Stacking effective ASR and APR rust genes for multiple disease resistance in bread wheat cultivars

Abstract

Rusts and powdery mildews pose serious threats to wheat and have caused substantial yield losses worldwide. Host resistance is the most economical and sustainable approach for managing such diseases. In this study, an effective leaf rust resistance gene Lr45-derived from Secale cereale L. and a linked stem rust and powdery mildew resistance gene Sr36/Pm6-derived from Triticum timopheevii were successfully pyramided. They were validated into well-adapted Indian wheat cultivars that were already carrying the APR stem rust gene Sr2/Lr27/Yr30 through marker-assisted backcross selection (MABC) following two parallel backcrossing schemes. Three efficiently linked microsatellite markers, G372₁₈₅ (Lr45), Stm773-2(Sr36), and Xgwm533 (Sr2), were used to confirm introgression of these genes. Lines with resistance genes in each background showed improved agronomic traits in comparison to their recurrent parents. These lines could be used in wheat improvement programs as potentially resistant stocks for leaf, stem rusts and powdery mildew to develop new wheat cultivars.

Keywords:
T. aestivum; Lr45; Sr36/Pm6; Sr2; pink awns

INTRODUCTION

Wheat (Triticum aestivum L.) is the most widely grown crop worldwide, covering approximately 222.14 million hectares (USDA 2023). Rusts caused by Puccinia sp. and powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt) are key constraints on global wheat production. Of the three types of rust, leaf or brown rust were caused by Puccinia triticina Eriks. (Pt) is one of the most disruptive diseases affecting wheat crops worldwide because of its widespread occurrence (Bhardwaj et al. 2021) causing yield losses of up to 65% (Chhuneja et al. 2011). The stem rust caused by Puccinia graminis f. sp. tritici (Pgt) has caused yield losses of between 10% and 50% in recent years (Roelfs et al. 1992). However, the recent emergence and spread of the Ug99 race and its variants have been reported to cause upto100% damage (Amulaka et al. 2013). In addition to rust, PM is an emerging disease that causes considerable yield losses of up to 35% (Sharma et al. 1996). Recently, the incidence of PM has increased in several wheat-growing regions worldwide, including India (Vikas et al. 2020).

The development of disease-resistant varieties by stacking effective genes is one of the most effective and eco-friendly methods for combating rust and PM (Singh et al. 2020). Currently, over 80, 60, and 100 genes for resistance to leaf, stem rust, and PM, respectively, have been formally cataloged in wheat (McIntosh et al. 2020) and many genes offer all-stage resistance (ASR). ASR/seedling resistance is governed by a single major gene that is often race-specific (Rosewarne et al. 2013). The effectiveness of such genes can be detected in the early stages of plant growth (Bariana et al. 2022). Generally, ASR fails within a few years of deployment, owing to the emergence of virulent variants (Chen 2005). Conversely, adult plant resistance (APR) is effective either at the post-seedling or adult plant stage and is usually race-specific, governed by minor genes with additive effects. A few also have pleiotropic effects in conferring resistance to multiple diseases (Herrera-Foessel et al. 2014). However, the number of APR genes identified and reported to date is limited. As the pathogen continues to evolve, stacking resistance genes is being pursued by wheat breeders to extend the resistance offered by these genes.

The translocation of T2AS-2RS.2 RL from Petkus rye (Secale cereale L.) postulated to carry the ASR geneLr45 (Naik et al. 2015) is reported to be effective in the seedling and adult plant stages against leaf rust pathotypes worldwide (Zhang et al. 2006) and has not been widely used. A distinct morphological trait known as “pink awns or glumes” is tightly linked to this gene (Sivasamy et al. 2010). It appears during the early anthesis and then gradually disappears as maturity progresses. It was observed that its expression differs across varied environments. Triticum timopheevii derived stem rust ASR gene Sr36 originally transferred to wheat chromosome 2B and is effective in all the prevalent stem rust races, including the lineages of the Ug99 (Chemayek et al. 2017) except TTTSK (Jin et al. 2009). Although virulence has been reported for this gene, it is still effective on the Indian subcontinent (Sai Prasad et al. 2014) and continues to be widely exploited in combination with other minor or major genes (Jin et al. 2009). Sr36 is also closely linked to the effective powdery mildew resistance gene, Pm6 (Jorgensen and Jensen 1973). In India, the linked gene Sr36/Pm6 confers effective resistance to stem and powdery mildew (Sivasamy et al. 2017).

