Genome-wide Mapping of Quantitative Trait Loci Conferring Resistance to Stripe Rust in Spring Wheat Line PI 660072

doi:10.21203/rs.3.rs-3752526/v1

Download PDF

Research Article

Genome-wide Mapping of Quantitative Trait Loci Conferring Resistance to Stripe Rust in Spring Wheat Line PI 660072

https://doi.org/10.21203/rs.3.rs-3752526/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating diseases of wheat worldwide. Identifying resistance genes is crucial for developing resistant cultivars to control the disease. Spring wheat PI 660072 (Triticum aestivum) has been identified to possess both adult-plant resistance (APR) and all-stage resistance (ASR) to stripe rust. To elucidate the genetic basis of the resistance in PI 660072, a mapping population consisting of 211 F5 - F7 recombinant inbred lines (RILs) was developed from a cross of PI 660072 with susceptible spring wheat Avocet S. The mapping population was phenotyped for stripe rust responses across five field environments from 2020 to 2022 and genotyped using the 15K SNP (single nucleotide polymorphism) array to map stripe rust resistance loci. The mapping population was also tested at the seedling stage with predominant Chinese Pst races CYR31, CYR32, CYR34 and PST-YX1-3-1 in the greenhouse. Stripe rust resistance genes were identified using the quantitative trait locus (QTL) mapping approach. Two QTL were identified with QYrPI660072.swust-2BL mapped on the long arm of chromosome 2B for ASR and QYrPI660072.swust-4BL on the long arm of chromosome 4B for APR. To facilitate marker-assisted selection breeding, Kompetitive allele specific PCR (KASP) markers, KASP-1269 for QYrPI660072.swust-2BL and KASP-3209 for QYrPI660072.swust-4BL, were developed. These markers could be used to introgress the effective resistance QTL into new wheat cultivars.

Two major QTL for resistance to stripe rust were mapped on chromosome 2BL and 4BL in spring wheat PI 660072, and their KASP markers were developed.

Wheat stripe rust is a highly contagious disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst). The pathogen is capable of spreading rapidly over long distances and causing large-scale epidemics (Wellings 2011; Chen 2020). When wheat crops in a large geographic region are affected by stripe rust, the grain yield can be reduced by 5% − 20% (Wellings 2021). In an individual field grown with a highly susceptible wheat cultivar, severe stripe rust can cause a complete loss of grain yield (Chen 2014; Zhou et al. 2022). The most effective approach for controlling stripe rust is to breed resistant cultivars. Unfortunately, many wheat varieties that were once resistant to the disease have become susceptible in China and many other countries (Chen et al. 2009; Kang et al. 2015). It is urgent to identify wheat germplasm and genes for resistance to be used in developing wheat cultivars with effective and durable resistance to stripe rust (Zhou et al. 2015b).

There are two types of stripe rust resistance genes, all-stage resistance (ASR) and adult-plant resistance (APR). ASR refers resistance to the disease throughout all growth stages, whereas plants with only adult-plant resistance (APR) are susceptible at the seedling stage, but become resistant after the seedling stage and the resistance level can increase as plants grow older and often when the weather becomes warmer (Chen 2005, 2013). ASR can be easily detected in the seedling stage and easily transferred into new cultivars. When effective, an ASR gene can provide complete control for the cultivar or cultivars carrying the gene, but the gene can be circumvented by new virulent races of the pathogen. In contrast, APR is usually non-race specific and therefore durable. However, APR is usually partial, and some APR genes may not provide adequate resistance if stripe rust starts early in the plant growth season and the weather is not warm enough for the full expression of the resistance genes (Chen 2013, 2014). The best approach is to combine both effective ASR and APR genes in wheat cultivars to achieve adequate and durable resistance for control of stripe rust (Chen 2013).

To date, 86 officially named Yr genes (Klymiuk et al. 2022; Feng et al. 2023; Zhu et al. 2023) and more than 300 provisionally named genes or quantitative trait loci (QTL) in wheat and its wild relatives have been identified for resistance to stripe rust (Wang and Chen 2017; Pakeerathan et al. 2019; Li et al. 2020). Of the 86 permanently designated genes, about 30 genes confer APR, including Yr11, Yr12, Yr13, Yr14, Yr16, Yr18, Yr29, Yr30, Yr34, Yr36, Yr39, Yr46, Yr48, Yr49, Yr52, Yr54, Yr56, Yr58, Yr59, Yr60, Yr62, Yr68, Yr71, Yr75, Yr77, Yr78, Yr79, Yr80, Yr83 and Yr86; and the others confer ASR. Six ASR genes (Yr5, Yr7, Yr15, YrAS2388, YrSP and YrU1) (Klymiuk et al. 2018; Marchal et al. 2018; Wang et al. 2020; Zhang et al. 2020) and three APR genes (Yr18, Yr36 and Yr46) (Fu et al. 2009; Krattinger et al. 2009; Moore et al. 2015) have been cloned. Although the number of reported stripe rust resistance genes is quite large, many of the race-specific ASR genes are no longer effective. It is still needed to identify more effective genes and develop molecular markers for more efficiently breeding stripe rust resistant wheat cultivars.

The utilization of marker-assisted selection (MAS) enables breeders to incorporate multiple resistance genes into new cultivars (Zhou et al. 2015a). With the advancing high-throughput sequencing and molecular marker technologies, more effective and user-friendly markers have been developed for various traits, including stripe rust resistance and used in breeding programs through MAS (Barendse et al. 2009; Kump et al. 2011). Simple nucleotide polymorphisms (SNPs) offer an extremely promising approach for exploring genetic variations in crop germplasms and permit the identification of markers closely linked to the target genes or QTL (Wu et al. 2018). By converting SNPs into Kompetitive Allele Specific PCR (KASP) markers, individual markers can be used to screening breeding lines for the specific traits (Rasheed et al. 2016).

