Improving Stripe Rust Resistance and Agronomic Performance in Three Elite Wheat Cultivars by Using a Combination of Marker Detection and Phenotypic Selection

Wheat is an important cereal crop globally and improvement programs reach a plateau from ever-evolving stripe rust pathogen. Stripe rust, caused by Puccinia striiformis f. sp. Tritici (PST), is a fungal disease that devastates global wheat production. This disease is most effectively restrained by developing and deploying cultivars with highly durable resistance. The utilization of resistant genes is essential for breeding resistant cultivars. To improve stripe rust resistance and achieve favorable agronomic performance in commercial cultivars, an adult plant resistance gene Yr48 was introgressed from PI610750 into three Chinese elite wheat cultivars Chuanmai 42, Lunxuan 987, and Bainongaikang 58 using a combination of marker detection and phenotypic selection.The three Chinese elite cultivars as the recipient parent were used to cross with the donor parent PI610750. Eighty-seven introgression lines (ILs) were gained from all three crosses in the F 5 generation. To validate the introgression lines possessing Yr48, SSR markers previously reported to be linked to the resistance gene loci were used to detect its presence or absence in different genetic backgrounds. Resistance and effect of introgressed Yr48 were studied using F 6 and F 7 grain grown in the eld. The ILs from three crosses were evaluated the agronomic traits and the stripe rust reaction following stringent phenotypic criteria. Both phenotype and genotype data conrmed the presence of Yr48. A combination of phenotypic selection and marker-assisted breeding revealed that 9 selected lines had more application prospects. Those ILs had increased TGW (4.5–31.1%) in infested environments compared to the recipient parents. New lines with improved disease resistance and better agronomic performance should have more advantages than the original cultivars in wheat breeding programs.


