Genetic mapping and comparative analysis of heading date in hexaploid oat

doi:10.21203/rs.3.rs-4837955/v1

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Genetic mapping and comparative analysis of heading date in hexaploid oat

https://doi.org/10.21203/rs.3.rs-4837955/v1

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Heading date is a critical stage in the growth and development of oats. The objectives of this study were to identify DNA markers associated with quantitative trait loci (QTL) affecting heading date in hexaploid oat, and to locate and characterize heading date QTL by comparative analysis using an oat reference genome. Genetic linkage maps were constructed using single nucleotide polymorphism (SNP) markers in RIL lines from oat populations FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett. Days to heading were collected in field trials, and QTLs for heading date were identified in several genomic regions of the two maps. Oat loci with the most significant additive effects were associated with linkage groups Mrg02, Mrg06, and Mrg05 in the FL0206B-S-B-S1 × UFRGS 078030-1 population and with Mrg12, Mrg24, and Mrg11 in the URS Taura × Leggett population. Major QTLs affecting heading date were mapped to locations consistent with known QTLs and candidate genes that play critical roles in regulating flowering. These genes include Hd3a and FT, which are orthologs of the Vrn3 gene and integrate the vernalization and photoperiod response pathways. The gene PRR37 is an ortholog of Ppd1, the main gene determining photoperiod sensitivity in wheat and barley. The COL13 and GI genes are involved in the regulation of flowering by photoperiod. In addition, novel QTLs were identified that are valuable for molecular selection in oat breeding programs aimed at developing new and improved oat cultivars adapted to specific growing conditions.

Heading date

Flowering time

Hexaploid oat (Avena sativa)

SNP markers

Quantitative trait loci (QTL)

Comparative analysis

Cultivated oat (Avena sativa L.) is an important cereal crop, providing high-quality grain for human consumption and animal feed. Avena sativa is an allohexaploid species (2n = 6x = 42) that arose through natural interspecific hybridization of the AACCDD genomic constitution, a member of the Poaceae family, and has a large genome of approximately 12.5 billion nucleotides (Chaffin et al. 2016; Fu 2018). This cereal crop is grown on over nine million hectares annually, primarily for soil fertility improvement, crop rotation, and as a source of animal feed and human food. Adapting oat genotypes to specific environments directly relates to their ability to flower under conditions that optimize reproductive success and seed production. Flowering time (here equated with heading date) is a complex biological event in oats. Most of its natural variation is related to photoperiod (here equated with day length) and vernalization pathways. Oat is a long-day crop species, with many genotypes showing earlier flowering under longer photoperiods (Sorrells and Simmons 1992). In wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), the response to photoperiod is mainly controlled by dominant alleles of the Ppd-1 (Photoperiod-1) and Ppd-H1 genes, respectively (Kamran et al. 2014; Royo et al. 2016). In these species, Ppd-1 and Ppd-H1 stimulate flowering by upregulating the CO (CONSTANS) and Vrn3 (Vernalization 3) genes under long-day conditions (Distelfeld et al. 2009). In wheat, the Ppd-1 gene is regulated by the PHYB (Phytochrome B) and PHYC (Phytochrome C) genes, which act as red and far-red light receptors (Chen et al. 2015; Pearce et al. 2016; Kippes et al. 2020).

Oat genotypes also differ in their response to vernalization, which involves prolonged exposure to low temperatures to induce flowering. Differences in the nucleotide sequences of the Vrn1 (Vernalization 1), Vrn2 (Vernalization 2), Vrn3 and Vrn-D4 (Vernalization-D4) genes have been linked to differences in vernalization response in wheat (Kippes et al. 2016). In oats, the Vrn3 gene was found in collinear chromosomal regions in three mapping populations (Nava et al. 2012). In addition, oat sequences associated with the vernalization genes Vrn1, Vrn2, and Vrn3 were identified by orthology with other grass species. Oat sequences showed 78–87% similarity to Vrn1 genes from Lolium multiflorum, Lolium perenne, Festuca arundinacea, Festuca pratensis, Hordeum vulgare, Secale cereale, Triticum timopheevii, Triticum turgidum, Triticum monococcum, Triticum Urartu and Aegilops tauschii. Oat sequences also showed 91–96% similarity to FT (Flowering locus T) genes from L. perenne, Lolium temulentum, L. multiflorum and F. pratensis, and Vrn3 genes from T. turgidum, Triticum aestivum, T. monococcum and H. vulgare. While the oat sequences did not closely resemble grass Vrn2 genes, they showed 89–90% similarity to a retrotransposon (OARE-1) derived from Avena sativa. This suggests that the presence of OARE-1 in the oat genome results in non-functional alleles of the Vrn2 gene. Since the Vrn2 gene suppresses flowering by requiring vernalization, the absence of functional Vrn2 alleles allows oats to be grown in regions without natural vernalization conditions, such as subtropical environments like southern Brazil (Nava et al. 2012).

