Molecular Characterizations of the er1 Alleles Conferring Resistance to Erysiphe pisi in Three Chinese Pea (Pisum sativum L.) Landraces

Powdery mildew caused by Erysiphe pisi DC. is a major disease affecting pea worldwide. This study aimed to confirm the resistance genes contained in three powdery mildew-resistant Chinese pea landraces (Suoshadabaiwan, Dabaiwandou, and Guiwan 1) and to develop the functional markers of the novel resistance genes. The resistance genes were identified by genetic mapping and PsMLO1 gene sequence identification. To confirm the inheritance of powdery mildew resistance in the three Landraces, the susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8 were crossed with Suoshadabaiwan, Dabaiwandou, and Guiwan 1 to produce F1, F2, and F2:3 populations, respectively. All F1 plants were susceptible to E. pisi, and phenotypic segregation patterns in all the F2 and F2:3 populations fit the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, respectively, indicating powdery mildew resistance in the three Landraces were controlled by a single recessive gene, respectively. The analysis of er1-linked markers and genetic mapping in the F2 populations suggested that the recessive resistance genes in three landraces could be er1 alleles. The cDNA sequences of 10 homologous PsMLO1 cDNA clones from the contrasting parents were obtained. A known er1 allele, er1-4, was identified in Suoshadabaiwan. Two novel er1 alleles were identified in Dabaiwandou and Guiwan 1, which were designated as er1-13 and er1-14, respectively. Both novel alleles were characterized with a 1-bp deletion (T) in positions 32 (exon 1) and 277 (exon 3), respectively, which caused a frame-shift mutation to result in premature termination of translation of PsMLO1 protein. The co-dominant functional markers specific for er1-13 and er1-14, KASPar-er1-13, and KASPar-er1-14 were developed and effectively validated in populations and pea germplasms. Here, two novel er1 alleles were characterized and their functional markers were validated. These results provide powerful tools for marker-assisted selection in pea breeding.


Introduction
Pea (Pisum sativum L.) is an important and widely distributed cool season legume crop, which frequently suffers from abiotic and biotic stresses during the whole growth season [1,2]. Among the biotic factors, the disease is the main cause affecting pea production [2]. Powdery mildew caused by Erysiphe pisi DC. is a major constraint for pea yield and quality worldwide [3]. E. pisi infections of peas can lead to yield losses of up to 80% in regions that are suitable for disease infection [3]. To date, the use of resistant cultivars carrying the E. pisi-resistant gene er1 has been considered to be the most effective and environmentally friendly way to control this disease [4,5].
To date, the recessive gene er1 is the most widely used gene in pea production due to er1 conferring high resistance or immunity to E. pisi in most pea germplasms [21]. In contrast, resistance conferred by er2 is unstable and easily affected by leaf development stage and plant location [7,[21][22][23]. er2 is only found in a few pea germplasms [21]. Er3 was known from wild pea (P. fulvum), and there have not been any extensive studies conducted to date [8,24].
E. pisi severely affects the yield and quality of pea crops in China [2]. The disease infects up to 100% of pea plants in some regions of planting susceptible cultivars. In our previous studies, we have focused on the identification of pea germplasms resistant to E. pisi [31,37]. A novel er1 allele er1-6 had been identified in a Chinese pea germplasm [17] and new alleles er1-7, er1-8, and er1-9 were identified in pea germplasms from India, Afghanistan, and Australia, respectively [17,32]. er1-6 was also identified in some pea landraces from Yunnan Province of China [18]. Thus, a natural mutation of the er1 gene conferring E. pisi-resistance has been observed in some Chinese pea landraces, which provides rich resistant sources that can be used to improve the E. pisi resistance of Chinese pea cultivars. The allelic diversity of this locus in the cultivated pea has been well characterized; however, relatively few studies have investigated and characterized the E. pisi-resistant gene in Chinese pea landraces. Thus, this study aimed to identify and characterize the E. pisi-resistant gene in three E. pisi-resistant Chinese pea landraces by genetic mapping and homologous PsMLO1 gene sequence cloning. Additionally, any novel er1 alleles were performed to develop their functional markers to improve marker-assisted selection in E. pisi-resistant pea breeding programs.