Introduction
Wheat, as one of the three most important cereal crops in the world, is the staple food of about 40% of the world's population . The global people will exceed 9.5 billion by 2050, and global wheat yield will need to grow by 60% to satisfy food requirements (Savadi et al. 2017;Tian et al. 2003). However, wheat production is often affected by pathogenic fungi, resulting in huge yield loss. Stripe rust or yellow rust (Yr) of wheat caused by Puccinia striiformis f. sp. Tritici (PST), is one of the most globally devastating fungal diseases that signi cantly reduces yield and quality (Tao et al. 2018). It damages virtually all winter wheat varieties and limits the realization of the full genetic potential of modern improved wheat cultivars considered the most important fungal disease to decrease wheat production in China (Wan et al. 2004;Zhou et al. 2019).As the adaptability and rapid evolution of the stripe rust pathogen, previously used resistant varieties usually last only a few years. Then the ineffective resistance and heavy yield losses will render popular wheat varieties phased out of farmers' elds. Due to the ineffectiveness of ASR genes, many stripe rust epidemics have taken place all over the world, resulting in enormous yield losses Wellings et al. 2007). In general, effective fungicides are available for preventing or reducing yield losses from stripe rust. However, the use of fungicides adds a signi cant cost for wheat production and adversely affects the environment and human health. Hence, it is compulsory to produce more high level, durable resistance resource to reduce the threat of the widespread epidemic. The deployment of new effective varieties has always been the most economical, effective, and environmentally friendly approach for combating the disease (Chen. 2005). Breeders across the whole world are engaged in the creation and cultivation of new resources of resistance to control the continuously evolving disease (Bariana et al. 2006). Improving resistance to the fungal disease with stripe rust-resistant genes is usually the highest priority in various regions of the world where stripe rust epidemics infest frequently (Qie et al. 2018).
Seeding/all stage resistance (ASR) and adult plant resistance (APR) are the most common types of stripe rust resistance (Chen. 2005;Rosewarne et al. 2013). Seeding resistance is characterized by racial speci city and qualitative inheritance (Carter et al. 2009). This type of resistance is prone to be detected during the seedling stage of the wheat and is frequently expressed at all ranges of temperatures, and continues throughout the life cycle of all growth stages of plant development. As the speci c nature and the strong selection pressure placed on the pathogen, all-stage resistance is frequently defeated by race changes in the pathogen population (Chen. 1995). On the other hand, APR which expresses at the post-seedling growth stages and often is effective to a broader range of races, confer partial resistance and encode a more varied set of proteins (Chen. 2013;Fu et al. 2009;Lowe et al. 2011a). New virulent variants with constant evolution damage resistance of popular cultivars especially for which carry ASR genes. Therefore, it is necessary to continuously identify and deploy more effective resources in wheat breeding. Naturally, the introgression of the APR gene into modern and popular cultivars seems to be favorable to combat the fungal disease and yield loss. Recent developments in the wheat genome facilitate the identi cation of molecular markers associated with resistant genes that can be deployed to develop more durable resistant cultivars.
A typical application of molecular markers is the marker-assisted breeding (MAB), which is based on the concept of molecular biology. This process has been practiced to improve disease resistance in various crops including barley, cotton, pea, rice, soybean, and wheat (Chemayek et al. 2017;Chen. 2013;Cox et al. 1994;Gahan et al. 2005). MAB has gained considerable attraction as a breeding tool in wheat improvement. Over 60 previously reported genes/quantitative trait loci (QTL) have been used in wheat breeding (Gupta et al. 2010).
Many wheat cultivars, such as Clear white 515, Expresso, Patwin 515, Scarlet 09, Summit 515, and Seahawk grown in the western United States, were developed by MAB. Moreover, there have been many successful cases in peanuts where disease-resistant QTLs have been introduced into advanced varieties that have lost resistance (Varshney et al. 2014). Likewise, three elite wheat cultivars successfully improved resistance to stripe rust in China by MAB (Hu et al. 2020). Here, we describe MAB progress in a wheat breeding program aimed at improving stripe rust resistance and agronomic traits in three Chinese advanced cultivars. Markerassisted breeding for disease resistance is performed routinely in many crop-breeding programs. Phenotypic selection and marker-assisted methods have been used for breeding stripe rust-resistant wheat cultivars in the Southwest University of Science and Technology wheat-breeding program. The MAB methodology has proved more successful in producing cultivars with introgression of adult plant resistance genes. Yr48, as an APR gene, was located in the long arm of chromosome 5A by Lowe et al Lowe et al. (2011b). Despite the effectiveness of Yr48 was clearly con rmed by the previous report (Lan et al. 2017;Lowe et al. 2011b), this APR gene has not been adequately adopted in a wheat breeding program. Gene-linked markers recommended for marker-assisted breeding programs are BE495011 (0.09 cM proximal of Yr48) and CFA2149 (0.06 cM distal of Yr48) (Lowe et al. 2011b).
