetic wheat (AABBDD). Synthetic wheat can then be used in wheat breeding by crossing with bread wheat. Synthetic wheat lines were first developed in the United States and Japan in 1940s (Kihara, 1944, 1946; McFadden and Sears, 1944). During the next few decades, a number of synthetic wheat lines were developed by various groups (Kihara and Lilienfeld, 1949; Tanaka, 1961; Dyck and Kerber, 1970; Kerber and Dyck, 1979; Hatchett et al., 1981; Chèvre et al., 1989; Valkoun et al., 1990; Lange and Jochemsen, 1992; Lutz et al., 1994; Wang et al., 2006). Later in the 1980s, CIMMYT started a large-scale production of synthetic wheat, developing more than 1,000 lines (Das et al., 2016; Li et al., 2018). Matsuoka et al. (2007) also reported another set of “Langdon” synthetic wheat lines, and Zeng et al. (2016) produced synthetic wheat using local Chinese land races that were more adaptable to China.

FIGURE 4

The use of Ae. tauschii in wheat breeding. (A) indirect crossing pathway (synthetic hexaploid pathway) through durum × Ae. tauschii crosses; (B) direct crossing pathway through bread wheat × Ae. tauschii crosses.

In the direct crossing pathway, Ae. tauschii (DD) is crossed with bread wheat (AABBDD) to make an F1 (ABDD). These F1s are then backcrossed with the same bread wheat (AABBDD) to generate BC1, where the plant selection process begins. Gill and Raupp (1987) and Cox et al. (1992) reported this method as successful for transferring Hessian fly and rust resistance. The merit of this method is that it will only change the D genome, making it easy to perform some analyses, as well as directly improving the “best” line without contribution from durum wheat. One of disadvantage of this method may be sterility of the F1 plants even as females, and as such, it is necessary to backcross a large number of spikes to have enough BC1 seeds to introgress the whole genome (Cox et al., 1990; Fritz et al., 1995; Olson et al., 2013). It is important to note that the seed setting rates in F1 plants also depend on Ae. tauschii accessions (Matsuoka and Takumi, 2017).

Octaploid synthetic wheat is another way to utilize Ae. tauschii in wheat breeding, in which an F1 (ABDD) from bread wheat (AABBDD) × Ae. tauschii (DD) has its chromosome number doubled to produce an octaploid synthetic wheat (AABBDDDD) (Chèvre et al., 1989). Sehgal et al. (2011) and Zhang D. et al. (2018) reported the production of five and one AABBDDDD lines, respectively. CIMMYT has also produced a few hundred octaploid synthetic wheat lines (Supplementary Figure S3). This research resulted in the successful transfer of a dormancy QTL (Dale et al., 2017) and Septoria tritici Blotch resistance (Figure 5).

FIGURE 5

Improvement of Septoria disease resistance through synthetic octaploid wheat. The derivative shows resistance while wheat parent line “NAVOJOA” is susceptible.

Table 2 summarizes the resistance genes identified and/or transferred into wheat, including leaf rust, stem rust, stripe rust, powdery mildew, and Hessian fly resistance. It is difficult to identify genes related to abiotic stress (drought and heat) and yield potential, as these traits are not obvious by sight. However, synthetically derived lines have shown up to a 30% yield increase under rain-fed conditions, and a 45% yield increase under drought condition over their wheat parents (Narasimhamoorthy et al., 2006; Dreccer et al., 2007; Trethowan and Mujeeb-Kazi, 2008; Li et al., 2014) and better performance under heat (Iehisa and Takumi, 2012; Jafarzadeh et al., 2016). The percentage of synthetic derivative lines (SDLs) in the Semiarid Wheat Yield Trial reached 52% in 2010, with a five-year average (2010–2015) of 35%. At least, 62 wheat varieties were released using CIMMYT synthetic wheat in their pedigree around the world since 2003 (Li et al., 2018). Elbashir et al. (2017) reported that synthetic derivative lines are promissive for improving heat tolerance in the analysis of multiple synthetic derivative (MSD) lines that cover the whole diversity of Ae. tauschii.