Phenotypic Evaluation and Inheritence Analysis for Resistance
Six parental cultivars and contrasting controls were evaluated for their resistance to the E. pisi isolate EPYN. At 10 days post-inoculation, the E. pisi disease severity of the susceptible control was rated as score 4, indicating susceptibility to E. pisi. As expected, the three resistant pea parents, Suoshadabaiwan, Dabaiwandou, and Guiwan 1, and resistant control (Xucai 1) were immune to E. pisi infection (disease severity 0), while the three susceptible parents (Bawan 6, Longwan 1, and Chengwan 8) were susceptible to E. pisi (disease severity 4) ( Figure 1). The segregation patterns of E. pisi resistance in the F 1 , F 2 , and F 2:3 populations derived from the crosses Bawan 6 × Suoshadabaiwan, Longwan 1 × Dabaiwandou, and Chengwan 8 × Guiwan 1 are presented in Table 1.  "R", "Rs", and "S" stand for resistant, segregating, and susceptible, respectively.
Five F 1 plants produced from the cross Bawan 6 × Suoshadabaiwan were susceptible to E. pisi ( Table 1). One of the five plants generated 102 F 2 and F 2:3 offspring through selfpollination. Of these 102 F 2 plants, 26 were resistant (R) to E. pisi, and 76 were susceptible (S) to E. pisi. This indicates that the segregation ratio (resistance: susceptibility) in the F 2 population was 1:3 (χ 2 = 0.02; p = 0.88), indicating the inheritance of a single recessive gene. Moreover, a segregation ratio of 26 (homozygous resistant):51 (segregating):25 (homozygous susceptible) in the F 2:3 population fitted well with the genetic model of 1:2:1 ratio (χ 2 = 0.03, p = 0.99) ( Table 1), confirming that the E. pisi resistance in Suoshadabaiwan was controlled by a single recessive gene.
The cross of Longwan 1 × Dabaiwandou generated six F 1 plants, which showed E. pisi-susceptibility ( Table 1). One of six F 1 plants generated 121 F 2 offspring. Of 121, 29 were resistant, and 92 of 121 were susceptible to E. pisi. The segregation ratio in the F 2 population of resistance to susceptibility fitted a genetic model ratio of 1:3 (χ 2 = 0.07; p = 0.79), also indicating the inheritance of a single recessive gene. Moreover, a segregation ratio of 29 (homozygous resistant):56 (segregating):36 (homozygous susceptible) in the F 2:3 population (121 families) fitted well with the genetic model of 1:2:1 ratio (χ 2 = 1.41; p = 0.49), indicating that E. pisi resistance in Dabaiwandou was also controlled by a single recessive gene ( Table 1).
The cross of Chengwan 8 × Guiwan 1 generated eight F 1 plants which showed E. pisi-susceptibility ( Table 1). One of eight F 1 plants generated 131 F 2 offspring. Of 131, 36 were resistant, and 95 of 131 were susceptible to E. pisi. The segregation ratio in the F 2 population of resistance to susceptibility fitted a genetic model ratio of 1:3 (χ 2 = 0.43; p = 0.51), also indicating the recessive inheritance of a single gene. Moreover, a segregation ratio of 36 (homozygous resistant):61 (segregating):34 (homozygous susceptible) in the F 2:3 population (131 families) fitted well with the genetic model of 1:2:1 ratio (χ 2 = 0.67; p = 0.71), indicating that E. pisi resistance in Guiwan 1 was also controlled by a single recessive gene (Table 1).