Spring wheat PI 660072 was developed by the Wheat Health, Genetics and Quality Research Unit of the US Department of Agriculture, Agricultural Research Service (USDA-ARS) and Washington State University and deposited in the USDA-ARS National Small Grains Collection (Wang et al. 2012). The line was selected from the progeny from a cross of stripe rust susceptible spring wheat Avocet S (AvS) and resistant spring wheat line PI 180957 originally from India. In the previous study, PI 660072 was resistant to US Pst races PST-114 and PST-127 and moderately resistant to PST-43 and PST-100 in the seedling tests and highly resistant in the fields under natural Pst infection in Washington State before its registration, and it was thus concluded to have both ASR and high-temperature adult-plant resistance (Wang et al. 2012). PI 660072 has continually shown high resistance to stripe rust in the United States (Wang MN and Chen XM, unpublished data). In China, PI 660072 was also highly resistant to the predominant Pst races in both greenhouse seedling and field adult-plant tests (Zhou et al. 2015b). However, the genetic basis of the stripe rust resistance in PI 660072 was not clear. The objectives of this study were to: 1) genetically characterize the stripe rust resistance in PI 660072 and map its resistance genes using a whole-genome QTL mapping approach, 2) assess the stability of the resistance genes across different environments, 3) determine if the genes confer ASR and APR and 4) to develop KASP markers to be used in MAS.

Plant materials

Used as the male parent, PI 660072 was crossed to AvS for developing a mapping population. AvS, an Australian spring wheat selection, is susceptible to most Pst races in Australia, China, the United States and many other countries, and was used as the recurrent parents in developing near-isogenic lines and mapping populations to identifying stripe rust resistance in many wheat genotypes (Wellings et al. 2004; Lin et al. 2007; Sui et al. 2009; Cheng et al. 2010; Li et al. 2011; Ren et al. 2012; Wang et al. 2012; Sharma-Poudyal et al. 2013; Xu et al. 2013; Cheng et al. 2014; Lu et al. 2014; Zhou et al. 2014; Zhou et al. 2015b; Xiang et al. 2016; Feng et al. 2023). From the cross AvS/PI 660072, a mapping population consisting of 211 recombinant inbred lines (RIL) was developed using the single-seed descent method, and its F₅ - F₇ generations were used in the phenotypic tests for stripe rust responses.

Field tests

The 211 RILs, together with PI 660072 and AvS, were evaluated for stripe rust response at Mianyang (MY) (31°33′N, 104°55′E, 485 m above sea level) in Sichuan province in 2020–2022 using the F₅ - F₇ generations, respectively and at Yangling (YL) (34°17′N, 108°04′E, 530 m above sea level) in Shaanxi province in 2021 and 2022 using the F₆ and F₇ generations, respectively. MY is an area where Pst is able to overwinter and occurs naturally with no need of artificial inoculation. The predominant Pst races in Shannxi and Sichuan provinces were CYR32, CYR33, and CYR34 in recent years (Ma et al. 2016; Wang 2017; Li et al. 2018; Zhao et al. 2023). In the YL nurseries, the plants were inoculated with a mixture of the prevalent races CYR32 and CYR34. The field tests were conducted in a completely randomized block design with three replications. In each replication, approximately 30 seeds for each line were sown in a row of 100 cm with 25 cm between rows. The parents and susceptible check were planted every 20 rows throughout the field. ITs were recorded based on the 0–9 scale, with 0–3 classified as resistant, 4–6 moderately resistant and 7–9 susceptible (Line et al. 1992; Wan et al. 2004), and disease severity (DS) was recorded as percentage of leaf areas infected. Both IT and disease severity (DS) of each row were recorded three times starting when DS on MX169 reached 80% and plants were at the adult stage with the second and third notes taken 7 and 14 days after the first note, respectively.

Seedling tests

Seedling tests were conducted in a greenhouse. Chinese predominate Pst races CYR31, CYR32 and CYR34 and a new local isolate (PST-YX1-3-1) were used for evaluating the stripe rust responses of PI 660072, AvS and selected F₇ RILs. CYR32, CYR34 and PST-YX1-3-1 were used in phenotyping the mapping populations. In the seedling phenotyping tests, the two parents and their F₈ RILs were seeded in a 9 × 9 × 9 cm pot with approximately 10 seeds and grown in the greenhouse with the temperature and light conditions optimal for wheat seedling growth. The seedlings were inoculated at the two-leaf stage with urediniospores of a single race. Inoculated plants were kept in a dew chamber in the dark at 8 ℃ for approximately 24 h. The seedlings were then moved to a growth chamber at 16 ℃ with a daily 16 h light for stripe rust development. Approximately 15 days later, the infection type (IT) data were recorded based on a 0–9 scale (Line and Qayoum 1992). Chinese wheat cultivar Mingxian 169 (MX169) was used as a susceptible check.

Genotyping the parental lines and RILs

Genomic DNAs of the parents and the 211 F₆ RILs were extracted from fresh seedling leaves using the cetyltrimethylammonium bromide (CTAB) method (Anderson et al. 1993). The stock DNA solutions were determined for their quality and concentration using a NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE, USA) and were diluted to 50 ng/µl using deionized distilled water (ddH₂O) to be used for genotyping. The parents and RILs were genotyped using the 15K Illumina® iSelect wheat SNP array by Zhongyujin Marker Biotechnology Co., Ltd. (Beijing, China).

Statistical analyses and QTL mapping

The IT data from greenhouse, and IT and DS data from each environment were used for the analysis of variance (ANOVA) and subsequent QTL mapping. ANOVA and Pearson’s correlation coefficients were calculated using QTL IciMapping V4.1 (Meng et al. 2015). The broad-sense heritability (\({ℎ}^{2}\)) of stripe rust resistance was estimated using the following formula: \({ℎ}^{2}={{\sigma }}_{g}^{2}/({{\sigma }}_{g}^{2}+{{\sigma }}_{ge}^{2}/e+{{\sigma }}_{{\epsilon }}^{2}/re)\), where \({{\sigma }}_{g}^{2}\), \({\sigma }_{ge}^{2}\) and \({{\sigma }}_{{\epsilon }}^{2}\) are estimates of the genotype, genotype × environment interaction and error variances, respectively, and e and r are the number of environments and number of replicates for each environment, respectively (Yang et al. 2005). For the plotting purpose, the step was set to 1 cM and the PIN (probability of SNP being included in the model) was set to 0.001. For QTL mapping, the threshold value of the logarithm of odds (LOD) score for each chromosome analysis was set to 2.5 to declare a significant QTL. To determine the additive effects of QTL, boxplots were used to demonstrate the effects of QTL combinations on the mean IT and DS of RILs that shared the same number of beneficial alleles.