Table 2

Identified or transferred biotic resistance genes of Ae. tauschii into wheat.

Disease/pest	Gene (s)	Method	References
Leaf rust	Lr21	SHW	Dyck and Kerber, 1970
	Lr22a	SHW	Rowland and Kerber, 1974
	Lr32	SHW	Kerber, 1987
	Lr41	Direct	Cox et al., 1994
	Lr42	Direct	Cox et al., 1994
Stem rust	Sr33	SHW	Dyck and Kerber, 1970
	Sr45	SHW	Marais et al., 1998
	Sr46	SHW	Yu et al., 2015
Stripe rust	Yr28	SHW	Singh et al., 2000
Powdery mildew	Pm2a	SHW	Lutz et al., 1995
	Pm19	SHW	Lutz et al., 1995
	Pm34	Direct	Miranda et al., 2006
	Pm35	Direct	Miranda et al., 2007
	Pm58	Direct	Wiersma et al., 2017
Septoria tritici	Stb5	SHW	Arraiano et al., 2001
	Stb16	SHW	Ghaffary et al., 2012
Septoria nodorum	Snb3	SHW	McIntosh et al., 2008
Tan spot	Tsr3 ( = tsn3)	SHW	Tadesse et al., 2006
Cyst nematode	Cre3	SHW	Eastwood et al., 1991
	Cre4	SHW	Eastwood et al., 1994
Root knot nematode	Rkn1	SHW	Kaloshian et al., 1990
Hessian fly	H13	SHW	Gill et al., 1987
	H22	Direct (D)	Raupp et al., 1993
	H23	Direct (D)	Raupp et al., 1993
	H24	Direct (D)	Raupp et al., 1993
	H26	SHW	Cox and Hatchett, 1994
Greenbug	Gb3	SHW	Hollenhorst and Joppa, 1983
	Gb4	SHW	Martin et al., 1982
	Gb7	SHW	Weng et al., 2005
	(Gba, Gbb, Gbc,	SHW	Zhu et al., 2005
	Gbd, Gbx2,	SHW	”
	Gbx1, Gbz)∗	Direct	”
Russian wheat aphid	Dn3	SHW (D)	Nkongolo et al., 1991
Wheat curl mite	Cmc1	Direct (D)	Thomas and Conner, 1986
	Cmc4	Direct	Malik et al., 2003
Soil-Borne Cereal Mosaic Virus	SBWMV ( = allelic of Sbm1?)	Direct	Hall et al., 2009

The Use of Tetraploid/Hexaploid Aegilops Species With a D Genome

Hybrids between tetraploid Aegilops species with the D genome can show meiotic pairing with the D genome chromosomes of bread wheat (Kimber and Zhao, 1983). These species include Ae. cylindrica, Ae. crassa, and Ae. ventricosa, Ae. juvenalis and Ae. vavilovii. Amphiploids of wheat with Ae. crassa have been reported (Jovkova et al., 1977; Xu and Dong, 1992), and a number of amphiploids of Ae. ventricosa × durum wheat were produced (Delibes et al., 1987). CIMMYT has also developed 20 amphiploid lines of these species using durum and bread wheat (Figure 6) for bread wheat D genome improvement (Supplementary Figure S4).

FIGURE 6

Amphidiploid lines derived from between durum and diploid or tetraploid Aegilops with a D genome. (A) durum wheat cv. CIRNO C 2008, (AABB); (B) durum cv. CIRNO C 2008 × Ae. tauchii (895), (AABBDD); (C) durum cv. CIRNO C 2008 × Ae. ventricosa (PI 542385), (AABBDDNN); (D) durum cv. ACONCHI 89 × Ae. crassa (PI 542385), (AABBDDMM); (E) durum cv. CIRNO C 2008 × Ae. crassa (PI 542385), (AABBDDMM); (F) durum cv. CIRNO C 2008 × Ae. cylindrica (PI 639294), (AABBDDCC). All amphidiploids were produced and maintained at CIMMYT.