The stem rust APR gene Sr2 located on the short arm of wheat chromosome 3B (Hare and McIntosh 1979), provides broad-spectrum protection against stem rust and is associated with pseudo-black chaff (PBC), a dark pigmentation around the stem internodes and glumes post-anthesis.

Marker-assisted backcross (MABC) breeding is a recent approach for effectively transferring multiple genes for resistance, given that conventional breeding is time-consuming (Singh et al. 2018). The availability of specific microsatellite markers linked to resistance genes makes MABC easy and relatively rapid. The recurrent parents chosen in this study were released during the post-green revolution period, which had the highest level of adaptability to the changing environmental scenario and easy combining ability. However, the advancement of new virulent races renders these varieties more susceptible. Therefore, combining ASR and APR in such cultivars is desirable for the development of resistant stocks with higher levels of adaptability. With this objective, stacking multiple resistance genes for rust and PM in adapted wheat cultivars was initiated by pyramiding the ASR leaf rust gene Lr45 and stem rust gene Sr36/Pm6 with the APR stem rust gene Sr2/Lr27/Yr30 through MABC in the background of eight well-adapted Indian bread wheat varieties.

MATERIAL AND METHODS

Plant material and breeding method

Leaf rust resistance gene Lr45 was introgressed from a near-isogenic line (NIL) of Thatcher (Thatcher*7/ST−1 = RL6144) (Naik et al. 2015) into the background of eight well-adapted Indian bread wheat varieties viz., HD 2329, HD 2402, LOK-1, MACS 2496, NIAW 34, PBW 343, PBW 502, and RAJ 3077 that already carried race non-specific stem rust APR gene Sr2/Lr27/Yr30/Pbc (Bhardwaj 2011). Sr2+ alone is ineffective against stem rust. The selected recurrent parents were susceptible to leaf rust and PM. Likewise, the Australian line Cook (Cook*6/C80-1) carrying the Triticum timopheevi-derived gene Sr36/Pm6 (Chemayek et al. 2017) was used as a donor to transfer the Sr36 gene into the same background. These genes were introgressed into wheat varieties by adapting a backcross breeding approach assisted by linked molecular markers at the ICAR-Indian Agricultural Research Institute (IARI), Regional Station, Wellington, Nilgiris, through two parallel backcrossing schemes. Appropriate agronomic practices were followed to raise crops in each generation.

The F₁s carrying individual genes were intercrossed to form a pyramid with Lr45 and Sr36. The resulting F₁s was then backcrossed with the respective recurrent parents for up to three generations to raise the BC₃F₁ populations and recover the parental genome. The backcross progeny with the target genes were selected based on phenotype and MABC in each generation. From BC₃ onwards, the marker-selected homozygous plants were selfed for six generations to obtain homozygosity of the alleles received from the donor. The stable pyramided lines constituting BC₃F₆ were named HW 3641, HW 3642, HW 3654, HW 3655, HW 3657, HW 3660, HW 3661, and HW 3662.

Marker-assisted backcross breeding

Gene-specific microsatellite markers viz. G372 ₁₈₅ linked to Lr45 (Naik et al. 2015), Stm773-2 linked to Sr36 (Tsilo et al. 2008), and Xgwm533 linked to Sr2 (Spielmeyer et al. 2003) were used for the selection of the genes in this study. DNA was extracted from 14-20 day old seedlings following a modified CTAB method (Doyle and Doyle 1990). PCR reactions were conducted in 20 µL reaction containing 25-50 ng of template DNA, 0.2 µM of each forward and reverse primer, Dream Taq Hot Start Green PCR mix (Thermo Fischer Scientific), and nuclease-free water in an Applied Biosystem thermocycler (Veriti). The details of the primers and their PCR conditions are given as Supplementary data (Table 1A). PCR products were resolved on a 3% agarose gel and visualized using a gel documentation unit (Syngene, Gene Genius Bioimaging System, UK). PCR markers were used to select heterozygous plants from the BC₁ to BC₃ generations and homozygous resistant plants from the BC₃F₆ generation.

