Genetic characterization and deployment of a major gene for grain yield on chromosome arm 1BS in winter wheat

Winter bread wheat (Triticum aestivum L., 2n = 6 × =42, AABBDD) cultivars “Duster” and “Billings” have occupied significant acreages in the Southern Great Plains for their outstanding yielding ability. In this study, we discovered a major quantitative trait locus (QTL) QYld.osu-1BS for grain yield in a population of 260 doubled haploid (DH) lines derived from the cross of Duster and Billings. When the population was tested under field conditions for 2 years, QYld.osu-1BS explained 13.9% and 23.5% of the total phenotypic variation. However, no crossover was observed among 40 genotyping-by-sequencing markers covering the region from the telomere to 25.3 Mb in the population of 260 DH lines. Furthermore, no crossover was observed in the region from the telomere to 18.4 Mb, when up to 4146 individual plants within F2:4 lines derived from the cross of Duster and Billings were screened. The 1BL-1RS translocation was not observed in the region with the abnormal recombination rate in Duster or Billings. Duster is a unique haplotype in the whole exome capture dataset, compared with 57 cultivars and breeding lines with various genetic backgrounds. Unique sequences of the QYld.osu-1BS allele for the higher grain yield in Duster were identified, and kompetitive allele specific PCR (KASP) markers for the unique sequences were developed for breeding of novel cultivars with increased grain yield in winter wheat.


Introduction
Wheat (Triticum aestivum L., 2n = 6 × =42, AABBDD) is one of the most important crops in the world. Winter wheat, which requires a period of low temperature to accelerate the transition from vegetative to reproductive development, occupies 75% of total wheat in worldwide (Pugsley 1971;Li et al. 2013). To maximize the grain yield of the winter wheat cultivars by conventional plant breeding and biotechnology is vital to wheat improvement of the most important economic trait (Rajaram 2005;Ray et al. 2013). Numerous quantitative trait loci (QTLs) or genomic regions have been mapped for yield traits by genetic analyses on segregated biparental populations, such as recombinant inbred lines (RILs) and doubled haploid (DH) lines, as well as association analyses on germplasm from landraces to modern varieties and breeding lines (Zhang et al. 2010;Bennett et al. 2012;Liu et al. 2012;Mir et al. 2012;Bordes et al. 2014;Cui et al. 2014;Zanke et al. 2014a, b;Gao et al. 2015;Lopes et al. 2015;Guo et al. 2016;Zhai et al. 2016;Assanga et al. 2017;Liu et al. 2018;Ma et al. 2018;Pradhan et al. 2019). Clustered QTLs were also found on the short arm of chromosome 1B, where pleiotropic effects on yield per plant, spikes per plant, spike length, grains per spike, thousand grain weight, and dry matter were observed (Quarrie et al. 2005;Wang et al. 2009;Sukumaran et al. 2015;Zhai et al. 2016). For grain yield as a trait, a major QTL was also located on chromosome 3AS (Baenziger et al. 2011;Rustgi et al. 2013), chromosome 5A in a diverse panel of spring wheat under drought stress , to the Vrn-B1 locus on chromosome 5B (Guedira et al. 2016;Sehgal et al. 2017). Vrn-B1 is a major vernalization gene that plays a crucial role, by using RIL populations derived from a cross between two soft winter wheat lines or by using CIMMYT wheat GWAM panel of 720 lines to test across diverse environments (Yan et al. 2003(Yan et al. , 2004Guedira et al. 2016;Sehgal et al. 2017). However, due to multiple loci following complex genetic interactions with environments, almost all of the previous yield QTLs account for only small parts of the phenotypic variation in grain yield, which has inhibited rapid utilization of the yield-related genes in wheat breeding (Quarrie et al. 2005;Bolot et al. 2009;Liu et al. 2012;Mir et al. 2012).