The eye spot resistance gene Pch1 (one of two strong seedling resistance genes) was transferred from an amphiploid (AABBDDNN) between tetraploid wheat (AABB) × Ae. ventricosa (DDNN) (Table 2). This amphiploid was crossed with bread wheat (AABBDD) to have a derivative line named “VPM1” (Maia, 1967; Doussinault et al., 1983). It was later determined that the location of the transferred Pch1 is chromosome 7D (Mena et al., 1992).

The Use of Other Aegilops Species and Chromosome Pairing

For the use of other Aegilops species, a reduced chromosome pairing frequency is more problematic. Ae. cylindrica, Ae. crassa, Ae. ventricosa, Ae. Juvenalis, and Ae. vavilovii are also categorized in this group due to the presence of non-D genomes.

Because of the lack of recombination, it is common to produce so-called alien chromosome addition or substitution lines in which one pair of Aegilops (or alien) chromosomes is added to or substituted for a pair of wheat chromosomes, respectively (Supplementary Figure S5). A number of addition lines have been produced from 14 different Aegilops species (For a review see; Schneider et al., 2008; Kilian et al., 2011). These addition lines are very useful for analysis and locating useful genes at the chromosomal level. However, addition lines have less breeding value, because they have many negative factors and the presence of an extra alien chromosome disrupts the genetic harmony of a genome. To be more appropriate for breeding, it is necessary to produce introgression lines (small Aegilops chromosome segment transfers) or Robertsonian/centromeric translocation lines (Robertson, 1916), in which one of the Aegilops chromosome arms is translocated to a wheat chromosome, replacing an arm of that wheat chromosome (Supplementary Figure S5). These translocations can be obtained spontaneously from addition/substitution lines or amphiploids in backcrossing populations, or using wheat monosomic lines (2n = 41; with one of the homoeologous chromosomes is missing). All of these can lead to the occurrence of univalent chromosomes during meiosis. Then, the meiotic spindle fiber will attach to the both sides of univalent chromosomes, which then causes chromosome breakages through the centromeric regions at high frequency. The broken chromosome arms are sticky and may fuse to other broken chromosomes to produce centromeric translocations (Supplementary Figure S6).

Homoeologous meiotic pairing between chromosomes of wheat and Aegilops species is inhibited mostly by the Ph1 gene (5BL) (Okamoto, 1957; Riley and Chapman, 1958; Sears and Okamoto, 1958; Riley, 1960). Therefore, the meiotic barrier can be overcome by suppressing of Ph1 activity. Sears (1977) produced the ph1b mutant in which the Ph1 locus is missing and this is the most widely used Ph1 gene mutant in wheat breeding. Another gene which affects homoeologous chromosome pairing was identified as Ph2 (3D) (Mello-Sampayo, 1971) and has a mild inhibition effect on Ph1 (Sears, 1982). Additional mutants, ph1c (Giorgi, 1983) and ph2 (Sears, 1982) are also available, even though they have been rarely used in the breeding. It is also known that the presence of Ph1 suppressors or promotors of homoeologous chromosome paring are present in some accessions of Aegilops species: Ae. speltoides (Feldman and Mello-Sampayo, 1967; Dover and Riley, 1977; Dvorak et al., 2006), Ae. longissima (7), Ae. mutica (Dover and Riley, 1972), Ae. umbellulata (Riley et al., 1973), Ae. Peregrina, and Ae. kotschyi (Fernandez-Calvin and Orellana, 1991) and Ae. geniculata (Koo et al., 2017). Therefore, the transfer of traits may be easier in accessions that have the suppressive effects. The Ae. speltoides genes are considered to be suppressants, because they can promote more meiotic pairing in the presence of the Ph1 gene (Dover and Riley, 1972). A couple of suppressor genes of Ae. speltoides has been transferred into wheat and designated as PhI (Chen et al., 1994) and Su1-Ph1 (7S) and Su2-Ph1 (3S) (Dvorak et al., 2006; Li et al., 2017). Since these genes are dominant, they can be faster and easier to utilize in breeding. Yet the effects of PhI have been shown to be lower than that of ph1b (Aghaee-Sarbarzeh et al., 2000).