Developing KASP markers

To find more close markers for stripe rust resistance QTL, the parents were genotyped using the 660K SNP chip. SNP markers near or at the peaks of the identified resistance QTL based on the results obtained from both 15K and 660K SNP chips were selected for developing KASP markers by Zhongyujin Marker Biotechnology Co., Ltd. (Beijing, China). The developed KASP markers were validated using the parents and RILs.

Phenotypic evaluation

The infection types of AvS and PI 660072 tested with the Pst races or isolates were shown in Table 1. AvS was susceptible (IT 8–9), whereas PI 660072 was resistant to CYR31 (IT 2), CYR32 (IT 3), CYR34 (IT 2) and PST-YX1-3-1(IT 2). In the field tests, AvS had IT 9 across the three years and two locations, and its final DS values ranged from 90–100%. In contrast, PI 660072 was highly resistant (IT 2) in all field tests with DS 10–20%. The mean values of IT and DS of the RILs ranged from 0 to 9 and 0 to 100%, respectively, suggesting that the stripe rust resistance in PI 660072 was quantitatively inherited (Fig. 1a, b). The broad-sense heritability values of IT and DS were 0.86 and 0.87, respectively. The ANOVA analysis revealed significant differences (P < 0.001) among RILs, environments, and line × environment interactions, indicating that the resistance genes in the RIL population are the primary source of phenotypic variation (Table 2). The correlation coefficients for IT and DS ranged from 0.73–0.95 and 0.68–0.95 (P < 0.001) based on the YL and MY data, respectively (Table 3).

Table 1

Infection types of AvS, PI 660072, and 15 selected F₇ recombinant inbred lines tested with *Puccinia striiformis* f. sp. *tritici* (*Pst*) races or isolates CYR32, CYR34 and PST-YX1-3-1 in the seedling stage under controlled greenhouse conditions
		Infection types produced by Pst race isolate
Wheat line	QTL	CYR31	CYR32	CYR34	PST-YX1-3-1^a
AvS	None	9	9	9	9
PI 660072	2BL, 4BL	2	3	2	2
RIL-5	2BL	2	2	3	2
RIL-19	2BL	1	2	4	2
RIL-32	2BL	1	2	5	4
RIL-53	2BL	1	2	2	2
RIL-94	2BL	1	2	5	1
RIL-166	2BL	2	2	5	3
RIL-179	2BL	2	2	5	2
RIL-182	2BL	1	2	5	1
RIL-209	2BL	1	2	3	3
RIL-18	4BL	7	7	7	7
RIL-40	4BL	7	7	7	7
RIL-44	4BL	7	7	7	7
RIL-76	4BL	7	7	7	7
RIL-128	4BL	7	7	7	7
RIL-131	4BL	7	7	7	7
^a PST-YX1-3-1 is an isolate obtained from stripe rust collection in Zhongxing Town, Youxian District, Mianyang, Sichuan in 2023, and its race based on the Chinese differentials has not been determined.

Table 2

Analysis of variance and the estimates of broad-sense heritability (h²) of the infection type (IT) and disease severity (DS) in the recombinant line (RIL) population of AvS × PI 660072 tested at Mianyang (MY) and Yangling (YL) in 2020–2022
Source	IT			DS
Source	df^a	MS^b	F value	df	MS	F value
Line	210	25.38	12.59***^c	210	3322.62	12.65***
Environments	3	268.91	133.40***	3	14068.55	53.57***
Line/environment	630	38.86	19.28***	630	9110.39	34.69***
Error	840	2.02		840	262.63
h²	0.86			0.87
^a df, degree of freedom
^b MS, Mean square
^c “***” denotes the significant level of P < 0.001

Table 3

Correlation coefficients (r) of infection type (IT) and disease severity (DS) of the recombinant inbred lines of AvS × PI 660072 tested in different environments
Trait	Environment^a	20MY	21MY	21YL	22MY	22YL
IT	20MY	1.00***^b
	21MY	0.73***	1.00***
	21YL	0.75***	0.75***	1.00***
	22MY	0.75***	0.68***	0.71***	1.00***
	22YL	0.95***	0.74***	0.79***	0.78***	1.00***
DS	20MY	1.00***
	21MY	0.75***	1.00***
	21YL	0.78***	0.92***	1.00***
	22MY	0.73***	0.68***	0.73***	1.00***
	22YL	0.95***	0.76***	0.79***	0.77***	1.00***
^a MY and YL denote Mianyang and Yangling, respectively. 20, 21 and 22 denote 2020, 2021 and 2022, respectively
^b The r values based on the DS data are given in parentheses. “***” denotes the r value is significant at P < 0.001

Inheritance of stripe rust resistance

To determine the number of genes conferring stripe rust resistance in PI 660072, genetic analysis was performed. In the F₅ generation tested in Mianyang in 2020, 170 lines were resistant and 41 lines susceptible, fitting a 3:1 ratio (χ² = 3.20; P = 0.06). In the F₆ generation at Mianyang in 2021, 174 lines were resistant and 37 lines susceptible, marginally fitting the 3:1 ratio (χ² = 5.88; P = 0.01). In the F₆ generation tested at Yangling, 172 lines were resistant and 39 lines susceptible, also marginally fitting the 3:1 ratio (χ² = 4.44; P = 0.03). In the F₇ generation tested at Mianyang in 2022, 167 lines were resistant and 44 lines susceptible, fitting the 3:1 segregation ratio (χ² = 1.72; P = 0.16). In the F₇ generation tested at Yangling, 170 lines were resistant and 41 lines susceptible, fitting the 3:1 segregation ratio (χ² = 3.20; P = 0.06). When the F₈ generation tested at the seedling stage in the greenhouse with CYR32, 167 lines were resistant and 44 lines susceptible, fitting the 3:1 segregation ratio (χ² = 1.72; P = 0.16); with CYR34, 167 lines were resistant and 44 lines susceptible, fitting the 3:1 segregation ratio (χ² = 1.72; P = 0.16); and with PST-YX1-3-1, 174 lines were resistant and 37 lines susceptible, fitting the 3:1 segregation ratio (χ² = 5.88; P = 0.01) (Table 4). The genetic analyses indicated two genes for stripe rust resistance.