While the genetic effects of the genes/QTLs on grain yield are minor or genetic source for dramatic increases of grain yield in a gene pool in common wheat is lacking, alien genes from distant and closely related species of common wheat were exploited to improve grain yield (Huang et al. 2003). A genome fragment involving the short arm of rye chromosome 1R (1RS) was translocated into wheat, because the wheat-rye translocation provided resistance to insects and diseases while improved water-use efficiency and grain yield potential. The resulting 1BL.1RS translocation was used intensively in wheat breeding programs worldwide including those in the Southern Great Plains (Singh et al. 1998;Foulkes et al. 2002;Sukumaran et al. 2015). The 1BL-1RS translocation improved grain yield by increasing root and shoot dry weight under drought treatments, and increasing efficiencies of light conversion hence biomass (Singh et al. 1998;Foulkes et al. 2002;Ehdaie et al. 2003;Hoffmann 2008). However, the introgressed chromosome fragment carrying the desirable gene often introduces undesirable traits resulting in reduction of grain quality (Howell et al. 2014). New QTLs/genes that approach or even exceed the yield benefit of 1BL-1RS are greatly needed in the winter wheat.
The precise location of the gene for a QTL on a chromosome remains unknown in previous studies due to the lack of whole genome reference sequence, hence making it difficult to effectively utilize in wheat breeding. A draft sequence of the wheat genome was recently released by the International Wheat Genome Sequencing Consortium (IWGSC 2018), and the chromosomebased sequences provided a powerful tool for identification of physical locations of the markers developed using genotyping-by-sequencing approaches (Poland and Rife 2012;. The identified markers linking with grain-related traits are useful to screen germplasm for favorable alleles. This study reported a major gene for grain yield and its deployment by kompetitive allele specific PCR (KASP) markers in winter wheat.

Winter wheat population derived from Duster × Billings
In a previous study (Li et al. 2015), a doubled haploid (DH) population was developed from two locally adapted winter wheat cultivars, "Duster" (PI 644016) and "Billings" (PI 656843), both of which are released by the Oklahoma Agricultural Experiment Station. Duster and Billings have occupied significant acreages in the Southern Great Plains due to their outstanding yielding ability (Edwards et al. 2012;Hunger et al. 2014). While Duster offers yield protection across a diverse set of environmental conditions (Edwards et al. 2012), Billings shows larger kernel size and superior yield ability in high-yielding environments (Hunger et al. 2014).
The Duster × Billings population of 260 DH lines was tested in the field at the Stillwater Agronomy Research Station in 2014 (a drought year) and 2015 (a year with wide precipitation swings). The DH lines were arranged in a replicates-in-sets design with two replications in six sets for measuring grain in the field experiments. Each line was planted in two rows, and the single-row plots were 1 m long, spaced 0.5 m apart.
The F 3 #68 lines were derived from a cross between Duster × Billings, and those plants carrying the homozygous Duster allele and those carrying the homozygous Billings allele at QYld.osu-1BS, a QTL mapped in this study, were tested in fall 2017 in the same field as tested for the DH population. Each line was planted in a single 1 m row, spaced 0.33 m apart with two replicate sets in randomized design. The seeding rate was 50 seeds/row. The spikelets per spike (SNS), grains per spike (KNS), and grain weight per spike (GWS) were determined from twenty main spikes in each line.
Identification of unique sequence representing the Duster allele at QYld.osu-1BS The Duster × Billings DH population was genotyped using genotyping-by-sequencing (GBS) approach, and a total of 2358 GBS markers were subjected to linkage mapping for the DH lines (Li et al. 2015). The GBS marker sequences were used to blast IWGSC RefSeq v1.0 (https://urgi.versailles.inra.fr/blast/?dbgroup= wheat_iwgsc_refseq_v1_chromosomes), and the resulting physical locations were ordered along each chromosome.
Wheat exome capture data for 58 accessions including Duster and Billings in the coordinated agricultural project in wheat (Wheat CAP) was used to analyze genetic diversity in diploid, tetraploid, and hexaploid wheat (T3/Wheat database, https://triticeaetoolbox. org/wheat). The exon capture assay was expected to target 90% of the genes with non-redundant low-copy sequence (He et al. 2019). The sequences in the 25.3 Mb region covering the QYld.osu-1BS locus were used to analyze for haplotypes.