Sometimes it is difficult to induce homoeologous recombination due to different homoeologous co-linearity between wheat and Aegilops chromosomes (Molnár et al., 2013, 2016). It is also known that centromeric and other chromosomal regions may have very low recombination rates, even in wheat × wheat crosses (Saintenac et al., 2009). In these cases, other methodologies become an alternatives. Sears (1956) demonstrated a successful transfer of Ae. umbellulata Lr9 gene into wheat using irradiation. Yet this is the only success story using irradiation for introgression of Aegilops chromatin for wheat breeding until recently. Singh et al. (2016) and Verma et al. (2016) recently reported the production of a translocation by irradiation of Ae. kotschyi hybrids. Mild effect Gc genes and some chemicals can also induce random translocations, much like irradiation. Even though it is not for breeding purposes, the Gc system has been used for producing translocations of wheat-rye and wheat-barley (Joshi et al., 2013; Li et al., 2013; Ishihara et al., 2014).

Useful Genes of Aegilops Transferred to Wheat

Through the use of the various techniques described above, a number of genes have been transferred from Aegilops (including Ae. tauschii) to wheat (Tables 3, 4). In terms of total number, leaf rust resistance genes are the most numerous (20), followed by powdery mildew (15), and green bug (12). Since more than 75 resistance gene loci have been identified and permanently designated as resistance genes by 2018 (Ponce-Molina et al., 2018). Aegilops provided more than 20% of them. For powdery mildew, 54 resistance loci were found by 2018 (Tang et al., 2018), and Aegilops contributed about 20%. For Cereal Cyst Nematodes (CCN) resistance genes, a total of 12 genes have been identified, including Cre1-8, CreR, CreV, CreX, CreY (Ali et al., 2019). Of them, two (Cre1 and Cre8) are indigenous to the wheat genepool. The others are from Ae. tasuchii (Cre3 and Cre4), Ae. ventricosa (Zhuk.) (Cre2, Cre5, and Cre6), Ae. triuncialis L. (Cre7); Ae. peregrina (CreX and CreY), Secale cereale (CreR) and Dasypyrum villosum (CreV) (Zhang et al., 2016), showing that two thirds of them are from Aegilops. In terms of actual species of origin, Ae. tauschii has provided the most number of genes, followed by Ae. speltoides and then Ae. ventricosa. It is worth noting that most of the disease resistances from Ae. ventricosa are provided by a single 2NS-2AS translocation, including Lr37, Sr38, Yr17, Cre5, Rkn3 (Bariana and McIntosh, 1993, 1994; Jahier et al., 1996, 2001; Helguera et al., 2003; Tanguy et al., 2005; Williamson et al., 2013); this translocation has originated from VPM1 (Maia, 1967) that also has Pch1 resistance on 7D (Mena et al., 1992).

Table 3

Identified or transferred biotic resistance genes in Aegilops (other than from Ae. tauschii) into wheat.