Table 4

The number of genes for resistance to stripe rust in the AvS × PI 660072 determined by genetic analysis based on the infection type data of adult plants in Mianyang (MY) and Yangling (YL) in 2020 (20), 2021 (21), 2022 (22) and seedling stage with CYR32, CYR34 and PST-YX1-3-1 in 2023
Test	R^a	S^b	Total	No. of genes	χ²	P-values
20MY(F₅)	170	41	211	2	3.20	0.06
21MY(F₆)	174	37	211	2	5.88	0.01
22MY(F₇)	167	44	211	2	1.72	0.16
21YL(F₆)	172	39	211	2	4.44	0.03
22YL(F₇)	170	41	211	2	3.20	0.06
CYR32	167	44	211	2	1.72	0.16
CYR34	167	44	211	2	1.72	0.16
PST-YX1-3-1	174	37	211	2	5.88	0.01
^a R, RILs showing IT < 7
^b S, RILs showing IT ≥ 7

Genetic linkage maps

A total of 13,946 SNPs were found between the two parents using the 15K SNP array, of which 13,198 SNPs had known chromosomal locations. After filtering the redundant markers based on “BIN”, 3,869 SNPs were used to construct genetic linkage maps. These markers were distributed across 21 linkage groups corresponding to the 21 wheat chromosomes, spanning a total length of 5,884.82 cM (Fig. 2). The number of markers per chromosome in the genetic linkage map ranged from 7 for chromosome 1D to 248 for chromosome 3A, with an average of 94 SNP markers (Table 5). The map lengths of individual chromosomes ranged from 35.10 cM (chromosome 6A) to 994.95 cM (chromosome 7D). The mean distance between adjacent SNP markers in the genetic linkage map varied across chromosomes, ranging from 0.37 cM for chromosome 6A to 29.93 cM for chromosome 1D. The genetic maps were subsequently used to locate the stripe rust resistance QTL.

Table 5

Summary of chromosome assignment, number of SNPs, map length and marker density of the genetic maps of the AvS × PI 660072 recombinant inbred line population
Chromosome	No. of SNPs	Map length (cM)	Mean SNP distance (cM)
1A 1B 1D 2A 2B 2D 3A 3B 3D 4A 4B 4D 5A 5B 5D 6A 6B 6D 7A 7B 7D	98 12 7 46 87 105 248 33 43 74 66 66 55 244 33 95 49 100 58 220 235	382.44 236.82 209.51 354.94 351.74 214.90 278.34 307.41 218.87 297.17 135.32 247.24 231.87 237.06 137.75 35.10 271.85 298.95 307.08 135.51 994.95	3.90 19.73 29.93 7.72 4.04 2.05 1.12 9.32 5.09 4.02 2.05 3.75 4.22 0.97 4.17 0.37 5.55 2.99 5.29 0.62 4.23

QTL mapping

To identify the resistance genes for stripe rust in PI 660072, QTL analysis was conducted using the genotypic data and the stripe rust phenotype data from the five field environments and the seedling tests with three races in the greenhouse. Two major QTL for resistance to stripe rust were identified in the mapping population and were located on chromosomes 2BL and 4BL, named QYrPI660072.swust-2BL and QYrPI660072.swust-4BL, respectively (Fig. 3a, b). Both QTL were from PI 660072. QYrPI660072.swust-2BL was located between SNP markers AX-109547533 and AX-111640532 at the 376,495,312 bp and 682,801,798 bp positions, respectively, about 21.0 cM apart. This QTL explained about 10.04–24.77% phenotypic variation explained (PVE), with the LOD value of 3.78 ‒ 11.63. QYrPI660072.swust-4BL was located between AX-109412222 and AX-108847266, at 362,135,412 bp and 429,339,058 bp positions, respectively, about 1.5 cM apart. This QTL explained 20.50 ‒ 28.18% PVE, and its LOD value ranged from 13.59 to 18.10 (Table 6).

Table 6

Summary of two quantitative trait loci (QTL) for stripe rust resistance identified based on mean disease severity (DS) and infection type (IT) of the 211 recombinant inbred lines from cross AvS × PI 660072 tested in Mianyang (MY) and Yangling (YL) in 2020 (20), 2021 (21) and 2022 (22) and seedling stage with CYR32, CYR34 and *Pst*-YX1-3-1 in 2023
QTL	Environment	Marker interval	IT			DS
QTL	Environment	Marker interval	LOD	PVE (%)	Add	LOD^a	PVE (%)^b	Add^c
QYrPI660072.swust-2BL	20MY	AX-109547533 AX-111640532	9.92	17.32	-0.98	7.97	14.53	-9.83
	21MY		10.54	18.44	-0.87	11.22	19.62	-12.12
	21YL		7.82	12.89	-0.74	11.63	19.43	-14.54
	22MY		6.05	10.04	-0.69	7.81	14.27	-9.11
	22YL		8.74	15.24	-1.03	6.61	12.42	-10.18
QYrPI660072.swust-4BL	CYR32	AX-108847266 AX-109412222	10.16	24.34	-0.87	10.33	24.77	-9.57
	CYR34		5.16	12.91	-0.60	5.31	13.14	-6.40
	Pst-YX1-3-1		3.78	11.10	-0.52	3.83	11.15	-5.19
	20MY		17.19	25.74	-1.20	15.58	24.61	-12.85
	21MY		15.53	22.83	-0.97	14.10	20.50	-12.42
	21YL		15.26	24.74	-1.04	16.48	22.99	-15.87
	22MY		13.59	23.33	-1.06	14.66	22.50	-11.51
	22YL		17.96	27.40	-1.39	18.10	28.18	-15.40
	CYR32		-	-	-	-	-	-
	CYR34		-	-	-	-	-	-
	Pst-YX1-3-1		-	-	-	-	-	-
^a LOD, logarithm of odds score
^b PVE, percentage of the phenotypic variance explained by individual QTL
^c Add, the additive effect of resistance allele. A negative value indicates that the resistance allele is from PI 660072