Mapping of Resistance Genes
Of the molecular markers tested, six (c5DNAmet, AD160, AC74, AD51, AD59, and AD60) were polymorphic between contrasting parents Bawan 6 and Suoshadabaiwan, and three (c5DNAmet, AA220, and AD51) were polymorphic between Longwan 1 and Dabaiwandou, Unfortunately, no polymorphic marker appeared between Longwan 1 and Dabaiwandou among the above markers tested. Thus, the additional eight SSR markers (16410, 28516, 26140, 23309, 29872, 26514, 23949, and 22903) developed recently were used to test the polymorphism between Longwan 1 and Dabaiwandou [38]. Two (26514 and 22903) were polymorphic between the contrasting parents, Longwan 1 and Dabaiwandou. All polymorphic markers between the parents were likely linked to the E. pisi resistance gene, respectively. Thus, the six, three, and the two parental polymorphic markers were used to confirm the genotypes of each F 2 plant derived from Bawan 6 × Suoshadabaiwan, Longwan 1 × Dabaiwandou, and Chengwan 8 × Guiwan 1, respectively. This genetic linkage analysis suggested that six markers (c5DNAmet, AD160, AC74, AD51, AD59, and AD60), three markers (c5DNAmet, AA220, and AD51), and two markers (26514 and 22903) were linked to the resistance gene er1 in Suoshadabaiwan, Dabaiwandou, and Guiwan 1, respectively ( Figure 2). Our results also indicated that the resistance genes in the three resistant cultivars were located in the er1 region. In Suoshadabaiwan, the linkage map indicated that the markers (AD59 and AD60) were mapped on both sides of the target gene with 3.4 cM and 8.3 cM genetic distances, respectively ( Figure 2A). In Dabaiwandou, two other markers (c5DNAmet and AA220) were located on both sides of the target gene with 2.6 cM and 11.6 cM genetic distances, respectively ( Figure 2B). In Guiwan 1, two markers (26514 and 22903) were located on both sides of the target gene with 12.8 cM and 19.3 cM genetic distances, respectively ( Figure 2C). Our linkage and genetic map analyses confirmed that an er1 allele controlled E. pisi resistance in Suoshadabaiwan, Dabaiwandou, and Guiwan 1, respectively ( Figure 2).

PsMLO1 Sequence Analysis
The PsMLO1 cDNA sequences of Bawan 6, Longwan 1, Chengwan 8, and the susceptible parents, were consistent with that of the wild-type PsMLO1 cDNA.
In landrace Suoshadabaiwan, a 1-bp deletion (A) was identified in a previously reported position 91 in exon 1 of the PsMLO1 cDNA sequence. This result is consistent with the mutation in the er1 gene carried by germplasm YI (JI1591), named er1-4. In landrace Dabaiwandou, a novel mutation pattern was found in the Dabaiwandou cDNA fragment homologous to PsMLO1: a 1-bp deletion (T) corresponding to positions 32 in exon 1 (the first exon) of the PsMLO1 cDNA sequence. This deletion caused a substitution of the amino acid leucine with tryptophan at position 11 of the PsMLO1 protein sequence ( Figure 3A). This change caused the early termination of protein translation, probably also resulting in a functional change of PsMLO1 ( Figure 3A). In Guiwan 1, a 1-bp deletion (T) was also identified in a previously unreported position 277 in exon 3 of the PsMLO1 cDNA sequence. This deletion caused a substitution of the amino acid tryptophan with glycine at position 93 of the PsMLO1 protein sequence ( Figure 3B). This change caused the early termination of protein translation, probably also resulting in a functional change of PsMLO1 ( Figure 3B). The two natural mutations differed from all known er1 alleles, indicating that the E. pisi resistance of Dabaiwandou and Guiwan 1 was controlled by the novel alleles of er1. These novel alleles were designated er1-13 and er1-14, respectively, following the accepted nomenclature [9,17,18,32,33,35,36]. Thus, a known and two novel er1 alleles were discovered in the three resistant cultivars, Suoshadabaiwan (from Chongqing), Dabaiwandou (from Yunnan), and Guiwan 1 (from Guangxi), respectively.

Discussion
Pea powdery mildew caused by E. pisi DC. is an important disease and reduces considerable yield in pea production worldwide. The deployment of resistant cultivars containing the er1 gene is the most effective way to control this disease The E. pisi resistance gene er1 is recessive in pea cultivars, which is the most widely deployed gene for controlling powdery mildew worldwide.