Since the expression of resistance to stripe rust of Yr48 and the reliability of closely linked molecular markers, this study was undertaken to select a set of signi cantly improved PST resistance wheat lines with Yr48 and important agronomic traits. These selected lines will be useful for cultivating new wheat varieties with highly durable resistance to stripe rust.
CM42 is a widely adapted, high-yielding variety. This variety, a derivative of the cross SynCD768/SW3243//C6415, was bred at Sichuan Academy of agricultural science, China, and released for cultivation in 2004. CM42 is China's rst accredited product bred from synthetic genetic resources Species SynCD768, an introduction from International Maize and Wheat Improvement Center (CIMMYT). LX987 is a cold-resistant and drought-tolerant variety with high and stable yields. It was cultivated at the Chinese Academy of Agricultural Sciences following the method of recurrent selection and pedigree method, released for agriculture during 2003 and has been very prevalent in China. BNAK58, a popular variety, is a high-yielding, cold-resistant and drought-tolerant variety with high harvest index is used extensively for confectionery purpose. All three elite varieties were popular in farmers' land for high yield and wide suitability in china. However, these elite cultivars succumbed to new races of CYR34 thus it is imperative to restructure the genetic makeup of these varieties to achieve better yield and stripe rust resistance. PI610750, spring wheat, is a synthetic derivative developed by CIMMYT. PI610750 is a resistant variety to stripe rust and was selected as a donor parent in the breeding program as it was used to identify the Yr48 gene for stripe rust resistance. The reliable markers and resistance of PI610750 were con rmed in America and Mexico made it a prominent resistant germplasm resource (Lan et al. 2017;Lowe et al. 2011b).
Developing three crosses presumably possessing Yr48 with favorable agronomic performance The schematic procedure for crossing, screening, and selection is illustrated in Fig. 1. To combine Yr48 and yield-related traits, all three target cultivars were used as female parents to make an independent cross (CM42 × PI610750, LX987 × PI610750, and BNAK58 × PI610750). Initially, The F 1 seeds were planted and harvested in the eld. Subsequently, the F 2 , F 3 , F 4 , and F 5 of each wheat cross combination were planted over 30 rows with about 80-100 seeds in a 2 m row with 25 cm between rows. We executed a modi ed bulk method before the F 5 for each cross to collect all promising genotypes (Hu et al. 2020), and after the accumulation of several generations, we selected the F 5 generation plants with resistance to stripe rust and important agronomic traits including plant height, productive tiller numbers, spike length, grain number per spike, and thousand-grain weight. These populations were selected at Mianyang in Sichuan province (31.682N, 104.663E) under a natural PST infection environment, together with MX 169, PI610750, CM42, LX987, and BNAK58 during the 2017-2018 cropping seasons.
Phenotyping for stripe rust reaction and agronomic traits Three populations of F 5 plants were selected and advanced to F 7 . Such F 6 and F 7 generations were subjected to a selection of lines with rust resistance and excellent agronomic traits. Phenotyping of parents, F 6 and F 7 lines for adult plant stripe rust response and agronomic traits were done in China elds in Mianyang (31.682N, 104.663E) of Sichuan province during the 2018-2019 and 2019-2020 cropping seasons (2019MY and 2020MY). The F 7 lines and their parents were also evaluated for APR to stripe rust in China elds in Yangling (34.292N, 108.077E) of Shanxi province during the 2019-2020 (2020YL) cropping seasons. These lines were arranged in randomized complete blocks with three replications. About 80-100 seeds per line were planted in a row of 2 m long with 25cm space between rows. Spreader rows containing the PST-susceptible MX169 were planted around the experimental plot to produce an epidemic environment. These experimental elds are ideal over-wintering and over-summering regions for stripe rust in China and nurseries naturally become infected without creating arti cial disease epiphytotic.
The stripe rust infection type (IT) of each plant or each line was rated based on the 0 to 9 scale (Line and Qayoum. 1992). Disease severity (DS) was scored by using the modi ed Cobb scale (Peterson et al. 1948). Both IT and DS were collected two times during each season. The rst note was recorded when the susceptible check MX169 exhibited approximately 80% severity and repeated about a week later (Nsabiyera et al. 2018). Mean IT and DS over test environments were used to analyze. The SSR markers CFA2149 and STS markers BE495011, which are closely linked to Yr48, were used to detect its presence or absence in the wheat genotypes. Plant genomic DNA was isolated from leaf tissues of the parental lines (PI61750, CM42, LX987, and BNAK58) and the F 6 and F 7 lines of CM42 × PI61750, BNAK58 × PI61750, and LX987 × PI61750, employing the modi ed cetyltrimethylammonium bromide (CTAB) extraction method (Riede et al. 1996). DNA quality and quantity were measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies) and diluted to a concentration of 50ng/ul for further genotyping with linked markers. The tightly linked markers were used for ampli cation using polymerase chain reaction (PCR) following conditions mentioned in the previous report (Lowe et al. 2011b). PCR reactions were prepared by mixing 2 ul of genomic DNA, 1 µl 10×PCR buffer (including Mg 2 +), 0.2 µl Taq DNA polymerase solution, 1 µl of each primer solution, 0.8 µl of each dNTP, and 4 µl sterilized ddH 2 O. The PCR reaction was performed at 94.0°C for 3 min, 40 cycles of (95.0°C for 30 sec, 59°C or 60°C (according to each primer pair) for 30 sec, 72°C for 30 sec), and the nal extension at 72°C for 5 min. The PCR products of the linked markers were separated in 6% polyacrylamide gel electrophoresis (PAGE).