Development and validation of KASP markers

The scanning of the 660K SNP chip for the two parents resulted in 65 different SNPs on chromosome 2B and 37 polymorphic SNPs on 4B between the parents. From the 65 SNPs on 2B, 24 were initially selected for converting to KASP markers, and 3 KASP markers were finally developed. From the 37 SNPs on chromosome 4B, 14 were initially selected for developing KASPs, and 4 KASP markers were eventually developed. The 3 KASP markers on chromosome 2BL were KASP-072-1269, KASP-072-7058 and KASP-072-4948, and the 4 on chromosome 4BL were KASP-072-3209, KASP-072-9918, KASP-072-2222 and KASP-072-1793. The corresponding SNPs of the three 2BL KASP markers were AX-108941269 (at the 682,743,322 bp position, AX-108757058 (640,086,397 bp) and AX-11114948 (655,225,005 bp); and those on 4BL were AX-109493209 (385,557,144 bp), AX-110399918 (376,431,384 bp), AX-109412222 (362,135,412 bp) and AX-111501793 (404,838,495 bp), respectively. KASP markers KASP-072-1269 on 2BL and KASP-072-3209 on 4BL were tightly linked to the target QTL (Fig. 3). The genotypes of seven recombinant RILs with their phenotypes in the genetic interval between markers AX-109412222 and AX-108847266 of QYrPI660072.swust-4BL are shown in Table 7, indicating that the QTL region has a high recombination frequency.

Table 7 SNP genotypes of seven recombinant inbred lines in the genetic interval AX-109412222 and AX-108847266 of QYrPI660072.swust-4BL (A, the resistant genotype from PI 660072; B, the susceptible genotype from AvS)

Marker^a	SNP genotype
Marker^a	PI 660072	RIL-146	RIL-33	RIL-31	RIL-81	RIL-117	RIL-181	RIL-80	AvS
AX-111022564	A	A	A	A	B	B	B	B	B
AX-110359713	A	A	A	A	B	B	B	B	B
AX-109412222	A	A	A	A	A	B	B	B	B
KASP-072-3209	A	A	A	A	A	A	B	B	B
AX-109015565	A	A	A	A	B	B	B	B	B
AX-108847266	A	A	A	B	B	B	B	B	B
AX-109368728	A	A	B	B	B	B	A	B	B
AX-109296842	A	B	B	B	B	B	A	A	B
Phenotype	R	R	R	R	R	S	S	S	S

^aThe eight markers were from the 15K SNP and KASP marker development

Effects of the individual QTL and their combination

To estimate the individual and combined effects of the two QTL on IT and DS, the 211 RILs were grouped into different genotypic groups based on the presence of markers highly associated with the two QTL. These genotypes were further grouped into 4 groups based on the number of QTL. RILs without any of the two QTL had a mean infection type (MIT) 6 and mean DS (MDS) 54.9%. RILs carrying only QYrPI660072.swust-2BL had MIT 4 and MDS 30%. RILs carrying only QYrPI660072.swust-4BL had MIT 3 and MDS 23%. RILs carrying the two QTL had MIT 2 and MDS 11%. As expected, these results demonstrated that RILs carrying the two QTL had the highest level of resistance. RILs carrying only QYrPI660072.swust-4BL had lower IT and DS than those carrying only QYrPI660072.swust-2BL (Fig. 4a, b). The results showed that combining different QTL could increase the level of resistance through additive interactions.

Determination of resistance types of the QTL

Based on the SNP marker data, 9 lines with QYrPI660072.swust-2BL and 6 lines with QYrPI660072.swust-4BL were identified from the 211 RILs. These 15 lines were either highly or moderately resistant at the adult-plant stage in all field tests. These lines were further tested with CYR31, CYR32, CYR34 and PST-YX1-3-1 at the seedling stage in the greenhouse, and their infection types produced by the four Pst races or isolates are presented in Table 1. The nine lines with QYrPI660072.swust-2BL were resistant to the four races (IT 0–4) similar to their field resistance (IT 1–4). The six lines with QYrPI660072.swust-4BL were intermediate to susceptible to the three races (IT 5–8), which were different to their high or moderate resistance (IT 2–5) in the field tests. Based on these results, we concluded that QYrPI660072.swust-2BL confers ASR, and QYrPI660072.swust-4BL confers APR. The results confirmed the conclusion of both ASR and HTAP resistance in PI 660072 in the previous study (Wang et al. 2012).

To achieve sustainable control of stripe rust, it is critical to identify and use effective genes to develop new cultivars with high-level and durable resistance (Chen 2013, 2020). In the present study, we identified two QTL, QYrPI660072.swust-2BL and QYrPI660072.swust-4BL, conferring resistance to stripe rust in wheat line PI 660072. The two major QTL were identified through QTL mapping using the stripe rust phenotypic data of five field environments. Two KASP markers, KASP-072-1269 for the 2BL QTL and KASP-072-3209 for the 4BL QTL, were developed and could be used in MAS for incorporating the QTL into wheat cultivars to improve resistance to stripe rust.

QYrPI660072.swust-2BL, mapped on chromosome 2BL, was effective against the tested predominant Chinese Pst races in the greenhouse seedling tests and consistently detected in all five field environments. This ASR QTL was mapped between SNP markers AX-109547533 and AX-111640532, corresponding to the 376,495,312 bp and 682,801,798 bp positions, respectively on 2BL of the Chinese Spring reference genome (IWGSC RefSeq v1.0 2BL). Many stripe rust resistance genes or QTL have been reported on 2BL (Wang and Chen 2017; Liu et al. 2019; Nicolas et al. 2019). Five permanently named stripe rust resistance genes, Yr5 (Yan et al. 2003), Yr7 (Yao et al. 2006), Yr43 (Sui et al. 2009), Yr44 (Cheng et al. 2010) and Yr53 (Xu et al. 2013) have been mapped to 2BL and conferred ASR. Yr5, Yr7 and YrSP have been cloned, and their physical position was 685.27 Mb on 2BL in the Chinese Spring reference genome (IWGSC RefSeq v1.0) (Marchal et al. 2018). Yr43 in a hard white spring wheat cultivar ‘IDO377s’ (PI 591045) is flanked between Xwgp103 and Xwgp110, with a genetic distance of 4.4 and 5.5 cM, respectively (Cheng et al. 2010). Yr43 was susceptible to the Pst CYR32, CYR33 and CYR34 at the seedling stage (Liu et al. 2017). Yr44 is originally from a soft white spring wheat cultivar ‘Zak’ (PI 607839). This gene is 3.9 and 9.4 cM to its frank markers Xwgp100 and XpWB5/N1R1, respectively (Sui et al. 2009). Xwgp100 is on the same side as Xgwm501 that is around 672,082,697 bp on 2BL of the Chinese Spring genome (IWGSC RefSeq v1.0 2BL). Yr53 in durum wheat PI 480148 originally from Ethiopia was mapped between XLRRrev/NLRRrev₃₅₀ and Xwmc441, about 5.6 cM to the latter marker (Xu et al. 2013). Xwmc441 is around 598,064,318 bp on 2BL in the Chinese Spring genome (IWGSC RefSeq v1.0 2BL). Because QYrPI660072.swust-2BL had no linked markers between the flanking markers, this interval also includes Yr44 and Yr53. However, according to a previous study, Yr44 was susceptible to Chinese Pst races CYR32, CYR33 and CYR34 at the seedling stage (Wang et al. 2019). Therefore, QYrPI660072.swust-2BL should be different from Yr44. Further research is needed to narrow the target interval and determine whether Yr53 and QYrPI660072.swust-2BL are the same gene.