Plant Material and E. pisi Inoculum
Previously, many Chinese pea germplasms had been screened for E. pisi and some were found to be E. pisi-resistant [31,37,39]. In this study, the three E. pisi-resistant Chinese pea landraces, Suoshadabaiwan, Dabaiwandou, and Guiwan 1, respectively, from the Chongqing, Yunnan, and Guangxi provinces of China were conducted to reveal their E. pisi-resistant genes. The three E. pisi-susceptible Chinese pea cultivars, Bawan 6, Longwan 1, and Chengwan8, were used as susceptible controls or cross susceptible parents for genetic analysis [15,40]. The Chinese pea cultivar Xucai 1 containing er1-2 was used as E. pisiresistant control [16].
The E. pisi isolate EPYN from Yunnan Province of China was used as the inoculum, which is highly virulent on pea [17,18]. The EPYN isolate was maintained through the continuous re-inoculation of healthy seedlings of Longwan 1 under controlled conditions. The inoculated plants were incubated in a growth chamber with controlled conditions [16].

Powdery Mildew Resistance Evaluation
Thirty-five seeds were planted from each of the three E. pisi-resistant germplasms (Suoshadabaiwan, Dabaiwandou, and Guiwan 1), three E. pisi-susceptible pea cultivars (Bawan 6, Longwan 1, and Chengwan 8), and from the resistant and susceptible controls (Bawan 6, Longwan 1 and Chengwan 8, and Xucai 1) [18]. The healthy seedlings were thinned to 30 per pot before the phenotypic evaluation. Three replications were planted. Seeded pots were placed in a greenhouse maintained at 18 to 26 • C. At the same time, the E. pisi inoculum was prepared by inoculating the 10-day-old seedlings of Longwan 1, which were incubated in a growth chamber at 20 ± 1 • C with a 12-h photoperiod. Two weeks later, all seedlings of the germplasms and controls were inoculated by gently shaking off the conidia of the Longwan 1 plants. Inoculated plants were incubated in a growth chamber at 20 ± 1 • C with a 12-h photoperiod. Ten days later, disease severity was rated based on a scale (0-4 scale) [17,18]. Plants with a score of 0 were considered E. pisi-immune, while those with scores of 1, 2 and 3, 4 were considered E. pisi-resistant and E. pisi-susceptible, respectively. For those identified as immune or resistant to E. pisi, repeated identification was performed.

Inheritance Analysis of Resistant Pea Cultivars
To reveal the inheritance controlled by E. pisi resistance genes in the three E. pisiresistant Chinese pea landrace, Suoshadabaiwan, Dabaiwandou, and Guiwan 1, they were crossed with the E. pisi-susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8, respectively, to generate genetic populations. The derived F 1 , F 2 , and F 2:3 populations from three crosses (Bawan 6 × Suoshadabaiwan, Longwan 1 × Dabaiwandou, and Chengwan 8 × Guiwan 1), which were used for the powdery mildew resistance genetic analysis of Suoshadabaiwan, Dabaiwandou, and Guiwan 1. The six parents and the derived F 1 and F 2 populations were planted in a propagation greenhouse to generate F 2 and F 2:3 family seeds, respectively.
The plants of the F 1 and F 2 populations at the fourth or fifth leaf stage were inoculated with the E. pisi isolate EPYN using the detached leaf method [16][17][18]. After inoculation, the treated leaves were placed in a growth chamber at 20 • C with a 14-h photoperiod. The six parents were also inoculated as controls. Ten days after inoculation, disease severity was rated based on a scale of 0-4 as described above. Plants with scores of 0-2 and 3-4 were classified as resistant and susceptible, respectively [16][17][18]. Those plants identified as E. pisi-resistant were tested again to confirm their resistance.
Twenty-five seeds were selected randomly from each of the 102, 121, and 131 F 2:3 families derived from Bawan 6 × Suoshadabaiwan, Longwan 1 × Dabaiwandou, and Chengwan 8 × Guiwan 1, respectively. These seeds were planted and cultivated together with their parents, following previously published protocols [25][26][27]. Disease severity was scored 10 days after inoculation using the 0-4 scale, as described above for the phenotypic identification of the pea germplasms. The F 2:3 families with scores of 0-2 and 3-4 were classified as homozygous resistant and homozygous susceptible, respectively. Families with scores of 0-2 and 3-4 were considered segregated to E. pisi resistance. The families identified as homozygous-resistant or resistance segregated were subjected to repeated testing.