Development of common wheat lines presumably possessing Yr48
Marker detection and phenotypic selection with elite cultivars including genotyping and phenotyping was undertaken as mentioned in Fig. 1. For the transfer of Yr48 to wheat varieties CM42, LX987, BNAK58, the crosses were made with PI610750 as a male parent. Before the F 5 generation, we executed a bulk harvest for each cross to maintain all potential genotypes. After several generations of accumulation, Eighty-seven F 5 plants according to their resistance to stripe rust, moderate plant height, and the high number of tiller and grain numbers were selected following stringent visual phenotypic criteria. Of these, 64 lines were selected from the cross of CM42/PI610750, 11 lines were selected from the cross of LX987/PI610750, and 12 lines were selected from the cross of BNAK58/PI610750. Progenies of the 87 selected lines from three crosses were advanced to further genotype and phenotype.

Stripe rust and marker detection
During the 2018-2019 and 2019-2020 screening season, Stripe rust IT of ILs ranged from 0 to 8, and stripe rust DS of ILs ranged from 0 to 80% over 2 years. The response of donor parent was consistently low (IT 1-3, DS < 20%), and the recipient parents CM42, LX987, and BNAK58 were higher (IT 7-8, DS 50-80%) (Fig. 2). The evaluation results of several elds revealed that PI610750 expressed excellent stripe rust resistance in different environments. The majority of the tested ILs showed a disease score of 3 or 6 in different environments, a lower score compared with that of the recipient parents. Similarly, the DS was much lower than the recipient parents. Disease reaction for stripe rust resistance on parents and introgression lines (ILs) possessing resistant gene introgressed through marker detection in wheat (Fig. S1). The donor parent (PI610750) and its ILs (CM42 + Yr48, CM42 + Yr48, and CM42 + Yr48) showed favorable resistant features.
According to the absence or presence of Yr48, the 87 lines from three crosses were divided into two groups: possessing Yr48, lacking Yr48. As a result, 45 lines were found to be positive lines carrying the Yr48. Among those, Thirty-six lines from CM42/PI610750, 4 lines were from the cross of LX987/PI610750, and 5 lines were from the cross of BNAK58/PI610750. We compared IT and DS between two groups in different environments and recorded the disease score of parents to illustrate the effectiveness of Yr48. The mean adult plant stage IT ranged and DS ranged from 1 to 5 and 5 to 50% for lines carrying Yr48, respectively, whereas it was 4 to 7 and 10 to 80% for lines lacking this resistance gene, respectively (Fig. 3A, 3B). To further assess stripe rust was successfully restrained by the wheat breeding program, we compared the disease score in different genetic backgrounds. Student's t-test results showed that the lines lacking the Yr48 alleles had much higher phenotypic values than those carrying Yr48 at all the three crosses (P < 0.05, Fig. 3C, 3D).

Phenotyping of agronomic traits of ILs under disease infection
During 2018-2019 and 2019-2020 cropping season same seasons, evaluation trials were conducted at Mianyang to compare agronomic traits including plant height, productive tillers, grain number per spike, spike length, and thousand-grain weight of ILs with their respective recipient and donor parent genotypes (Fig. 4). A good variation in TGW and other agronomic traits was observed among the 87 ILs. PTN of parents were 3 or 4 while the majority of the tested lines showed a much higher number (Fig. 4A). Similarly, the SL of parents at around 9 cm, which is much lower than the tested lines (Fig. 4B). The GNS ranged from 35 to 65 among the selected lines whereas their parents ranged from 35 to 45. More importantly, the majority of these lines recorded higher GNS than the recipient parents (Fig. 4C). The TGW in ILs ranged from 39-67 g in the genetic background of CM42, 34-54 g in the genetic background of LX987 while that of BNAK58 ranged from 36-46 g. The PH of CM42, LX987, and BNAK58 was 101cm, 91cm, and 85cm, respectively. Enhanced thousand-grain weight and better agronomic performance were observed in some ILs compared to their parent (Fig. 4).