QYrPI660072.swust-4BL, mapped on chromosome 4BL, was consistently associated with stripe rust APR in all five field environments. This QTL was mapped between markers AX-109412222 and AX-108847266 corresponding to the 362,135,412 bp and 429,339,058 bp positions, respectively on 4BL in the Chinese Spring genome (IWGSC RefSeq v1.0 4BL). So far, more than ten genes or QTL have been reported on chromosome 4BL. QYrus.vt-4BL for APR in wheat line USG 3555 was mapped between SSR markers gwm165 and gwm149 corresponding to the 412,716,441 bp and 544,649,745 bp positions respectively on 4BL in the Chinese Spring genome (IWGSC RefSeq v1.0) (Christopher et al. 2013). QYrhm.nwafu-4B in wheat Humai 15 was mapped to an interval of 3.4 cM on chromosome 4BL, flanked by SNP markers AX-111150955 and Xgwm251 corresponding to the interval between 523,448,600 and 568,556,138 bp, respectively on 4BL in the Chinese Spring genome (IWGSC RefSeq v1.0) (Yuan et al. 2018). Permanently named stripe rust resistance genes on 4BL include Yr50 (Liu et al. 2013) and Yr62 (Lu et al. 2014). Yr50 is an ASR gene mapped between SSR markers Xbarc1096 and Xwmc47, about 8.0 cM and 7.2 cM apart to these markers, respectively (Liu et al. 2013). The two markers correspond to the positions 105,145,886 and 644,865,926 bp, respectively on 4BL of the Chinese Spring genome (IWGSC RefSeq v1.0). The HTAP resistance gene Yr62 in PI 192252 was mapped between SSR markers Xgwm251 and Xgwm192 (Lu et al. 2014). The Xgwm251 is around the 568,556,138 bp position on 4BL in the Chinese Spring genome (IWGSC RefSeq v1.0). Based on the physical positions, some of the above-mentioned genes are different from QYrPI660072.swust-4BL, but QYrus.vt-4BL is close to QYrPI660072.swust-4BL. The relationship between QYrPI660072.swust-4BL and QYrus.vt-4BL needs a further study.

The present study showed that the combination of QYrPI660072.swust-2BL and QYrPI660072.swust-4BL increased the level of resistance through additive interactions. The selected RILs carrying the two genes, as well as PI 660072, are valuable sources to be used in wheat breeding programs for developing cultivars resistant to stripe rust. The two QTL can be used in combination and also with other genes for stripe rust resistance. The KASP markers for the two QTL should be useful for MAS in pyramiding them with other resistance genes.

Author contribution statementXZ made the cross, participated in field experiments and contributed to the genotyping experiment, analyzed the data, detected the QTL and prepared the first draft of the manuscript. XZ, YW and JS contributed to the collection of samples and phenotypic data. YW, YL and HC tested the resistant parental and single-gene lines. JS, YW, GJ, LZ and HC assisted in analyzing the data. JS, XL, SY, GL, KH and YR participated in field experiments. MW provided seed of the resistant parent and assistance to the study. XC and XZ conceived and directed the project and revised the manuscript. All authors revised and approved the manuscript for submission.

AcknowledgmentsThis work was financially supported by the Key Research and Development Program of International Science and Technology Innovation Cooperation of Science and Technology Department of Sichuan Province, China (No. 2022YFH0032), and was partially funded by the National Natural Science Foundation of China (No. 32101707), Major Program of National Agriculture Science and Technology of China (NK20220607), PhD Foundation of Southwest University of Science and Technology (No. 19zx7146, 19zx7116, 18zx7159).

Conflict of interest The authors declare that they have no conflict of interest.

