Investigation of Imidazolinone Herbicide Resistance Gene with KASP Markers for Japonica/Geng Rice Varieties in the Huanghuaihai Region of China

Rice is a staple food for more than half of the global population due to its food security and sustainable development. Weeds compete with crops for sunlight and indispensable nutrients, affecting the yield and quality of crops. Breeding herbicide-tolerant rice varieties paired with herbicide application is expected to help with weed control. In this study, 194 Japonica/Geng rice varieties or lines collected from the Huanghuaihai region of China were screened by Kompetitive Allele-Specific PCR (KASP) markers based on four mutation sites within OsALS1 (LOC_Os02g30630), which is the target of imidazolinone (IMI) herbicides. Only the OsALS1627N haplotype was identified in 18 varieties, including the previously reported Jingeng818 (JG818), and its herbicide resistance was validated by treatment with three IMIs. To investigate the origin of the OsALS1627N haplotype in the identified varieties, six codominant PCR-based markers tightly linked with OsALS1 were developed. PCR analysis revealed that the other 17 IMI-tolerant varieties were derived from JG818. We randomly selected three IMI-tolerant varieties for comparative whole-genome resequencing with known receptor parent varieties. Sequence alignment revealed that more loci from JG818 have been introduced into IMI-tolerant varieties. However, all three IMI-tolerant varieties carried clustered third type single nucleotide polymorphism (SNP) sites from unknown parents, indicating that these varieties were not directly derived from JG818, whereas those from different intermediate improved lines were crossed with JG818. Overall, we found that only OsALS1627N from JG818 has been broadly introduced into the Huanghuaihai region of China. Additionally, the 17 identified IMI-tolerant varieties provide alternative opportunities for improving such varieties along with other good traits.