PCR markers for six genes were developed to confirm the physical location of QYld.osu-1BS or to genotype germplasm. The six genes are annotated i n I W G S C R e f S e q v 1 . 0 , i n c l u d i n g P LT ( p h o s p h o l i p i d -t r a n s p o r t i n g AT P a s e ) f o r Tr a e s C S 1 B 0 1 G 0 0 1 8 0 0 a t t h e p o s i t o n o f 1,430,072 bp, Pm3-B1 for TraesCS1B02G012000 at the positon of 5,851,440 bp, ZFP4 (zinc finger protein) for TraesCS1B02G022700 at the positon of 10,104,627 bp, NAK (serine/threonine-protein kinase-NAK) for TraesCS1B01G038800 at the positon o f 1 8 , 4 2 1 , 4 4 7 , a n d d C A P 3 f o r Tr a e s C S 1 B 0 2 G 0 4 5 4 0 0 a t t h e p o s i t o n o f 25,260,567 bp. Primers specific to the genes at the QYld.osu-1BS locus were designed, and PCR products representing each of Duster and Billings alleles were sequenced. Allelic variation between Duster and Billings were determined. The information for the primers and the expected sizes of PCR products is provided in Table 1.A genomic region with an extremely low recombination rate was found in the Duster × Billings DH population, and it was thus speculated that one of the parental lines might carry the 1RS/1B translocation. The 1RS/1B translocation has been observed in different wheat lines (Koebner 1995), and PCR markers for specific repetitive sequences can effectively detect small 1RS fragments from rye genome (Koebner 1995;Saal and Wricke 1999). The pSaD15 and pSc20H are two 1RS specific markers that are used to detect the translocated fragment from 1RS in the long arm of chromosome 1B (Liu et al. 2008;Zhai et al. 2016). In this study, three PCR markers, RIS, RYE-NOR, and SCM9 (Koebner 1995;Saal and Wricke 1999), were used to test if there is any translocated fragment from 1RS in the short arm of chromosome 1B in Duster or Billings. The information for the primers and the expected sizes of PCR products is provided in Table 1. PCRs for the above markers were performed in Quickload Master Mix (BioLabs) under the following conditions: denature at 94°C for 5 min, amplification for 35 cycles at 94°C for 30 s, 55-60°C (depending on Tm of primers) for 30 s, and 72°C for 0.5-2 min (depending on the lengths of PCR products) per cycle, and final extension at 72°C for 10 min.

Development of KASP-based assays for QYld.osu-1BS
In order to speed up the selection process, three KASPbased markers for unique sequence representing the Duster allele for higher grain yield were developed in this study. KASP primers were designed following standard KASP guidelines (LGC Genomics, Hoddeson, UK), and the primer sequences are provided in Table 1. The allele-specific forward primers were designed to carry the standard FAM (5′-GAAGGTGACCAAGT TCATGCT-3′) and HEX (5′-GAAGGTCGGAGTCA ACGGATT-3′) tails with the targeted SNP at the 3′ end, and a specific reverse primer was designed. FAM tail was added to Duster allele-specific forward primer for all three markers. The total amplicon length of expected PCR products was less than 200 bp. The primer mixture comprised 46 μl ddH 2 O, 30 μl common primer (100 μM), and 12 μl of each tailed primer (100 μM). Assays were tested in 96-well formats and set up as around 10 μl reactions (4.83 μl DNA at 40 ng/ μl, 5 μl of 1 × KASP master mixture, and 0.14 μl of primer mixture). PCR cycling was performed using the following protocol: hot start at 94°C for 15 min, followed by ten cycles of touchdown PCR (94°C for 20 s; touchdown at 61°C initially and decreasing by − 0.6°C per cycle for 60 s), followed by 40 additional cycles (94°C for 20 s; 55°C for 60 s). PCRs were performed in a real-time PCR cycler (ABI-7500) and PCR products were read in a fluorescence scanner following manufacturer's instruction (http://www.kbioscience.co. uk/reagents/KASP_manual.pdf). Testing was performed following the guideline of "Guide to running KASP genotyping on the ABI 7500 instrument" from LGC.