Anderson JA, Churchill GA, Autrique JE, Tanksley SD, Sorrells ME (1993) Optimizing parental selection for genetic linkage maps. Genome 36:181–186
Barendse W, Harrison B, Bunch R, Thomas M, Turner L (2009) Genome wide signatures of positive selection: The comparison of independent samples and the identification of regions associated to traits. BMC Genomics 10:178–180
Chen WQ, Wu LR, Liu TG, Xu SC, Wang BT (2009) Race dynamics, diversity, and virulence evolution in Puccinia striiformis f. sp tritici, the causal agent of wheat stripe rust in China from 2003 to 2007. Plant Dis 93:1093–1101
Chen XM (2005) Epidemiology and control of stripe rust [Puccinia striiformis f. sp. tritici] on wheat. Can J Plant Pathol 27:314–337
Chen XM (2013) Review Article: high-temperature adult-plant resistance, key for sustainable control of stripe rust. Am J Plant Sci 4:608–627
Chen XM (2014) Integration of cultivar resistance and fungicide application for control of wheat stripe rust. Can J Plant Pathol 36:311–326
Chen XM (2020) Pathogens which threaten food security: Puccinia striiformis, the wheat stripe rust pathogen. Food Sec 12:239–251
Cheng P, Chen XM (2010) Molecular mapping of a gene for stripe rust resistance in spring wheat cultivar IDO377s. Theor Appl Genet 121:195–204
Cheng P, Xu LS, Wang MN, See DR, Chen XM (2014) Molecular mapping of genes Yr64 and Yr65 for stripe rust resistance in hexaploid derivatives of durum wheat accessions PI 331260 and PI 480016. Theor Appl Genet 127:2267–2277
Christopher MD, Liu SY, Hall MD, Marshall DS, Griffey CA (2013) Identification and mapping of adult plant stripe rust resistance in soft red winter wheat VA00W-38. Crop Sci 53:871–879
Feng JY, Yao FJ, Wang MN, See DR, Chen XM (2023) Molecular mapping of Yr85 and comparison with other genes for resistance to stripe rust on wheat chromosome 1B. Plant Dis 107:3585–3591
Fu D, Uauy C, Distelfeld A, Blechl A, Epstein L, Chen XM, Sela H, Fahima T, Dubcovsky J (2009) A kinase-START gene confers temperature-dependent resistance to wheat stripe rust. Science 323:1357–1360
Kang ZS, Wang XJ, Zhao J, Tang CL, Huang LL (2015) Advances in research of pathogenicity and virulence variation of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. Sci Agric Sin 48:3439–3453
Klymiuk V, Chawla HS, Wiebe K, Ens S, Fatiukha A, Govta L (2022) Discovery of stripe rust resistance with incomplete dominance in wild emmer wheat using bulked segregant analysis sequencing. Commun Biol 5:826
Klymiuk V, Yaniv E, Lin H, Raats D, Fatiukha A (2018) Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family. Nat Commun 9:3735
Krattinger SG, Lagudah ES, Spielmeyer W, Singh SR, Huerta-Espino JH, McFadden H, Bossolini E, Selter, Liselotte, Keller Beat (2009) A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science 323:1360–1363
Kump KL, Bradbury PJ, Wisser RJ, Buckler ES, Belcher AR, Oropeza Rosas MA (2011) Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population. Nat Genet 43:163–168
Li J, Kang XH, Chen WQ, Zhang H, Yang H, Jiang XD (2018) Identification and cluster analysis of stripe rust in 22 wheat varieties in Sichuan Province. Guangdong Agric Sci 45:82–86
Li JB, Dundas L, Dong CM, Li GR, Trethowan R, Yang ZJ, Hoxha SM, Zhang P (2020) Identification and characterization of a new stripe rust resistance gene Yr83 on rye chromosome 6R in wheat. Theor Appl Genet 133:1095–1107
Li Q, Chen XM, Wang MN, Jing JX (2011) Yr45, a new wheat gene for stripe rust resistance mapped on the long arm of chromosome 3D. Theor Appl Genet 122:189–197
Lin F, Chen XM (2007) Genetics and molecular mapping of genes for race-specific all-stage resistance and non-race specific high-temperature adult-plant resistance to stripe rust in spring wheat cultivar Alpowa. Theor Appl Genet 114:1277–1287
Line RF, Qayoum AN (1992) Virulence, aggressiveness, evolution and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968–87 USDA-ARS. Tech. Bull 1788:44
Liu B, Liu TG, Zhang ZY, Jia QZ, Wang BT, Gao L, Peng YL, Jin SL, Chen WQ (2017) Discovery and pathogenicity of CYR34, a new race of Puccinia striiformis f. sp. tritici in China. Acta Agron Sin 47:681–687
Liu J, Chang ZJ, Zhang XJ, Yang ZJ, Li X, Jia JQ et al (2013) Putative Thinopyrum intermedium-derived stripe rust resistance gene Yr50 maps on wheat chromosome arm 4BL. Theor Appl Genet 126:265–274
Liu L, Wang MN, Feng JY, See DR, Chen XM (2019) Whole-Genome mapping of stripe rust resistance quantitative trait loci and race specificity related to resistance reduction in winter wheat cultivar Eltan. Phytopathology 109:1226–1235
Lu Y, Wang MN, Chen XM, See D, Chao SM, Jing JX (2014) Mapping of Yr62 and a small–effect QTL for high–temperature adult–plant resistance to stripe rust in spring wheat PI 192252. Theor Appl Genet 127:1449–1459
Ma LJ, Kong XY, Qiao JX, An F, Hu XP, Xu XM (2016) Overwintering of Puccinia striiformis f. tritici on winter wheat at varying altitudes in Gansu and Qinghai provinces. Plant Dis 100:1138–1145
Marchal C, Zhang J, Peng Z, Fenwick P, Uauy C (2018) BED-domain containing immune receptors confer diverse resistance spectra to yellow rust. Nat Plants 4:662–668
Meng L, Li HH, Zhang LY, Wang JK (2015) QTL IciMapping: Integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. The Crop J 3:269–283
Moore JW, Herrera-Foessel S, Lan C, Schnippenkoetter W, Ayliffe M, Huerta-Espino J, Lillemo M, Viccars L, Milne R, Periyannan S, Kong X, Spielmeyer W, Talbot M, Bariana H, Patrick JW, Dodds P, Singh R, Lagudah E (2015) A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat Genet 47:1494–1498
Nicolas CB, Humphrey WG, Evans L, Jorge D (2019) A high-resolution map of wheat QYr.ucw-1BL, an adult plant stripe rust resistance locus in the same chromosomal region as Yr29. The Plant Genome 12:180055
Pakeerathan K, Bariana H, Qureshi N, Wong D, Hayden M, Bansal U (2019) Identification of a new source of stripe rust resistance Yr82 in wheat. Theor Appl Genet 132:3169–3176