Introduction
It is estimated that the world population will increase to over 9 billion by 2050 and determining how to feed this population is a serious challenge [1].Rice (Oryza sativa L.), an important cereal crop, is the staple food for more than half of the global population, providing 21% of the world's calories [2][3][4].Maintaining and increasing rice production is crucial for food security.Weeds are a serious biotic stress that pose a threat to rice yield, aggressively compete with crops for nutrients, light and other important resources, leading to greater than 40% yield loss in some regions [5][6][7].The shortage of labor in rice-planting areas is worsening because of the large number of people flowing to cities with the acceleration of the urbanization process, especially in China.To address this issue, rice cultivation methods are gradually shifting towards direct-seeded rice (DSR), and chemical herbicides are preferred for broad-spectrum weed control due to their low cost and high efficiency [8].However, improper use of herbicides usually results in indiscriminate hindrance to crop growth and ultimately leads to a decrease in crop yield.Additionally, breeding and planting herbicide-tolerant crops are highly important for easing weed infestations and maintaining sustainable crop production.Researchers have obtained herbicide-resistant rice through modern methods, such as transgenic technology, mutagenesis, breeding and gene editing, but restrictions on genetically modified plants make them difficult to plant and commercialize globally [9][10][11][12].Therefore, the genetic improvement of cultivated rice via natural selection or non-transgenic mutagenesis can ameliorate its tolerance to certain herbicides, along with the safe application of such herbicides to selectively kill weeds [8].
Plant resistance or tolerance to herbicides mainly comes from target site resistance (TSR) caused by mutations in a single gene, and most herbicides are designed to target specific enzymes or proteins.The acetyl-lactate synthase (ALS) protein is involved in the first synthesis of branched-chain amino acids (valine, leucine and isoleucine), which are vital for protein synthesis and plant growth.ALS inhibitors are Group B herbicides and are favored because of their broad spectrum, extreme activity, strong selectivity and high biosafety [13].To date, more than 50 commercial ALS inhibitors have been developed for weed control and essential crop protection [13,14].They include five main families of compounds with different structures: imidazolinones (IMIs), sulfonylureas (SUs), triazolo-pyrimidines (TPs), pyrimidine salicylic acids (PBs) and sulfonyl-aminocarbonyl-triazolinones (SCTs) [13].IMIs, including imazameth, imazamox, and imazethapyr, are among the most widely utilized groups of inhibitors of ALS [13].Moreover, mutations at specific sites within the ALS coding region have been verified as increasing crop and weed tolerance to IMIs [12,[15][16][17][18].The amino acid substitution resulting from the base mutation generates a novel ALS isoform that is less sensitive to IMIs but does not affect its enzyme activity compared with that of the wild type [19].Currently, mutations at four amino acid sites, A179V [20], W548M [21], S627N [22] and G628E [12], have been reported to confer IMI resistance in rice.Some of these plants have been widely planted in paddies [12].
Advances in high-throughput sequencing technology have spawned plentiful single nucleotide polymorphism (SNP) and insertion-deletion (InDel) sites in many crops, promoting the development of genotyping arrays based on these variants [23].The abundance of tools has accelerated the pace of molecular genomic breeding.Kompetitive Allele-Specific PCR (KASP) is a homogeneous, fluorescence-based genotyping technology based on allele-specific oligo extension and fluorescence resonance energy transfer (FRET) for signal generation [24].Two upstream primers in the KASP reaction system that differ in only 3 ′ terminal bases have the ability to discriminate between different haplotypes [24].Due to its advantages of high throughput, low cost, high reproducibility and easy operation, KASP has great application potential in the fields of agronomic trait improvement, variation screening and map-based cloning [25,26].
The annual planting area of Japonica/Geng rice in the Huanghuaihai region is approximately 1.73 million hectares, accounting for 18.6% of the rice planting area in China.The abundant sunshine, flat land and intensive and meticulous farming habits of locals make it one of the primary regions for high-quality rice production in China [27].However, Gramineae weeds, including Echinochloa crusgalli (L.) P. Beauv., Oryza sativa Linnaeus, Alopecurus aequalis, etc., have become serious biotic stresses that have plagued the rice-wheat rotation agricultural model and yield in this region [28].The adoption of light and simple cultivation and DSR technology has experienced rapid development and widespread promotion in this region due to its water and labor-saving benefits.IMI-tolerant rice varieties that originated from mutagenesis by ethyl methyl sulfonate (EMS) or the cultivar Jingeng818 (JG818) have been introduced into this region to cooperate with the cultivation system [29].Therefore, screening and breeding herbicide-tolerant varieties are of prime importance for the sustainable development of rice in this region.In this study, we selected IMI-targeted OsALS1 as the research object and screened 194 collected Japonica/Geng varieties with four KASP markers.Eighteen OsALS1 627N haplotype varieties were identified to be tolerant to IMI and originated from the same donor, JG818, based on PCR analysis with six developed InDel markers.Resequencing and sequence analysis were performed on three IMI-tolerant varieties and one of their parent plants.

Imidazolinone Herbicide Resistance Assay for OsALS1 627N Rice Varieties
As JG818 contains the OsALS1 627N mutation, to validate JG818 in our collections, we cloned OsALS1 and sequenced from JG818 as well as from the other three Japonica/Geng rice varieties, Nipponbare, Zhonghua11 and Kenxiang48, and two Indica/Xian rice varieties, 93-11 and Minghui63 (Figure S1).Sequence alignment revealed that only one specific

Imidazolinone Herbicide Resistance Assay for OsALS1 627N Rice Varieties
As JG818 contains the OsALS1 627N mutation, to validate JG818 in our collections, we cloned OsALS1 and sequenced from JG818 as well as from the other three Japonica/Geng rice varieties, Nipponbare, Zhonghua11 and Kenxiang48, and two Indica/Xian rice varieties, 93-11 and Minghui63 (Figure S1).Sequence alignment revealed that only one specific nucleotide difference (1880 G to A) existed between JG818 and the other five varieties.This mutation caused an amino acid substitution from serine to asparagine at position 627, which were named OsALS1 627S and OsALS1 627N , respectively.Another mutant site (1927 G to A) is jointly owned by JG818 and Minghui63, which cause the amino acid to change from valine to methionine at position 643; these are referred to as OsALS1 643V and OsALS1 643M , respectively.To probe whether the two substitution sites affect tolerance to IMIs, a phenotype validation experiment on six varieties was performed, and only JG818 was completely resistant to imazethapyr (Figure 2a).Moreover, Minghui63, which carries another variation type, OsALS1 643M , showed weak resistance to IMIs (Figure 2a).Taken together, the JG818 strains from the collections carried OsALS1 627N , which confers IMI tolerance.
Plants 2024, 13, x FOR PEER REVIEW 5 of 14 lotypes showed complete resistance to all three commercial IMI herbicides, including imazameth, imazamox and imazethapyr, while the other 15 OsALS1 627S varieties were sensitive and completely withered (Figure 2b).These results indicate that rice varieties harboring the OsALS1 627N variant identified from KASP are resistant to IMI herbicides.Water was used as a control.