Genetic map of QYld.osu-1BS
The average of grain yield of 12 Duster plots was 1834.3 kg/ha, whereas the average of grain yield of 12 Billings plots was 1675.7 kg/ha, which showed a significant difference in 2014 (P < 0.05). The two parental lines showed a highly significant difference in the average of grain yield of 12 plots tested in 2015, 1834.3 kg/ ha for Duster and 1643.7 kg/ha for Billings (P < 0.001). The average of grain yield of the 260 Duster × Billings DH lines was 1564.91 kg/ha ranged from 793.55 to 2427.73 kg/ha in 2014, whereas the average of grain yield of the same population was 1600.55 kg/ha ranged A total of 2358 GBS markers was assembled into 26 linkage groups, forming genetic maps for the Duster × Billings DH population (accession number SRP051982). On the basis of whole-genome QTL scanning using interval mapping (IM) analysis, a QTL for grain yield was found in linkage group 8 (Fig. 1a). The linkage group 8, consisting of 197 GBS SNP markers, was assigned to chromosome 1B in the winter wheat. The total length of the linkage group was 120.32 cM in genetic distance, with a marker density of 0.61 cM per marker. The linkage group almost covered the whole chromosome The QTL on the short arm of chromosome 1B for grain yield was referred to as QYld.osu-1BS. The logarithm of the odds (LOD) value at the peak position of QYld.osu-1BS for grain yield was 7.9 in 2014 and 13.2 in 2015, and this QTL explained 13.9% and 23.5% of the total phenotypic variation in field-based grain yield tested in 2014 and 2015, respectively. At the QYld.osu-1BS locus, Duster carried the allele for higher grain yield whereas Billings carried the allele for lower grain yield. At QYld.osu-1BS is located in the distal region of chromosome arm 1BS (Fig. 1a). The peak of QYld.osu-1BS was linked with 40 clustered GBS markers, and no crossover was observed among 260 DH lines (Fig. 1a). The clustered markers cover the region from GBS12138 at the 3.6 Mb position to GBS9911 at the 22.3 Mb position. A gap was observed between GBS9911 at the 22.3 Mb and GBS10313 at 168.6 Mb (Fig. 1b), and 33 crossovers were found in the 146 Mb gap between the two neighbor markers.
PCR markers for four genes confirmed the physical location of QYld.osu-1BS, including PLT at 1.4 Mb, Pm3-B1 at 5.8 Mb, ZFP4 at 10.1 Mb, and NAK at 18.4 Mb (Fig. 2a-d). A PCR marker, dCAP3 for TraesCS1B02G045400 at the position of 25,260,567 bp, was developed (Fig. 2e). The same pair of primers were used to perform PCRs with Duster and Billings, and PCR products with expected sizes were directly sequenced and the sequence reactions worked well, indicating a single copy of the PCR products. The PCR products were distinguished between the two alleles by using restriction enzyme DpnII to do digestion. The dCAP3 marker was used to narrow the QTL region from 168.6 Mb down to 25.3 Mb from the telomere of the short arm of chromosome 1B.
A large mapping population was developed from a cross between Duster and Billings to narrow down the 25.3 Mb region of QYld.osu-1BS. A total of 4146 individual plants within F 2:4 lines were genotyped using flanking markers, and 28 recombinant events were observed, including two from 196 F 2 plants, two from 1390 F 3 plants, and 24 plants from 2560 F 4 plants (Fig. 1c). After fine mapping of internal markers, no recombination event was observed within the region from the telomere to the marker NAK at the position of 18.4 Mb and all of the recombinant events were found to have occurred between NAK at 18.4 Mb and dCAP3 at the position of 25.3 Mb. These results indicated abnormal recombination rate in the distal region of the short arm of chromosome 1B between Duster and Billings.