Rasheed A, Wen W, Gao F, Zhai S, Jin H, Liu J, Guo Q, Zhang Y, Dreisigacker S, Xia X (2016) Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theor Appl Genet 129:1843–1860
Ren RS, Wang MN, Chen XM, Zhang ZJ (2012) Characterization and molecular mapping of Yr52 for high-temperature adult-plant resistance to stripe rust in spring wheat germplasm PI 183527. Theor Appl Genet 125:847–857
Sharma-Poudyal D, Chen XM, Wan AM, Zhan GM, Kang ZS, Cao SQ, Jin SL, Morgounov A, Akin B, Mert Z, Shah SJ (2013) Virulence characterization of international collections of the wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici. Plant Dis 97:379–386
Sui XX, Wang MN, Chen XM (2009) Molecular mapping of a stripe rust resistance gene in spring wheat cultivar Zak. Phytopathology 99:1209–1215
Wan AM, Zhao ZH, Chen XM, He ZH, Jin SL, Jia QZ, Yao G, Yang JX, Wang BT, Li GB, Bi YQ, Yuan ZY (2004) Wheat stripe rust epidemic and virulence of Puccinia striiformis f. sp. tritici in China in 2002. Plant Dis 88:896–904
Wang H, Zou S, Li Y, Lin F, Tang D (2020) An ankyrin-repeat and WRKY-domain-containing immune receptor confers stripe rust resistance in wheat. Nat Commun 11:1353
Wang LC, Tang XR, Wu JH, Shen C, Dai MF, Wang QL, Zeng QD, Kang ZS, Wu YF, Han DJ (2019) Stripe rust resistance to a burgeoning Puccinia striiformis f. sp. tritici race CYR34 in current Chinese wheat cultivars for breeding and research. Euphytica 215:68–76
Wang MN, Chen XM (2017) Stripe rust resistance. In: Chen XM, Kang ZS (eds) Stripe Rust. Springer, Dordrecht, pp 353–558
Wang MN, Chen XM, Xu LS, Cheng P, Bockelman HE (2012) Registration of 70 common spring wheat germplasm lines resistant to stripe rust. J Plant Regist 6:104–110
Wang SH, Chu BY, Liu Q, Luo Y, Ma ZH (2017) Development of a sequence-characterized amplified region marker using inter-simple sequence repeats for detection of Puccinia striiformis f. sp. tritici. J Plant Pathol 165:442–447
Wellings C (2011) Global status of stripe rust: a review of historical and current threats. Euphytica 179:1–13
Wellings CR, Singh RP, McIntosh RA, Pretorius ZA (2004) The development and application of near isogenic lines for the stripe (yellow) rust pathosystem. Page A1.39 in: Proc. 11th Intl. Cereal Rusts and Powdery Mildew Conf., Norwich, England, Cereal Rusts and Powdery Mildews Bull
Wu JH, Zeng QD, Wang Q, Liu S, Kang ZS (2018) SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene Yr26. Theor Appl Genet 131:1481–1496
Xiang C, Feng JY, Wang MN, Chen XM, See DR, Wan AM, Wang T (2016) Molecular mapping of Yr76 for resistance to stripe rust in winter club wheat cultivar Tyee. Phytopathology 106:1186–1193
Xu LS, Wang MN, Cheng P, Kang ZS, Hulbert SH, Chen XM (2013) Molecular mapping of Yr53, a new gene for stripe rust resistance in durum wheat accession PI 480148 and its transfer to common wheat. Theor Appl Genet 126:523–533
Yan GP, Chen XM, Line RF, Wellings CR (2003) Resistance gene-analog polymorphism markers co-segregating with the Yr5 gene for resistance to wheat stripe rust. Theor Appl Genet 106:636–643
Yang J, Bai GH, Shaner GE (2005) Novel quantitative trait loci (QTL) for Fusarium head blight resistance in wheat cultivar Chokwang. Theor Appl Genet 111:1571–1579
Yao ZJ, Lin RM, Xu SC, Li ZF, Wan AM, Ma ZY (2006) The molecular tagging of the yellow rust resistance gene Yr7 in wheat transferred from differential host Lee using microsatellite markers. Sci Agric Sin 39:1146–1152
Yuan FP, Zeng QD, Wu JH, Wang QL, Yang ZJ, Liang BP, Kang ZS, Chen XH, Han DJ (2018) QTL Mapping and Validation of Adult Plant Resistance to Stripe Rust in Chinese Wheat Landrace Humai 15. Front Plant Sci 9:968
Zhang CZ, Huang L, Zhang H, Hao Q, Fu D (2020) An ancestral NB-LRR with duplicated 3′UTRs confers stripe rust resistance in wheat and barley. Nat Commun 10:4023
Zhao YY, Huang XL, Li Q, Huang LL, Kang ZS, Zhao J (2023) Virulence phenotyping and molecular genotyping reveal high diversity within and strong gene flow between the Puccinia striiformis f. sp. tritici populations collected from barberry and wheat in Shaanxi province of China. Plant Dis 107:701–712
Zhou XL, Fang TH, Li KX, Huang KB, Ma CH, Zhang M, Li X, Yang SZ, Ren RS, Zhang PP (2022) Yield losses associated with different levels of stripe rust resistance of commercial wheat cultivars in China. Phytopathology 112:1244–1254
Zhou XL, Han DJ, Chen XM, Mu JM, Xue WB, Zeng QD, Wang QL, Huang LL, Kang ZS (2015a) QTL mapping of adult-plant resistance to stripe rust in wheat line P9897. Euphytica 205:243–253
Zhou XL, Wang MN, Chen XM, Lu Y, Kang ZS, Jing JX (2014) Identification of Yr59 conferring high-temperature adult-plant resistance to stripe rust in wheat germplasm PI 178759. Theor Appl Genet 127:935–945
Zhou XL, Zhan GM, Huang LL, Han DJ, Kang ZS (2015b) Evaluation of resistance to stripe rust in eighty abroad spring wheat germplasms. Sci Agric Sin 48:1518–1526
Zhu ZW, Cao Q, Han DJ, Wu JH, Wu L, Tong JY, Xu XW, Yan J, Zhang Y, Xu KJ, Wang FJ, Dong YC, He ZH, Xia XC, Hao YF (2023) Molecular characterization and validation of adult-plant stripe rust resistance gene Yr86 in Chinese wheat cultivar Zhongmai 895. https://doi.org/10.1007/s00122-023-04374-2. Theor Appl Gene

Download PDF

Version 1

posted

You are reading this latest preprint version

Genome-wide Mapping of Quantitative Trait Loci Conferring Resistance to Stripe Rust in Spring Wheat Line PI 660072

Status:

Version 1

Abstract

Figures

Key Message

Introduction

Materials and methods

Plant materials

Field tests

Seedling tests

Genotyping the parental lines and RILs

Statistical analyses and QTL mapping

Developing KASP markers

Results

Phenotypic evaluation

Inheritance of stripe rust resistance

Genetic linkage maps

QTL mapping

Development and validation of KASP markers

Effects of the individual QTL and their combination

Determination of resistance types of the QTL

Discussion

Declarations

References

Status:

Version 1