Development of Molecular Markers Tightly Linked to OsALS1 627N from Sequenced JG818
JG818 was the first IMI-tolerant rice variety introduced into the Huanghuaihai region.The other 17 KASP-positive varieties exhibited OsALS1 627N variation, which is reminiscent of the same origin as that of the donor JG818.To confirm this speculation, Illumina second- To further evaluate the impact of changes in amino acids at the OsALS1 627N site in rice, 21 rice varieties, including 6 OsALS1 627N and 15 OsALS1 627S haplotypes, were randomly selected from 194 rice populations for the IMI tolerance assay.All six OsALS1 627N rice haplotypes showed complete resistance to all three commercial IMI herbicides, including imazameth, imazamox and imazethapyr, while the other 15 OsALS1 627S varieties were sensitive and completely withered (Figure 2b).These results indicate that rice varieties harboring the OsALS1 627N variant identified from KASP are resistant to IMI herbicides.

Development of Molecular
Markers Tightly Linked to OsALS1 627N from Sequenced JG818 JG818 was the first IMI-tolerant rice variety introduced into the Huanghuaihai region.The other 17 KASP-positive varieties exhibited OsALS1 627N variation, which is reminiscent of the same origin as that of the donor JG818.To confirm this speculation, Illumina secondgeneration sequencing was performed on JG818.Subsequently, the sequence was aligned with the reference Nipponbare genome sequence to determine the linkage and flanking of InDel markers with OsALS1.We developed 5 InDel and one cleaved amplified polymorphic sequence (CAPS) marker flanking OsALS1 (Figure 3a, Table 1).Electrophoresis revealed that the PCR products of markers M1~M6 were clearly different between JG818 and Nipponbare (Figure 3b). of InDel markers with OsALS1.We developed 5 InDel and one cleaved amplified po morphic sequence (CAPS) marker flanking OsALS1 (Figure 3a, Table 1).Electrophore revealed that the PCR products of markers M1~M6 were clearly different between JG and Nipponbare (Figure 3b).After validating the polymorphism between JG818 and Nipponbare, six mark were subjected to PCR analysis for all 17 OsALS1 627N varieties and 15 randomly selec OsALS1 627S varieties.As shown in Figure 3c, for all 17 positive varieties, JG818-spec DNA bands were amplified, whereas for 15 OsALS1 627S varieties, Nipponbare-spec DNA bands were amplified for all six markers (M1 to M6).It contains approximately Kb flanking the OsALS1 region and ranges from M4 to M5 (Figure 3a).These results in cate that the above 17 OsALS1 627N varieties share the same OsALS1 locus originating fr JG818.After validating the polymorphism between JG818 and Nipponbare, six markers were subjected to PCR analysis for all 17 OsALS1 627N varieties and 15 randomly selected OsALS1 627S varieties.As shown in Figure 3c, for all 17 positive varieties, JG818-specific DNA bands were amplified, whereas for 15 OsALS1 627S varieties, Nipponbare-specific DNA bands were amplified for all six markers (M1 to M6).It contains approximately 598 Kb flanking the OsALS1 region and ranges from M4 to M5 (Figure 3a).These results indicate that the above 17 OsALS1 627N varieties share the same OsALS1 locus originating from JG818.