Ambient temperature also has a variable effect on the flowering time of oat genotypes. Differences in flowering time were demonstrated in a previous study where six oat genotypes were evaluated. The genotypes were grown under controlled environmental conditions with a 12-hour photoperiod and temperatures of 17 ^oC and 28 ^oC. At 17 ^oC, the oat genotypes UFRGS 8 and Amagalon/4*Ogle took 112 and 128 days, respectively, from emergence to heading, while at 28 ^oC the number of days decreased to 71 and 93, respectively. For the oat genotype UFRGS 881971, the number of days from emergence to heading remained constant at both temperatures (138 days) (Locatelli et al. 2008). In Arabidopsis thaliana, an increase in temperature from 23 ^oC to 27 ^oC induced flowering under a short photoperiod of eight hours of light (Balasubramanian et al. 2006). The temperature response in Arabidopsis has been linked to the PIF4 (PHYTOCHROME INTERACTING FACTOR 4) gene. PIF4 binds to the promoter region of the FT gene, stimulating its transcription, and facilitating flowering under short photoperiod (Kumar and Wigge 2010; Kumar et al. 2012). Ambient temperature also affects flowering time by regulating gene expression at the post-transcriptional level. Small non-coding RNAs (microRNAs) such as miR156 and miR172 play a crucial role in flowering regulation (Capovilla et al. 2015). The highest transcription rate of the miR172 gene was observed at 23 ^oC compared to 16 ^oC, while the transcription rate of the miR156 gene showed the opposite trend (Lee et al. 2010). miR172 promotes flowering by post-transcriptionally regulating target genes of the AP2 (APETALA 2) family, which function as flowering repressors (Aukerman and Sakai 2003).

The development of linkage maps has been challenging in oat, mainly due to physical rearrangements between chromosomes involving translocations and inversions (Tinker et al. 2022). However, quantitative trait loci (QTL) associated with flowering time have been mapped in recent decades. For example, QTLs associated with early flowering and response to vernalization have been identified in the Kanota × Ogle and Ogle × TAMO-301 populations (Holland et al. 1997; Holland et al. 2002). QTLs for vernalization and photoperiod response were also identified in three Brazilian oat populations, UFRGS 8 × Pc68/5*Starter, UFRGS 881971 × Pc68/5*Starter, and UFRGS 8 × UFRGS 930605 (Locatelli et al. 2006; Nava et al. 2012). QTLs influencing heading date were identified by genome-wide association studies (GWAS) performed by Klos et al. (2016). Several linkage groups of the map were found to contain QTLs influencing heading date, including Mrg01, Mrg02, Mrg06, Mrg11, Mrg12, Mrg13, Mrg17, Mrg20, and Mrg 24. However, most of these QTLs did not show consistency across different locations and years of testing, indicating a significant QTL x environment interaction. Among these, Mrg02, Mrg12, Mrg13, and Mrg14 were the most consistent across locations and years. QTLs influencing heading date located on Mrg02, Mrg12, Mrg20, Mrg21, and Mrg28 were also reported by Sunstrum et al. (2019), where Mrg20 and Mrg21 are homeologous chromosomes carrying the vernalization gene Vrn1 (Sunstrum et al. 2019). Recently, QTLs influencing the heading date trait were detected and located in linkage groups Mrg02, Mrg05, Mrg06, Mrg12, and Mrg21 (Zimmer et al. 2022).

In addition to QTL mapping, recent advances have been made in oat genomics. Some of these advances include the development of a high-density consensus linkage map, improved understanding of chromosomal rearrangements in hexaploid oat relative to its ancestral diploid genomes, application of genomic selection for heading date using tag-level genetic variant haplotypes and SNP markers, genome sequencing and annotation of diploid oat relatives, and the development of a fully annotated reference genome for spring oat (Chaffin et al. 2016; Bekele et al. 2018; Maughan et al. 2019; Kamal et al. 2022). All this information allows the identification of QTLs affecting important agronomic traits, such as heading date, in the context of comparative genomics. It will also allow molecular selection in oat breeding programs aimed at developing new and improved cultivars adapted to specific growing conditions. The objectives of this study were to identify loci associated with quantitative trait loci (QTL) affecting heading date in hexaploid oat, and to locate and characterize QTLs affecting heading date by comparative analysis using an oat reference genome.