Disease/pest		Genome	Gene	References
Eyespot	Ae. ventricosa	DN	Pch1	Doussinault et al., 1983
		(recombination between two D genomes)
Leaf rust	Ae. umbellulata	U	Lr9	Sears, 1956
			Lr76	Bansal et al., 2017
	Ae. speltoides	S	Lr28	McIntosh et al., 1982
			Lr35	Kerber and Dyck, 1990
			Lr36	Dvorak and Knott, 1990
			Lr37	Bariana and McIntosh, 1993
			Lr47	Helguera et al., 2000
			Lr51	Helguera et al., 2005
			Lr66	Marais et al., 2009a
	Ae. kotschyi	US	Lr54	Marais et al., 2005
	Ae. sharonensis	Ssh	Lr56	Marais et al., 2010
	Ae. geniculata	UM	Lr57	Kuraparthy et al., 2007
	Ae. triuncialis	UC	Lr58	Kuraparthy et al., 2011
	Ae. peregrina	US	Lr59	Marais et al., 2008
	Ae. neglecta	UM	Lr62	Marais et al., 2009b
Stem rust	Ae. speltoides	S	Sr32	McIntosh, 1988
			Sr39	Kerber and Dyck, 1990
			Sr47	Klindworth et al., 2012
	Ae. comosa	M	Sr34	McIntosh et al., 1982
	Ae. ventricosa	DN	Sr38	Bariana and McIntosh, 1993
	Ae. searsii	Ss	Sr51	Liu et al., 2011a
	Ae. geniculata	UM	Sr53	Liu et al., 2011b
Stripe rust	Ae. comosa	M	Yr8	Riley et al., 1968
	Ae. ventricosa	DM	Yr17	Bariana and McIntosh, 1993
	Ae. kotschyi	US	Yr37	Marais et al., 2005
	Ae. sharonensis	Ssh	Yr38	Marais et al., 2010
	Ae. geniculata	UM	Yr40	Kuraparthy et al., 2007
	Ae. neglecta	UM	Yr42	Marais et al., 2009b
	Ae. umbellulata	U	Yr70	Bansal et al., 2017
Powdery mildew	Ae. speltoides	S	Pm1d	Hsam et al., 1998
			Pm12	Jia et al., 1996
			Pm32	Hsam et al., 2003
			Pm53	Petersen et al., 2015
	Ae. longissima	Sl	Pm13	Donini et al., 1995
	Ae. geniculata	UM	Pm29	Zeller et al., 2002
	Ae. umbellulata	U	Pm57	Liu et al., 2017
Cyst nematode	Ae. ventricosa	DN	Cre2	Delibes et al., 1993
			Cre5	Jahier et al., 1996
			Cre6	Ogbonnaya et al., 2001
	Ae. triuncialis	UC	Cre7	Romero et al., 1998
	Ae. peregrina	US	(CreX)	Barloy et al., 2007
			(CreY)	Barloy et al., 2007
Root knot nematode	Ae. peregrina	US	Rkn2	Yu et al., 1990
	Ae. ventricosa	DN	Rkn3	Williamson et al., 2013
Hessian fly	Ae. ventricosa	DN	H27	Delibes et al., 1997
	Ae. triuncialis	UC	H30	Martin-Sanchez et al., 2003
Green bug	Ae. speltoides	S	Gb5	Friebe et al., 1991

Table 4

List of resistance gene against stem rust Ug99 race.

Origin of Sr genes	Effective Sr genes
Triticum aestivum	Sr9h	Sr15∗1	Sr28	Sr42	+ 2 temporal∗2
(Partial; APR∗a)	Sr55	Sr56	Sr57	Sr58
Triticum dicoccum	Sr2	Sr13
Triticum timopheevi	Sr36	Sr37
Triticum araraticum	Sr40
Triticum monococcum	Sr21	Sr22	Sr35	Sr60	+ 2 temporal∗3
Aegilops tauschii	Sr33	Sr45	Sr46		+ 3 temporal∗4
Aegilops speltoides	Sr32	Sr39
Ae. sharonensis					+ 3 temporal∗5
Aegilops searsii	Sr51
Aegilops triuncialis	Sr47
Aegilops geniculata	Sr53
Aegilops umbellulata					+ 1 temporal∗6
Thinopyron ponticum	Sr24	Sr25	Sr26	Sr43
Thinopyrum intermedium	Sr44
Secale cereale	Sr27	Sr50	Sr59		+ 1 temporal∗7
Dasypyrum villosum	Sr52