Comparative Analysis of the Genome Sequence of Herbicide-Resistant Rice
To further confirm whether JG818 was the donor of the other OsALS1 627N varieties, we selected Runnong11 and Runnong11v2 (IMI-tolerant improved line of Runnong11), Huageng5 and Huageng5v2 (IMI-tolerant improved line of Huageng5) and Jifeng105 (one parent of which is Huageng5) for IMI tolerance tests and whole-genome sequencing.Run-nong11 and Huageng5 carry the OsALS1 627S genotype, while Runnong11v2, Huageng5v2 and Jifeng105 carry the OsALS1 627N genotype.Consistent with their genotypes, Run-nong11v2, Huageng5v2 and Jifeng105 were tolerant to imazethapyr treatment, whereas Runnong11 and Huageng5 were sensitive (Figure 4).nts 2024, 13, x FOR PEER REVIEW

Comparative Analysis of the Genome Sequence of Herbicide-Resistant R
To further confirm whether JG818 was the donor of the other O we selected Runnong11 and Runnong11v2 (IMI-tolerant improved l Huageng5 and Huageng5v2 (IMI-tolerant improved line of Huageng5 parent of which is Huageng5) for IMI tolerance tests and whole-genom nong11 and Huageng5 carry the OsALS1 627S genotype, while Runnon and Jifeng105 carry the OsALS1 627N genotype.Consistent with the nong11v2, Huageng5v2 and Jifeng105 were tolerant to imazethapyr Runnong11 and Huageng5 were sensitive (Figure 4).After resequencing, 4.09 to 10.18 Gb of mapped reads were generated that covered 96.85% to 97.55% of the rice genome for all six rice varieties or lines (Table 2).Through comparative analysis of the genome sequences of Runnong11v2 with those of JG818 and Run-nong11, we obtained 344,551 SNPs distributed evenly across 12 chromosomes.Among the total SNPs, 59,163 SNPs (17.17% of the total) were identified for JG818, while 258,674 SNPs were identified for Runnong11 (Table 2).In addition to the enrichment of SNPs from JG818 in the OsALS1 locus in the chromosome 2 (Chr) cluster, we also found that JG818-specific SNPs were clustered in the centromeric region of Chr 6, the short arms of Chr 5 and Chr 12, and the long arms of Chr 1, 3 and 7 (Figure 5a).In particular, 25384 SNPs (7.37% of the total) were not detected in either JG818 or Runnong11 (Figure 5a).Similar findings were also identified for the other two groups.According to the resequencing analysis of Huageng5, Huageng5v2 and JG818, 347,376 SNPs were evenly distributed on 12 rice chromosomes.Among them, 280,909 SNPs were consistent with Huageng5.Approximately 44,277 SNPs represented 12.75% of the total number of JG818-specific SNPs and were enriched on the long arms of Chr 1, 5, 7, 8, 9, and 12, the short arms of Chr 4, 6, 7 and 11, and the OsALS1 locus on Chr 2 (Figure 5b).Furthermore, 20,609 SNPs, which represented 5.93% of the total SNPs, were not identified in either JG818 or Huageng5 (Figure 5b).Additionally, the rice line Jifeng105 was also analysed and aligned with JG818 and Huageng5.A total of 391,478 SNPs were identified on all 12 chromosomes.There were 289,763 SNPs associated with Huageng5 and 79,139 SNPs (20.21% of the total) associated with JG818 (Figure 5c).Furthermore, there were also 20,510 SNPs identified in neither JG818 nor Huageng5 (Figure 5c).From the cluster enrichment of JG818-specific SNPs, in addition to the OsALS1 locus, Chr 1, 6, 7, 8, 9, 11, and 12 contained 1 to 3 enriched regions highlighted in green (Figure 5c).Overall, we found that there were long fragments of most chromosomes consistent with JG818 in all three OsALS1 627N genotype rice genomes.In particular, three improved lines (Runnong11v2, Huageng5v2, and Jifeng105) flanking the OsALS1 locus were 5-10 Mb in length on chromosome 2.In addition to this locus, many other clustered JG818-specific SNPs, represented by JG818-containing DNA fragments, have been identified on several other chromosomes.These results further suggested that OsALS1 627N in the Huanghuaihai region originated from the same donor.It has been employed in the genetic improvement of Japonica/Geng rice.
Mb in length on chromosome 2.In addition to this locus, many other clustered specific SNPs, represented by JG818-containing DNA fragments, have been ident several other chromosomes.These results further suggested that OsALS1 627N Huanghuaihai region originated from the same donor.It has been employed in the improvement of Japonica/Geng rice.