An F 3 population derived from the #68 F 2 plant was tested in the field, and the F 2 plant showed the homozygous Duster allele at dCAP3 (25.3 Mb) but heterozygous allele at NAK (18.4 Mb) and PLT (1.4 Mb). Those plants carrying the homozygous Duster allele and those carrying the homozygous Billings allele at QYld.osu-1BS were compared for phenotypes. As a result, the homozygous lines carrying the Duster allele at both PLT and NAK increased 2.94% in SNS, 7.20% in KNS, and 8.87% in GWS, compared with the lines carrying the homozygous Billings allele at the two markers (Table 2).

No alien fragment was detected in Duster or Billings
The 1BL.1RS translocation contributes 9% greater yield in NE in the absence of disease bias, and the near iso-chromosome stocks developed in the late 1980s were used intensively in wheat breeding programs in the Southern Great Plains (Moreno-Sevilla et al. 1995). In order to investigate if the 1BL.1RS was unexpectedly introduced into either Duster or Billings or their pedigrees, available markers for 1RS translocations, including RIS, NOR, and SCM9, were used to genotype the two parental lines. When the 1BL.1RS translocation was observed in Gallagher, it was not detectable in Duster or Billings using these PCR markers (Fig. 2f-h).
The exome capture assay was used to re-sequence the coding sequences of 58 wheat accessions including Duster and Billings in the Wheat CAP project. The variant calling using the GATK-based pipeline was performed, resulting in 1454 polymorphisms between Duster and Billings spanning over QYld.osu-1BS. Either Duster or Billings did not have identity to any of diploid wheat lines used in the project, suggesting neither of the two cultivars has translocation from any of the known genomes.

Identification of unique sequences in Duster and development of KASP markers for QYld.osu-1BS
Eighteen genes at the QYld.osu-1BS locus were selected for sequencing. Duster showed unique sequences in three genes, ZFP4, CLP for cyclophilin-like protein (TraesCS1B02G011100), and NMR for nitrogen metabolic regulator (TraesCS1B02G037100). In the NMR-B1 gene sequence, whereas Billings was 100% identical to CS, Duster was unique compared with the orthologous and homoeologous genes in diploid, tetraploid, and hexaploid wheat (Fig. 3). A PCR marker for the unique sequence at NMR in Duster was used to screen up to 135 accessions, only five accessions showed the same allele as Duster (Supplementary  Table S1).
Duster was a unique haplotype in the targeted QYld.osu-1BS region, compared with the other 57 cultivars and breeding lines with various genetic backgrounds that were used for exome capture. Among 6820 SNPs in the 25.3 Mb QYld.osu-1BS region, Duster had 1102 bp of unique SNPs compared with Billings and Chinese Spring. In comparison, Billings had 432 bp of unique SNPs compared with Duster and Chinese Spring. The 1064 bp (96.5%) out of the 1102 SNPs in Duster occurred in the region from the telomere to the marker NAK at the position of 18.4 Mb (Fig. 4a). In addition, among 58 accessions, Duster has 928 SNPs representing minor allele, and 718 (77%) out of the 928 SNPs in Duster were found to be located at the genomic region between 5 and 12.5 Mb (Fig. 4b).
The unique sequences in Duster were converted into high-throughput KASP assays. KASP-12 was designed for the SNP at 1,253,260 bp (Fig. 5a), KASP-17 was designed for the SNP at 10,104,175 bp (Fig. 5b), and KASP-8 was designed for the SNP at 18,386,168 bp (Fig. 5c). The unique markers representing the Duster

Discussion
In this study, QYld.osu-1BS, as a major QTL for grain yield, was discovered in Duster, which accounted for 18.7% of the phenotypic variation in grain yield of the entire population tested for 2 years. The Duster allele at QYld.osu-1BS increased 15.9% in field-based grain yield, compared with the Billings allele. QTLs for yield and yield components have been reported to account for only small parts of the phenotypic variation, because the QTLs may be subject to large genotype-environment interactions Kirigwi et al. 2007).
The major gene at QYld.osu-1BS in Duster is one of the largest QTL influencing grain yield identified to date.