A combination of marker detection and phenotypic selection
Selection for stripe rust resistance was done in a combined phenotypic and genotypic strategy. Yr48 positive plants were selected by amplifying gene-speci c markers BE495011 and CFA2149. Screening for stripe rust was also done in the eld along with marker-assisted selection to validate the effectiveness of Yr48. F 6 and F 7 of 87 ILs were screened against stripe rust at the adult plant stage in the eld during season 2018-2019 and 2019-2020. Initially, 45 positive ILs for Yr48 were selected by marker detection. According to the disease reaction for stripe rust over two years, IT less than 5 and DS lower than 50% were selected from promising uniform stripe rust resistant lines. Thirty-three lines were selected following the standard disease score.
Subsequently, 33 lines were also evaluated TGW and other agronomic traits to select better lines. The three popular recipient parents in china have very good agronomic traits, which are regarded as related references.
Yield parameters for nine of the best ILs in three genetic backgrounds are given in Table 2. The thousand-grain weight in ILs ranged from 50.4-56.1 g in the genetic background of CM42, 42.1-45.8 g in the genetic background of LX987 while that of BNAK58 ranged from 42.3-46.4 g. enhanced-thousand grain weight was observed in these ILs compared to their respective recipient parent genotypes (Table 2). More accurately, 3 ILs of CM42/PI610750 recorded 17.7-31.1% higher TGW than the recipient parent genotype CM42 (42.8 g). Three ILs of LX987/PI610750 recorded a higher TGW of 4.5-13.6% compared to LX987 (40.3 g). Three ILs of BNAK58/PI610750 recorded higher TGW of 9.9-20.2% compared to BNAK58 (38.6 g). These selected lines showed lower disease score to stripe rust, appropriate plant height, more productive tiller and grain number, better spike length and thousand grain weight compared with their recipient parent, respectively. The new developed lines will be good for wheat breeders to cultivate stripe rust resistant wheat varieties with high yield.