Table 1
Disease response in the seedling and adult plant stage and marker analysis data of the donor, recurrent parent and the pyramided lines

Parent/ Cross	Details	Seedling response leaf rust pathotypes					Seedling response stem rust pathotype					Adult plant response			Marker confirmation
		12−5	77−1	77−5	77−8	77−9	104−2	11	40A	40−2	117−6	Leaf rust	Stem rust	PM	Lr45 (G372 185)	Sr36/ Pm6 (stm773)	Sr2 (Xgwm533)
HD 2329	RP		-	3+	3+	3+	1	2=	2C	-	-	80S	60S	6	-	-	+
HW 3641	HD2329 (Lr45, Sr36/Pm6, Sr2+)	-	0	-	0	-		0	0		-	F	F	0	+	+	+
HD2402	RP	-	1	33+	-	3+	3+	12	12	0	-	40S	20S	6	-	-	+
HW 3642	HD2402 (Lr45, Sr36/Pm6, Sr2+)	-	0	0	-	-		0	-	0		F	F	0	+	+	+
LOK-1	RP	33+	3+	3+	3+	3+	3+	3+	3+	0	3+	60S	60S	7	-	-	+
HW 3654	LOK-1 (Lr45, Sr36/Pm6, Sr2+)		0	1		-	1	12	0	-	0	F	F	0	+	+	+
MACS2496	RP	22+	12	3+	0	-	3+	2=	-	0	2=	20S	F	6	-	-	+
HW 3655	MACS2496 (Lr45, Sr36/Pm6, Sr2+)	0	-	0	0	-	-	-	-	0	0	F	F	0	+	+	+
NIAW 34	RP	23	3+	X+3	-	3+	3+	-	12	2=	-	40S	20S	6	-	-	+
HW 3657	NIAW34 (Lr45, Sr36/Pm6, Sr2+)	-	-	0	-	-		0	0	-	0	F	F	0	+	+	+
RAJ 3077	RP	-	1	23	-	3+	33+	-	-		-	80S	20S	7	-	-	+
HW 3662	RAJ3077 (Lr45, Sr36/Pm6, Sr2+)	0	-	0	-	-	-	2+	12	0	-	F	F	0	+	+	+
PBW 343	RP	-	3+	3+	0	3+	3+	2=	0			40S	F	7	-	-	+
HW 3660	PBW343 (Lr45, Sr36/Pm6, Sr2+)	-	-	0	-	-		0	0	0	0	F	F	0	+	+	+
PBW 502	RP	33+	3+	3+	0	3	3+	-	-	-		40S	F	7	-	-	+
HW 3661	PBW502 (Lr45, Sr36/Pm6, Sr2+)	-	-	0	0	-	-	-	2=	0	-	F	F	0	+	+	+
RL6144 (Lr45)	Donor	0	-	0		-	-	0	3+	0	0	F	60S	4	+	-	+
COOK (Sr36/Pm6)	Donor	3+	-	0	3+	3+	3+	-	2−	1	-	80S	F	0	-	+	-

• * F - Free; S - Susceptible; RP - Recurrent Parent

Phenotypic selection

Phenotypic selection was performed for each generation to select plants resistant to leaf rust, stem rust, and powdery mildew under natural epiphytotic conditions. Wellington, being a natural hotspot for rusts and powdery mildew allows a natural selection of resistant lines. In 2021 in the summer and winter seasons, gene-pyramided plants in the BC₃F5 and BC₃F₆ generations were planted in one meter row of five lines, each with a line spacing of 23 cm between the rows. To ensure early disease onset and adequate disease pressure, infector rows were sown around the population. An aqueous suspension of viable mixed uredospores of prevalent Pt (77-1, 77-5,77-9, etc.) and Pgt (40A and 40-1) pathotypes in Wellington was sprayed at regular intervals of 15 d with a drop of Tween 20 (0.75 µL mL^-1).The field response to leaf and stem rust was recorded at the adult plant stage (Z80) (Zadoks scale) (Zadoks et al. 1974) according to the modified Cobb scale (Peterson et al. 1948) as the percentage of leaf and stem area covered with uredospores combined with the type of infection response. PM severity was scored on a 0-9 scale (Sheng and Duan 1991) where 0-3 was considered resistant.