As the present study advanced, a draft sequence of the wheat genome was recently released in the International Wheat Genome Sequencing Consortium (IWGSC 2018). The chromosome-based sequences provided a powerful tool for identification of chromosomal locations of the GBS markers. QYld.osu-1BS was located in a genomic region where recombination rate was extremely low. A hypothesis accounting for the abnormal recombination rate was that the 1RS translocation used extensively in the breeding programs in the region was unexpectedly introduced into either Duster or Billings. The 1RS genomic fragment is mostly utilized in spring wheat cultivars, but it has also been introduced into "Gallagher," a new winter wheat cultivar that was released in 2013 (PI 667569, PVP201300134). However, the results from PCR markers and exome capture sequences indicated that the low recombination at QYld.osu-1BS was not caused by 1BL-1RS or any alien fragment from accessions of diploid wheat species tested in the exome capture data.
Based on the phenotypes and genotypes of the F 3 -68 population, the gene for QYld.osu-1BS for grain yield could be located in the region from the telomere to the marker NAK at the position of 18.4 Mb. However, it should be cautious that the result was from the only one recombinant line. A final conclusion should be drawn after more lines that have crossovers between NAK at 18.4 Mb and dCAP3 at 25.3 Mb are tested for the phenotypes.
Positional cloning of the gene in the genomic region with low recombination rate is a difficult task, though the draft of genomic sequences in wheat has been available. If the low recombination region was resulted from the presence of an alien genomic fragment in Duster, probably in the region between the telomere to 18.4 Mb, the problem could be solved by introduction of Ph1 Unique sequences were found throughout 2150 bp of the complete NMR-B1 gene including conserved exon regions in Duster. The partial sequences including exon 2 of the NMR-B1 genes are aligned to indicate difference between Duster and other sources available in the IWGSC RefSeq v1.0 database. The AG splicing site at the 3′end of intron 1 of the NMR-B1 genes is indicated with a red rectangle gene (Dover and Riley 1972). Increased recombination events could be used to clone the gene responsible for QYld.osu-1BS, but it would take several years to introduce the Ph1 gene and purify genetic backgrounds brought from genetic materials carrying the Ph1 gene. Another approach for cloning of the gene is to create mutants of the genes in the targeted region by using EMS and validate the functions of the genes (Krasileva et al. 2017), but it could be too difficult to validate up to 360 genes with high confidence in the QYld.osu-1BS locus. Before the gene is cloned, the unique sequences identified in the QYld.osu-1BS region can be used as molecular markers for accelerating deployment of the gene for QYld.osu-1BS in conventional wheat breeding programs.
While the genetic mechanisms underlying the high grain yields in the two winter wheat cultivars remain a mystery, there is still substantial potential in exploitation of wheat yield genes per se, in cultivars currently deployed in wheat production. The major QYld.osu-1BS for grain yield was found in the locally adapted cultivar, allowing it to readily breed new wheat varieties. The showing the Duster allele is indicated in blue color, KASP-HEX showing the Billings allele is indicated in green color, and NTC (non-template control) used for controls is indicated in black dots QYld.osu-1B gene has been introduced into numerous lines by marker-assisted backcrossing and to deliver those lines to the hub for evaluation. Duster, has the QYld.osu-1B gene, and its offspring or grand-offspring have been introgressed in the pedigrees of 25% of the elite germplasm resident to the wheat improvement program. The research results from this study can be used to accelerate deployment of a beneficial allele for a major gene increasing grain yield, leading to more productive winter wheat varieties in diverse environments worldwide.
Author contributions C-C Kan, H Jia, C Powers, and L Yan performed the experiments and the analyzed data. B Carver developed and phenotyped the population. B Carver and L Yan designed the experiments. C-C Kan and L Yan wrote the manuscript. All authors read and approved the manuscript.
Funding information This project was supported by the Agriculture and Food Research Initiative Competitive Grants (2017-67007-25932) from the USDA National Institute of Food and Agriculture (NIFA), and grants from the Oklahoma Center for Advanced Science and Technology (OCAST, AR17-020-03). This study was also supported by the Oklahoma Wheat Research Foundation and the Oklahoma Agricultural Experiment Station.

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Conflict of interest The authors declare that they have no competing interests.
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