Recently, Aegilops has gathered more attention for improving micro-nutrient content (such as Fe and Zn) in wheat grains. Zn deficiency affects 17.3% of the world’s population across Asia and Africa, leading to the deaths of more than 400,000 children each year (Cakmak, 2007; Black et al., 2013; Velu et al., 2018b). Micro-nutrient rich wheat, i.e., bio-fortified wheat, can improve the lives of these people. It is difficult to find high Zn and Fe content germplasm in the wheat genepool (Cakmak et al., 2010), even though some Aegilops species show three to four-fold higher Zn and Fe grain content, including Ae. longissima (Sl), Ae. kotschyi (US), Ae. peregrina (US), Ae. cylindrica (CD), Ae. ventricosa (DN), Ae. geniculata (UM) (Rawat et al., 2009). Amphiploid durum- Ae. longissima and partial amphiploids of wheat – Ae. kotschyi show two to three times higher levels of Zn and Fe grain content than the parental wheat line (Tiwari et al., 2008, 2010). Rawat et al. (2011) further reported Zn grain content three times higher in wheat- Ae. kotschyi addition/substitution lines than the wheat parent.

In addition to the benefit for wheat breeding mentioned above, it is also important to highlight that Aegilops introgression lines have a level of diversity and unique traits that wheat lacks. Even though these are of no immediate benefit at this moment, their value could be seen in the future, as exemplified by two recent global wheat production threats.

A Story of Aegilops Translocations on Stem Rust UG99 Race

A serious threat to global wheat production is the emergence of stem rust Ug99 race, which was recognized in Uganda in 1999 (Pretorius et al., 2000). This disease had the potential to develop into a global catastrophe, as more than 70% of wheat varieties around the world did not have resistance against Ug99 in the early 2000s (Singh et al., 2015). Many wheat breeders and pathologists, who had thought stem rust was no longer a problem, were caught unprepared and were then spurred to search for new resistant sources. The researchers realized that while the bread and durum wheat gene pools do not have many resistant sources, resistance is available outside the genepool from ancestral and alien species including many in Aegilops (Table 4, based on Yu et al., 2014; Kielsmeier-Cook et al., 2015; Randhawa et al., 2018 with updates by the author). This has also promoted various studies to identify new stem rust resistance genes, which led the identification of Sr46 (Ae. tauschii; Yu et al., 2015), Sr47 (Ae. triuncialis; Klindworth et al., 2012), Sr51 (Ae. searsii; Liu et al., 2011a), Sr53 (Ae. geniculate; Liu et al., 2011b) and three additional genes in Ae. tauschii (Rouse et al., 2011), three genes in Ae. sharonensis (Singh et al., 2015; Yu et al., 2017) and one gene in Ae. umbellulata (Edae et al., 2016). In addition, it has been reported that 81% of Ae. longissima (out of 394 accessions), 94% of Ae. neglecta (189 out of 202 accessions tested), 88% of Ae. cylindrica (DDCC) and Ae. peregrina (SSUU) were Ug99 resistant (Huang S. et al., 2018; Olivera et al., 2018).

Even though introgression lines of two Ug99 resistance genes (Sr32 and Sr39) from Ae. speltoides were available, they were not used in wheat breeding program due to the presence of large Ae. speltoides segments and associated negative factors on agronomy (Friebe et al., 1996). Fortunately, researchers in Australia and the United States started preparing for the possible appearance of dangerous new stem rust pathogen races back in the early 1990s and the reports of Ug99 just confirmed their expectations. Based on that work, shortened introgressions of chromosome 2S segments with Sr32 and Sr39 were already developed using the ph1b mutant and have been quickly distributed around the world (Mago et al., 2009, 2013; Niu et al., 2011).

It is notable that it has eight resistance genes (+ three temporary assigned genes) in the bread wheat gene pool are effective to Ug99, but four of them (Sr55, Sr56, Sr57, and Sr58) are partial or adult plant resistance genes (APR), so it is necessary to combine them with other genes to exert a higher level of resistance (Gustafson and Shaner, 1982; McIntosh et al., 1995, 1998, 2012).