Seedlings of the stable pyramided lines were evaluated for leaf and stem rust resistance under artificial glasshouse conditions at ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Regional Station, Flowerdale, Shimla in 2021. Seedlings that were approximately 10 d old sown in trays were inoculated with six pathotypes of Pt viz., 12-5 (29R45), 77-1 (109R63), 77-5 (121R63-1),77-8 (253R31), 77-9 (121R61-1) and 104-2 (21R55) and four pathotypes of Pgtviz., 11(79G31), 40A (62G29), 40-2 (58G13-3) and 117-6 (37G19). The infection type (IT) was recorded on a 0-4 scale as proposed by Stakman et al. (1962). The Infection types (ITs) 3, 3+ were considered susceptible, whereas lower ITs (“0”, “1,” “2,” and “X”) were considered resistant.

Agronomic evaluation of the advanced lines

Phenotypically and molecularly confirmed individual plants in the BC₃F₆ generation were planted with their respective recurrent parents in non-replicated plot trials in 2021-22. Ten uniformly resistant plants were selected from each background and data on plant height (PH) (cm), the number of productive tillers per plant (NPT), spike length (SPL) (cm), grain number per spike (GNS), and thousand grain weight (TGW) (g) were recorded.

RESULTS AND DISCUSSION

Genotypic Evaluation of gene stacked lines

Three microsatellite markers viz., G372 ₁₈₅ , Stm773-2, and Xgwm533 linked to Lr45, Sr36/Pm6, and Sr2 were used in MABC. The low availability of molecular markers limits the use of Lr45 in marker-assisted selection (Naik et al. 2015). Zhang et al. (2006) developed an amplified fragment length polymorphism (AFLP) marker for the detection of the Lr45 gene and Fein et al. (2009) developed a sequence-characterized amplified region (SCAR) marker. Naik et al. (2015) reported a highly polymorphic, co-dominant microsatellite marker (G372 ₁₈₅ ) that supported the efficient use of the Lr45 gene in the wheat improvement programs. Microsatellite marker G372 ₁₈₅ linked to Lr45 showed a single 185 bp allele in the homozygous resistant lines and a 127 bp allele or null allele in the lines devoid of the gene. Both alleles were present in the heterozygous lines (Figure 1A). Naik et al. (2015) validated the efficiency and specificity of this marker for Lr45 in Thatcher-based NILs.

Figure 1A
Molecular confirmation of leaf rust resistance gene Lr45 using marker G372 ₁₈₅ in the donor,recurrent parent, and pyramided lines (BC3F6). M: Marker; 1: RL 6144 (Positive control); 2: HD2329; 3-7: HW 3641, 8-HD 2402; 9-13: HW 3642, 14- Lok-1; 15-18: HW 3654; 19: MACS 2496; 20-22: HW 3655; 23: NIAW34; 24-28: HW 2657; 29: RAJ 3077; 30-33: HW 3662; 34: PBW343; 35-39: HW 3660; 40: PBW 502; 41-44: HW3661.

The STM (sequence-tagged microsatellite) marker Stm773-2 linked to Sr36/Pm6 developed a 155 bp amplicon in the pyramided lines homozygous for the presence of the gene and a 195 bp allele in the homozygous negative lines (Figure 1B). The codominant nature of this marker facilitates identification of heterozygotes (Tsilo et al. 2008). A microsatellite marker (Xgwm533) linked to Sr2 amplified a 120 bp allele in the lines positive for the gene and a 150 bp allele or null allele in the lines without the gene (Figure 1C). Similar screening of Sr2 in large populations using the microsatellite marker Xgwm533 was reported by Vishwakarma et al. (2019).

Figure 1B
Molecular confirmation of stem rust resistance gene Sr36/Pm6 using marker stm773 in the donor, recurrent parent, and pyramided lines (BC3F6). M-Marker (100bp); 1: Cook(Positive control); 2: HD2329; 3-6: HW 3641; 7: HD 2402; 8-13: HW 3642; 14: LOK-1; 15-17: HW 3654; 18: MACS 2496; 19-24: HW 3655; 25: NIAW 34; 26-30: HW 3657; 31: Raj 3077; 32-35: HW 3662; 36: PBW343; 37-39: HW3660; 40: PBW 502; 41-44: HW 3661

Figure 1C
Molecular confirmation of stem rust APR gene Sr2 using marker Xgwm533in the donor, recurrent parents, and pyramided lines (BC3F6). M: Marker; 1: Kingbird (Positive control); 2: Agralocal (Negative control); 3: HD2329; 4-7: HW 3641; 8: HD 2402; 9-14: HW 3642; 15: LOK-1; 16-10: HW 3654; 11: MACS 2496; 12-17: HW 3655; 18: NIAW34; 19-23: HW 3657; 24: RAJ 3077; 25-29: HW 3662, 30: PBW 343; 31-33: HW 3660; 34: PBW 502, 35-40: HW 3661.