Discussion
To feed up to 1.4 billion people, light and simple cultivation and DSR technology prevailed in the Huanghuaihai region, where the Japonica/Geng rice variety was mostly planted.Herbicide-tolerant Japonica/Geng rice varieties are closely linked with the cultivation system to be introduced and developed in this region.JG818 was the first non-transgenic IMI-tolerant Japonica/Geng rice variety identified from large-scale screening of more than 7000 varieties with IMI treatment [29].After the approval certificate, it was introduced into this region in 2015.In this study, we screened 17 other IMI-tolerant varieties or lines other than JG818 from 194 collections (Figure 1).The distribution frequency was approximately 9.28% (Table S1).All the other 17 IMI-tolerant varieties or lines were identified to be the same OsALS1 627N haplotype varieties derived from JG818 (Figure 3).There are still many improved IMI-tolerant varieties derived from crosses with JG818 or its improved lines that have not been listed in these collections in this region [22,30,31].In summary, the herbicide gene derived from JG818 has received much attention and has been favored and has spread to local breeders and farmers over the past nine years.
Changes in the key amino acids in OsALS1 can reduce the sensitivity of the target enzyme to the corresponding herbicide.The target site resistance of ALS involves one or more specific amino acid mutations, namely, Ala122, Pro197, Ala205, Asp376, Trp574, Ser653 and Gly654 (corresponding sites in Arabidopsis thaliana) [32].The resistance type and effect of herbicides were determined by the amino acid mutation site and type of ALS.For example, ALS1 122Y/V showed high resistance to SU and TP herbicides but low resistance to IMI herbicides, while ALS1 122T amino acid mutations showed high resistance only to IMI herbicides [33][34][35][36][37]. Since the first IMI-tolerant rice was marketed in the USA in 2001, it was developed from the EMS mutagenesis line AS3510 [38].To date, a series of ALS inhibitorresistant mutation sites containing amino acid mutations at Ala179, Trp548, Ser627, and Gly628 have been found in cultivated rice [12,[20][21][22]39].The long-term use of a single herbicide resistance gene, particularly a haplotype mutation, will easily increase the risk of generating herbicide-resistant weeds.To our surprise, only one variation, OsALS1 627N , was identified from a total of 194 collections (Figure 1).These findings suggest that donors containing different OsALS1 haplotypes should be used to improve IMI-tolerant rice in the Huanghuaihai region in the future.Recently, some rice strains with mutations conferring tolerance to acetyl-CoA carboxylase (ACCase) inhibitor herbicides and glufosinate have been generated via EMS mutagenesis or heavy ion beam treatment in China [40][41][42].The introduction of IMI-tolerant varieties into the Huanghuaihai region and rotational planting of IMI-tolerant varieties are important for sustainable weed control.
Various single-marker methods, including allele-specific PCR (AS-PCR), CAPS, temperature-switch PCR (TS-PCR), ultrahigh-throughput chips and gene resequencing, have been developed for SNP genotyping.KASP is a fluorescence-based homogeneous genotyping technique originally developed by KBioscience in the United Kingdom [24].KASP can be used for accurate biallelic genotyping of target SNPs due to its accuracy, specificity, flexibility, low cost and high efficiency [43].Additionally, the combination of multiple high-throughput sequences and KASP can compensate for the shortcomings of both methods and plays an important role in germplasm resource identification, markerassisted breeding, genetic map construction and QTL mapping [33,44,45].In this study, we developed four KASP markers based on OsALS1 variations, which facilitated the screening of Japonica/Geng rice in the Huanghuaihai region.Additionally, PCR-based marker linkage to OsALS1 is a convenient method for marker-assisted selection of IMI-tolerant rice [46].We developed six PCR-based markers linked to OsALS1 based on comparative analysis of JG818 resequencing data that will facilitate marker-assisted selection of IMI-tolerant rice (Figure 3).In particular, these markers showed no polymorphisms among 15 randomly selected IMI-sensitive varieties or lines (Figure 3c).These markers could be used instead of KASP markers to screen for OsALS1 variation in JG818.