Discussion
The conventional breeding method has been widely used to develop cultivars with improved disease resistance agronomic traits, but it is labor-intensive and time-consuming. Cultivating new cultivars in this method can take between 7 and 20 years, depending on plant species. The use of molecular markers improves the e ciency of breeding. A major application of molecular markers in this study is genotype screening. The development of selection technologies to achieve a genotype-based selection of resistance genes in the absence of bioassays has evolved rapidly in the last decades. Robust molecular markers are now available for several commercially important agronomic traits. Marker-assisted breeding has been proved to be a quick way to develop new cultivars with favorable agronomic traits and improved disease resistance (Bariana et al. 2007;Singh et al. 2000), but introgressing disease resistant genes into the existing preferred wheat cultivars has been rarely attempted.
In the present study, we rst developed three wheat populations by crossing a wheat line carrying Yr48 and another three leading wheat lines with special features such as high yield, enough grain number and moderate plant height, and others. Their progenies presumably carry Yr48 as some of them expressed lower disease scores than recipient parents. To con rm these lines carry the gene, we validated the presence of Yr48 by further phenotyping with the tightly linked markers (CFA2149 and BE495011). We genotyped the F 6 and F 7 lines to collect genotype data and evaluated the stripe rust reaction and other important agronomic traits to record phenotype data during 2018-2019 and 2019-2020 cropping seasons over multi-locations. Thus, the introgressing and selecting strategy practiced in the present study is accurate, effective, and economic. Both phenotype and genotype data con rmed the presence of Yr48. Though some QTLs for stripe rust resistance have also been reported in the PI610750 (Lowe et al. 2011b), the previously reported markers were found to be non-polymorphic between the receipient and donor parents used in the present study. Thus a conventional method can be a complement for marker assisted breeding.
Besides increase stripe rust resistance, progress was also made in improving TGW and some other agronomic traits. Replicated yield related traits assessment under infection environment showed an increase in thousand grain weight by up to 31.1 % higher than CM42, 13.6 % more than LX987 and 20.2 % higher than BNAK58. All these ILs showed a disease score on par with that of the donor parent genotype while displaying yield related traits better than that of the recipient parent genotypes. The higher thousand grain weight in ILs compared to that in the recipient parent may be partly attributed to the guaranty provided by the resistance gene against the fungal disease and stringent visual phenotypic criteria. The results against the theory that an active resistant reaction results in yield penalties for plants combating with pathogens. Thus, highlight the value of conventional phenotypic selection in crop breeding.
Also, we compared the agronomic traits in two groups (Possessing Yr48 and lacking Yr48) to illustrate the bene ts of combination using a marker assisted and phenotypic selection. Given the sheer magnitude of its effect reduction in disease severity (Fig. 5A), a clear evidence that Yr48 is an excellent resistance gene. Considering the function of Yr48, speci cally in stripe rust resistance at the adult plant stage, we wondered that introgressing the Yr48 into wheat may confer resistance in different genetic backgrounds whether affecting yield traits. As expected, the introgression of a short fragment on wheat 5AL resulted in wheat lines with broad resistance to stripe rust (Fig. S1). Signi cant thousand-grain weight reduction caused by Yr48 resistance was also not detected when it was introgressed into three different genetic backgrounds (Fig. 5B). Detailed characterization of the population (CM42 background) in eld conditions showed no signi cant difference in agronomic yield related traits (e.g., plant height, spike length, etc.; Fig. 5C). And, the stable agronomic performance and stripe rust resistance made the selected lines more reliable and valuable. These results demonstrated the high advantages of Yr48 resistance without yield reduction, and thus emphasized the practical utility of the Yr48 locus in wheat breeding for improved stripe rust resistance and good yield traits. So the improved agronomic performance is partially attributed to the phenotypic selection. Although, the Yr48 didn't clone yet. But with the tightly linked markers (less than 1cM), a preliminary diagnosis about the gene may be partly helpful to characterize Yr48 (Fig. 5C).
Yield penalty is a major concern in utilizing stripe rust resistance or any other favorable traits for wheat breeders. Disease resistance is frequently costly and physically detrimental for plant tness (Tian et al. 2003;Büschges et al. 1997;Denancé et al. 2013). For example, a previous found that one bene cial gene, PigmR, confers Pigm blast resistance in the rice variety Gumei4. However, the decreased yield was observed through a reduction of grain weight and size with the expression of PigmR and the absence of a gene named PigmS (Deng et al. 2017). In the present study, the yield difference caused by Yr48 resistance was also not detected when it was introgressed into three different genetic backgrounds, which appeared to be novel, as the usual negative correlation between yield and an active resistance response . The bene cial effects on TGW and other traits could be due to stringent visual phenotypic selection as reported in the previous study (Varshney et al. 2014, Hu et al. 2020).
The importance of genetic resistance to restrain fungal diseases was rst illustrated by Biffen (Biffen et al. 1905). Currently, the utilization of Yr genes has been widely practiced to develop cultivars with improved disease resistance. The introgression of the stripe rust resistance gene Yr15 has cultivated several commercial wheat cultivars (Yaniv et al. 2015 To our knowledge, this study is a rareness of its kind for developing elite cultivars for stripe rust resistance in a wheat breeding program. The introgressing and selecting strategy was practiced in the present study is effective and economic. It is helpful for molecular breeding study for developing Chinese superior lines in wheat. Nevertheless, the selected lines with Yr48 and bene cial agronomic traits in the present study should be more useful than the previously developed cultivars. It should be better to pyramid more useful genes through backcrossing and other breeding methods for increasing stripe rust resistance and wheat production. Advanced breeding of the selected lines can lead to the release of new cultivars and also serve as valuable recourse for crop breeders to be utilized in future breeding programs.

Conclusion
In the present study, Yr48 has been transferred into three Chinese elite wheat cultivars CM42, LX987, and BNAK58 from a resistant genotype, PI610750. In the background of CM42, LX987, and BNAK58, 9 ILs with an increase in TGW up to 31.1%, 13.6% and 20.2% respectively (with respect to recipient parents), other better yield trait and improved stripe rust resistance was selected, through a combination of marker detection and phenotypic selections in 2 years replicated eld trials.

Declarations
Ethics approval and consent to participate Not applicable.

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Data availability
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Con ict of interest
The authors declare that they have no con ict of interest.

Funding
This research received no external funding.
Author contribution statement QY detected the QTL, analyzed the data and prepared the rst draft of the manuscript. THF contributed to collected samples and phenotype data. XLZ and XL contributed to the crosses and revised the manuscript.
THF, and SZY contributed to the selected of target lines and evaluated the populations. XLZ and ZSK suggested the project and generated the nal version of the manuscript. All authors provided suggestions during revision of the manuscript.