Pyramiding multiple effective ASR genes with APR rust genes in the background of adapted cultivars is considered an effective strategy for enhancing resistance and durability (Jin et al. 2022). Stacking several disease-resistance genes in a single varietal background through phenotyping is difficult. Therefore, MABC is one of the most promising approaches for the incorporation and stacking of resistance genes while retaining the essential characteristics of the recurrent parent (Collard and Mackill 2008).

Phenotypic evaluation and screening of backcross population

Advanced lines stacked with Lr45, Sr36/Pm6, and Sr2+ were phenotypically evaluated in the field and were found to be completely resistant to leaf and stem rust. A susceptibility score of 20S-80S to both stem rust and leaf rust was recorded in recurrent parents (HD 2329, HD 2402, Lok-1, RAJ 3077, and NIAW 34). The recurrent parents, PBW 343, PBW 502, and MACS 2496, were resistant to stem rust because they carried the stem rust resistance gene Sr31+ and were only susceptible to leaf rust (20S-40S). The well-known 1BL/1RS translocation carrying the gene Sr31+ has contributed substantially to world wheat production, and several hundred cultivars possessing this gene locus were released during the mid-1990s and are continuing to be effective in the Indian subcontinent (Prasad et al. 2022). However, virulence has been reported extensively worldwide.

The APR gene Sr2+ in combination with other resistance genes is effective against the Ug99 lineage (Alabushev et al. 2019). However, phenotyping the resistance offered by Sr2 is difficult under field conditions because it confers partial resistance (Spielmeyer et al. 2003). Therefore, the microsatellite marker, Xgwm533 linked to Sr2 was used to accelerate the selection of lines carrying this gene.

Juvenile plants pyramided with the resistance genes showed a resistant infection response of 0-1 for the tested leaf and stem rust pathotypes whereas a susceptible infection type, ‘IT’ 33+ was recorded in the seedlings of the susceptible recurrent parents for leaf and stem rust races under controlled conditions. The rust and PM responses of the pyramided lines, donors, and recurrent parents are listed in Table 1.

In addition to phenotypic screening, morphological traits such as pink awns/glume linked to Lr45 (Sivasamy et al. 2010) which appear during the early flowering period (anthesis) and then gradually disappear as maturity progresses and PBC linked to Sr2+ further aided in the visual confirmation of the presence of the respective genes. However, direct selection based on these phenotypic traits is difficult because their expression varies across different environments.

Agronomic performance of the pyramided lines

Based on resistance to leaf and stem rust at the phenotypic and molecular level, ten plants from each background were selected and evaluated agronomically. The mean agronomic trait values are shown in Table 2. The gene-pyramided lines had agronomically superior traits, such as PH, NPT, SPL, GNS, and TGW, compared with their respective recurrent parents. There was a slight increase in spike length in the lax ear, which significantly increased TGW. From the data, it was observed that all gene-stacked lines carrying Lr45, Sr36/Pm6, and Sr2 showed a better TGW than the recurrent parent. The main components contributing to wheat yield are SPL, GNS, and TGW (Zheng et al. 2020). In certain backgrounds, such as Raj 3077, PBW 343, and PBW 502, there was an increase in GNS and SPL. In wheat varieties PBW 343 and PBW 502, the lowermost 2-3 spikelets remain sterile depending on the environmental conditions. However, the fertility of the lowermost spikelet also recovered after introgression of the Lr45 gene, which contributed to more GNS.