KASP Marker Development and Genotyping Assay
The OsALS1 sequences of Nipponbare, 93-11, and Minghui 63 were used as DNA templates.Primers were designed to amplify the allele sequence from JG818, ZH11, and Kenxi-ang48 (Table 3).The selection of mutation sites was based on alignment to the rice reference sequence and annotation of variations in A179V, W548M, S627N and G628E [12,[20][21][22].The KASP primers were designed and supplemented with the anchored sequence according to the instructions of the 2x Master Mix for ASPCR V1 (HC Scientific, Chengdu, China).The 5 µL KASP mixture consisted of 50 ng genomic DNA, 0.05 µM each upstream primer, 0.15 µM reverse primer, and 2.5 µL 2x Master Mix for ASPCR V1 (HC Scientific).KASP marker assessment was performed via Archimed X4 real-time fluorescent quantitative PCR system (Rocgene, Beijing, China) using the following thermal cycling program: predenaturing at 95 • C for 10 min; followed by 10 cycles of 95 • C for 20 s and 61~55 • C (gradient decrease of 0.

Conclusions
Based on the assessment of OsALS1-specific KASP markers, we identified 17 other rice varieties carrying the unique OsALS1 627N haplotype in the Huanghuaihai region of China, which provides important evidence that naturally mutated OsALS1 627N has been broadly introduced into cultivated Japonica/Geng rice varieties.These strains could serve as alternative donors to JG818 for improving IMI tolerance as well as improving other good agronomic traits.Additionally, it was first identified that all 17 IMI-tolerant varieties originated from the unique donor JG818.
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Figure 3 .
Figure 3. Development of PCR-based markers for linkage analysis of 18 OsALS1 627N varieties.(a Genetic location of the six developed markers on chromosome 2. (b) Polymorphism identified between Jingeng818 and Nipponbare.(c) Genotyping of 18 OsALS1 627N and 15 randomly selecte IMI-sensitive rice varieties or lines.Electrophoresis was performed on a 2.5% agarose gel with a Kb DNA ladder.

Figure 3 .
Figure 3. Development of PCR-based markers for linkage analysis of 18 OsALS1 627N varieties.(a) Genetic location of the six developed markers on chromosome 2. (b) Polymorphism identified between Jingeng818 and Nipponbare.(c) Genotyping of 18 OsALS1 627N and 15 randomly selected IMI-sensitive rice varieties or lines.Electrophoresis was performed on a 2.5% agarose gel with a 2 Kb DNA ladder.

6 •
C per cycle) for 40 s; followed by 30 cycles of 95 • C for 20 s and 55 • C for 40 s; and the fluorescence signals of FAM and HEM were collected during each program.The relative values of fluorescence intensity were exported for processing and genotyping generation by Microsoft Excel 2019 (Microsoft, Redmond, WA, USA).

Table 1 .
Primer sequence of six markers closely linked to OsALS1.

Table 1 .
Primer sequence of six markers closely linked to OsALS1.PCR product digested by Hinf I; Size of DNA fragment for 1 JG818 and 2 Nipponbare. *

Table 2 .
Re-sequencing data and mining SNPs for six rice varieties or lines.

Table 2 .
Re-sequencing data and mining SNPs for six rice varieties or lines.

Table 3 .
Primer sequence of four KASP markers for screening variations of OsALS1.highlighted sequences are complementary sequence of FAM and HEM labeled primers stored in PCR mix, respectively.Terminal base underlined represent as the variations. Italic