A Story of the 2NS Translocation in Relation to Wheat Blast Disease

Wheat blast caused by Pyricularia oryzae (Magnaporthe oryzae) is an emerging disease that was first recognized in Brazil in the 1980s (Igarashi et al., 1986). The pathogen gained an ability to infect the new host plant wheat through a mutation of an avirulence gene (Inoue et al., 2017). Since then, it has been a serious obstacle for wheat production in central and south Brazil, south-east Paraguay and eastern Argentina, affecting 300 million ha of wheat fields and reducing the yield of infected areas 100–10% (Kohli et al., 2011; Perello et al., 2015; Duveiller et al., 2016). The disease jumped to Bangladesh in 2016 and spread to 15,000 ha (Malaker et al., 2016). Because of this serious threat to the wheat production of South Asia, quick remedial action was required to prevent a devastating epiphytotic (Mottaleb et al., 2018). Eight different resistance genes against wheat blast (Rmg1-8) have been reported, and only two of them (Rmg7 and Rmg8) are effective in the field in Bangladesh (Anh et al., 2017). Since Rmg7 and Rmg8 recognize the same avirulence gene peptide of the pathogen, both resistance genes are functionally equivalent to a single gene for resistance (Anh et al., 2017). Despite of lacking resistance sources, a new resistance wheat variety, “BARI com” was released in Bangladesh within 2 years in 2018. This happened because of the existence of the 2NS-2AS translocation (Cruz et al., 2016; Velu et al., 2018a; Mahmud, 2019). This translocation has been utilized in wheat breeding programs because of rust resistances (Juliana et al., 2017), but it also happens to have a strong wheat blast resistance. If 2NS-2AS had not have been produced, the wheat blast issue would have been a much more serious problem in the last few years. Another amazing finding with the 2NS-2AS translocation is that nearly 90% of advanced lines of the CIMMYT bread wheat program have this translocation (Juliana et al., 2017; Philomin Juliana, Personal communication). As in the case of the T1BL.1RS translocation that dominated wheat cultivars for decades, a beneficial translocation can have a huge impact on wheat breeding and production.

The Use of Aegilops in the Genomic Age for Breeding and Pre-Breeding

During the last several decades, cytogenetic methods not only have been essential tools for screening and understanding the nature of translocations and alien introgressions from a number of progenies, but also possess the major constraint in handling large numbers of samples. But new cytogenetic FISH/GISH technology using oligo probes expands the capacity, proving a valuable tool in cytogenetics (Du et al., 2017; Huang X. et al., 2018). More importantly, recent progress in high through-put genotyping technology and availability of molecular methods makes it possible to detect alien segments very easily, which has been promoting the production of alien segment introgressions. Niu et al. (2011) screened about 1,000 plants and found 40 smaller alien recombinants of Ae. speltoides 2S chromosome using the ph1b mutant. The development of translocations which cover a whole genome have been demonstrated in Ambylopyrum mutica ( = Ae. mutica) (King et al., 2017) and Ae. speltoides (King et al., 2018). The number of estimated introgression segments obtained are about 200 of Am. mutica (King et al., 2017), and a map of about 600 cM was made with 540 plants in the case of Ae. speltoides (King et al., 2018), which allowed the construction of linkage maps even using wheat- Aegilops introgression lines and the Axiom 35K SNP array that was constructed on a wheat sequence based Axiom 820K SNP array by optimizing for finding polymorphism between wheat and Aegilops species. An increased number of whole genome linkage or physical maps in Aegilops species have been available (Table 5). A 4-gigabase physical map based on BAC clones of Ae. tauschii led the construction of a 10 K Ae. tauschii Infinium SNP array (Luo et al., 2013). Moreover, the draft sequence of Ae. tauschii has been recently reported (Luo et al., 2017), and a TILLING population of Ae. tauschii was reported (Rawat et al., 2018). It will be possible to have additional physical maps and draft sequences in another Aegilops species in near future that will facilitate their use in wheat breeding and gene identifications.