Genetic Mapping of the Resistance Gene
The Genomic DNA was isolated from the leaves of the F 2 populations and of their parents using a slightly modified cetyltrimethylammonium bromide (CTAB) extraction method [42]. The DNA solution was diluted and stored at −20 • C until use.
The segregation data of the polymorphic markers in the F 2 populations were evaluated for goodness-of-fit to Mendelian segregation patterns with a chi-squared (χ 2 ) test. Genetic linkage analyses were completed using MAPMAKER/EXP version 3.0b. A logarithm of odds (LOD) score > 3.0 and a distance < 50 cM were used as the thresholds to determine the linkage groups [45]. Genetic distances were determined using the Kosambi mapping function [46]. The genetic linkage map was constructed using the Microsoft Excel macro MapDraw [47].

RNA Extraction and PsMLO1 Sequence Analysis
The extraction of total RNA and synthesis of cDNA from Suoshadabaiwan, Dabaiwandou, and Guiwan 1 and controls were completed according to our previous studies [16][17][18].
To identify the resistance gene er1 alleles, the full-length cDNAs of the PsMLO1 homologs were amplified using the primers specific to PsMLO1 [9]. The PCR cycling conditions were as follows: 95 • C for 5 min; then 35 cycles of denaturation at 94 • C for 30 s, annealing at 58 • C for 45 s, and extension at 72 • C for 1 min; and a final extension at 72 • C for 10 min. The purified amplicons were cloned with a pEasy-T5 vector (TransGen Biotech, Beijing, China). The sequencing reactions of 10 clones per parental cultivars and controls were performed by the Shanghai Shenggong Biological Engineering Co., Ltd. (Shanghai, China). The resulting sequences were aligned with the wild-type PsMLO1 of pea (NCBI accession number: FJ463618.1) using DNAMAN v6.0 (Lynnon Biosoft, Vaudreuil, QC, Canada).

Development of Functional Markers for the Novel er1 Alleles
Primers flanking the mutation site were designed based on the PsMLO1 gene sequence (GenBank accession number KC466597), using Primer Premier v5.0, to develop a functional marker specific to allele er1-13 and er1-14 (Table 2). Table 2. Sequence information for the indel and Kompetitive allele-specific PCR (KASPar) markers specific to er1-13 and for the KASPar marker specific to er1-14.
KASPar markers were amplified with a Douglas Scientific Array Tape Platform (China Golden Marker Biotech Co., Ltd., (Beijing, China)) in a 0.8 µL Array Tape reaction volume with 10 ng dry DNA, 0.8 µL 2 × KASP master mix, and 0.011 µL primer mix (KBioscience, Hoddesdon, UK). A Nexar Liquid handling instrument was used to add the PCR solution to the Array Tape (Douglas Scientific). PCRs were performed on a Soellex PCR Thermal Cycler with the following conditions: initial denaturation at 94 • C for 15 min; followed by 10 cycles of denaturation at 94 • C for 20 s and 65 • C for 60 s at an annealing temperature that decreased by 0.8 • C per cycle; and then 26 cycles of denaturation at 94 • C for 20 s and 57 • C for 60 s; and a final cooling to 4 • C. A fluorescent end-point reading was completed with the Araya fluorescence detection system (part of the Douglas Scientific Array Tape Platform). Genotypes and clusters were visualized with Kraken (http: //ccb.jhu.edu/software/kraken/MANUAL.html (accessed on 5 August 2022)).
DNA was extracted from the 56 selected pea germplasms (resistant cultivars with known er1 alleles) and the six parents using the CTAB method [42]. The PCR amplification of the KASPar markers were performed as described above (in the section "Development of Functional Markers for the novel er1 alleles").