Table 2
Mean and Standard Deviation (SD) of agronomically significant traits of the recurrent parents and gene stacked lines

Parent/Cross	PH (cm)#	NPT	SPL (cm)	GNS	TGW (g)
HD 2329	70 ±2.07	8 ±0.54	9 ±0.25	51 ±3.42	26 ±0.83
HW 3641	80 ±1.65*	10 ±2.60*	9 ±0.13	48 ±2.12*	40 ±0.54*
HD2402	82 ±1.14	11 ±0.54	9 ±0.26	55 ±2.68	34 ±1.14
HW 3642	84 ±1.51*	12 ±1.67	12 ±0.54	56 ±1.64*	39 ±0.89*
LOK-1	89 ±0.65	7 ±1.09	9 ±0.2	45 ±2.12	32 ±1.22
HW 3654	75 ±2.75*	10 ±1.47*	10 ±1.03*	51 ±3.99*	34 ±2.45
MACS2496	89 ±0.68	8 ±0.89	11.2 ±0.22	65 ±3.87	32 ±0.54
HW 3655	90 ±1.03*	9 ±0.83	11.2 ±0.56	64 ±2.50	42 ±0.45**
NIAW 34	87 ±0.83	8 ±0.54	8.5 ±0.08	57 ±2.12	34 ±1.14
HW 3657	76 ±2.96*	12 ±1.64*	9 ±0.08	55 ±1.64	31 ±1.09**
RAJ 3077	83 ±2.12	9 ±0.89	7.7 ±0.17	45 ±2.12	24 ±1.14
HW 3662	82 ±1.51	14 ±1.30*	10.2 ±0.79*	49 ±1.64*	32 ±0.70*
PBW 343	83 ±2.38	12 ±1.09	8.6 ±0.11	54 ±2.68	28 ±2.07
HW 3660	80 ±2.02	14 ±0.70*	9.9 ±0.37*	61 ±2.68*	30 ±0.54*
PBW 502	74 ±1.14	9 ±0.54	9.8 ±0.20	51 ±2.12	26 ±1.92
HW 3661	82 ±1.17*	13 ±2.12*	10.6 ±0.65*	56 ±1.64*	32 ±0.70*

• * Recurrent parent and gene stacked line have significant differences (P < 0.05). ** Recurrent parent and gene stacked line have an extremely significant difference. # PH - Plant height, NPT - Number of productive tillers per plant, SPL - spike length, GNS - Grain number per spike, and TGW - Thousand grain weight.

Suppression of other diseases

The MABC-derived lines were resistant to leaf and stem rust and were also resistant to PM because of the presence of the Pm6 gene that inherited Sr36. Sivasamy et al. (2017) reported the effectiveness of Pm6 on Indian wheat. The recurrent parents in the study also carried other resistance genes, such as Lr13+, Lr10+, Lr23+, Lr1+, Sr8b+, Sr9b+, Sr11+, Yr2+, Yr18+, Yr2KS+ and Yr27+ (Bhardwaj 2011). Most of these genes were ineffective in the past. However, the accumulated residual effect of the defeated genes generally termed as the ghost effect (Martin and Ellingboe 1976) has also added to resistance when stacked with other effective ASR and APR resistance genes.

In conclusion, enhancing the genetic resistance of adapted or elite wheat cultivars to rust and PM is a highly decisive approach for tackling the rapid momentum of pathogen evolution. Resistance offered by a single ASR or major gene has an inherent risk of being overcome by newly evolving rust pathotypes. Therefore, the combination of the ASR and APR genes was found to have a more significant effect. In the process of stacking resistance genes, the synergistic effect of stacked genes has been reported to increase the lifespan of each gene (Klymiuk et al. 2018, Mundt 2018). Rapid stacking of genes could be conducted by taking advantage of the favorable environmental conditions prevalent in Wellington, which allows growing of two crop cycles per year. The gene-stacked lines developed with Lr45, Sr36 and Sr2 could be a potential source of new resistant varieties and could be suggested as potential germplasm resistant to leaf rust, stem rust, and PM. The co-dominant markers G372 _185, Stm773-2, and Xgwm533 allow the efficient detection of genes in the homozygous state and would serve as an important tool in the rapid transfer of these genes into adapted wheat cultivars.

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Publication Dates

• Publication in this collection
  29 May 2023
• Date of issue
  2023

History

• Received
  22 Nov 2022
• Accepted
  07 Mar 2023
• Published
  20 Mar 2023