Table 5

List of whole or semi-whole genome genetic or physical maps in Aegilops species.

Species name	Type of markers	Type of populations	References
Aegilops tauschii	RFLP∗1	F2 of Ae. tauschii	Gill et al., 1991
	RFLP; SSR∗2	F2 and F3 of Ae. tauschii	Boyko et al., 2002
	SSR	F2 of Ae. tauschii	Okamoto et al., 2013
	10K SNP array of Ae. tauschii	F2 of Ae. tauschii	Luo et al., 2013
	EST∗3; SSR; RJM∗4	RH∗10 of synthetic wheat	Kumar et al., 2012
	DArT∗5; SSR	RH of Ae. tauschii	Kumar et al., 2015
	SSR	F2 of Ae. tauschii	Nishijima et al., 2018
Aegilops speltoides	RFLP	F2 of Ae. speltoides	Dvorak et al., 2006
	Axiom 35K SNP array	Wheat/Ae. speltoides introgressions	King et al., 2018
Aegilops longissima	RFLP	F2 of Ae. longissima; CS/Ae. longissima addition	Zhang et al., 2001
	SSR	RIL∗11 of Ae. longissima	Sheng et al., 2012
	RNA-seq∗6	CS/Ae. longissima addition	Wang et al., 2018
Aegilops sharonensis	DArT; SSR	F2 of Ae. sharonensis	Olivera et al., 2013
	OPA∗7	RIL and F2 of Ae. sharonensis	Yu et al., 2017
Aegilops umbellulata	RFLP	CS/Ae. umbellulata addition	Zhang et al., 1998
	GBS∗8	F2 of Ae. umbellulata	Edae et al., 2016
	GBS	F2 of Ae. umbellulata	Edae et al., 2017
Aegilops caudata	SSR	CS/Ae. caudata addition	Niu et al., 2018
Aegilops comosa	PAUG∗9	CS/Ae. comosa addition	Liu et al., 2019
Amblyopyrum mutica	Axiom 35K SNP array	Wheat/Am. mutica introgressions	King et al., 2017

Yet the biggest limitation and challenge for the use of Aegilops is still reduced recombination rates between wheat and Aegilops chromosomes that is sometimes prohibitive in producing an Aegilops introgression segment. The new technologies such as MutChromSeq (mutant chromosome sequencing), MutRenSeq (Mutagenesis Resistance gene enrichment and sequencing) and AgRenSeq (Association Genetics R gene enrichment Sequencing) may provide an alternative to overcome gene identification obstacles. These techniques allow a rapid isolation of mutated genes with mutagenesis by sequencing sorted chromosomes (MutChromSeq) or enriching target gene families by exome capture (MutRenSeq) or a rapid isolation of natural variants by enriching target gene family (AgRenSeq) and sequencing for resistance gene homologs. Steuernagel et al. (2016) reported the cloning of Sr22 and Sr45 from bread wheat using MutRenSeq, Sánchez-Martín et al. (2016) reported the cloning of Pm2 using MutChromSeq, and, Arora et al. (2019) demonstrated the discovering and cloning of Sr33, Sr45, Sr46, and SrTA1662 from a panel of about 200 Ae. tauschii accessions using AgRenSeq. Development of new methodologies which can compensate the reduced recombination rate may overcome the biggest constrains of the use of Aegilops. Alternatively, we may be able to find new variations or genes to increase the recombination rate from Aegilops like PhI genes (Chen et al., 1994; Dvorak et al., 2006; Li et al., 2017).

As we can see from the stories of Ug99 and wheat blast, Aegilops species are important not only for pre-breeding but also for a proactive main-stream breeding. It is still necessary to induce a certain level of recombination between wheat and alien chromosomes for the use of Aegilops, but the new technologies are opening up a new era of Aegilops for wheat breeding.

Data Availability

All datasets generated for this study are included in the manuscript and/or the Supplementary Files.

Author Contributions

The author confirms being the sole contributor of this work and has approved it for publication.

Conflict of Interest Statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.