Population development

Two populations of recombinant inbred lines (RILs) resulting from the crosses FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett were evaluated. The RIL lines were developed by single-seed descent without artificial selection. The parents FL0206B-S-B-S1, UFRGS 078030-1, URS Taura, and Leggett originate from different oat breeding programs and show significant phenotypic differences in heading date. FL0206B-S-B-S1 (UFRGS 995088-3/LA9535D118-4) is a late-flowering genotype developed at the University of Florida, USA. UFRGS 078030-1 (UPF 93203-4/FL98061 F₁(IL90-1847/LA90122-BBB-7-1-1)) and URS Taura (UFRGS 970216-2/UFRGS 970461) are early-flowering genotypes developed at the Federal University of Rio Grande do Sul (UFRGS), Brazil. Leggett (OT294/Pc94) is a very late-flowering genotype developed at Agriculture and Agri-Food Canada, Canada. These phenotypes reflect the differences in heading date observed when the parents were grown under field conditions in southern Brazil.

Phenotyping

Heading date was measured in two large populations of self-pollinated F_4:5 and F_5:6 RIL lines in field trials. The FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations contained 460 and 326 RIL lines, respectively. Fifteen seeds of each RIL line and parent were sown in one meter long rows at 0.3-meter row spacing. The experiment was conducted in non-randomized, non-overlapping plots at the UFRGS Agronomy Experimental Station in Brazil (30º 05' 47.5" S and 51º 40' 26" W). In the F_5:6 generation, RIL lines from both populations and parents were tested in experiments to measure differences in the heading date for early and late planting in field trials. Early planting occurred on June 8, 2018 and the late planting on July 13, 2018. Ten plants of each F_5:6 RIL line and parent were grown in hill plots with 0.3-meter spacing between hills and rows. The experiments followed a randomized complete block design with two replications at the UFRGS Agronomy Experimental Station. Flowering time was estimated as the number of days from emergence to heading, recorded as the approximate time of panicle appearance at stage 55 of the Zadoks scale (Zadoks et al. 1974). Days to heading were further analysed by frequency distribution in both populations using the R/qtl software available at www.r-project.org. Supplementary Fig. S1 shows the environmental conditions, including mean daily temperature and photoperiod from sowing to heading date in the field trials. Mean daily temperatures were recorded using a Vantage Pro2™ (Davis Instruments) weather station at the UFRGS Agronomy Experimental Station, and photoperiod data were obtained from Time and Date at https://www.timeanddate.com/sun/@6317055.

Marker analysis

Single nucleotide polymorphism (SNP) markers were identified in 409 F_5:6 RIL lines in the FL0206B-S-B-S1 × UFRGS 078030-1 population, while the URS Taura × Leggett population included 325 F_5:6 RIL lines. Fifteen seeds from the same panicle of each RIL line and parent were germinated on wet germination paper and placed in a biochemical oxygen demand chamber at 25 ºC for seven days without light. Approximately 90 mg of fresh leaf tissue collected from each RIL line and parent was lyophilized for 48 hours and stored at -20 ^oC. DNA was isolated, purified, and genotyped at the University of Minnesota Genomic Center, Saint Paul, MN, USA. Genomic DNA was isolated using mechanical tissue disruption and the DNeasy® Plant DNA Isolation Kit (Qiagen, Valencia, USA). PicoGreen® dsDNA reagents and NanoDrop™ were used for DNA quantification. SNP markers were identified using the genotyping-by-sequencing (GBS) method, which uses endonucleases to reduce DNA complexity, according to Poland et al. (2012). Genomic DNA was digested with the rare-cutting endonuclease PstI (restriction site CTGCAG) and the frequent-cutting endonuclease MspI (recognition site CCGG), and the resulting single DNA molecules were barcoded. The GBS libraries were prepared and sequenced on a NovaSeq™ 6000 platform (Illumina, San Diego, USA), producing short reads of approximately 100 base pairs. Markers were then called using Haplotag software (Tinker et al. 2016), with polymorphic haplotype markers aligned to the high-density oat consensus map (Bekele et al. 2018) and filtered as biallelic markers with a minor allele frequency of 30%, minimum completeness of 95%, and maximum heterozygosity of 8%.

Map construction

Linkage maps were constructed for the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations using JoinMap 4.0 (van Ooijen 2006). Polymorphic SNP markers between the parents of each population were identified based on the criteria of the SNP calling and filtering steps described above. The genotypic data were converted into the coding system required by JoinMap. In this coding system, oat RIL lines with alleles from FL0206B-S-B-S1 and Leggett were assigned code a, RIL lines with alleles from UFRGS 078030-1 and URS Taura were assigned code b, and RIL lines with heterozygous loci were assigned code h. SNP markers with a segregation bias greater than 5% by chi-squared test and highly similar loci were excluded from further analysis. Linkage groups were formed with a logarithm of the odds score of seven and a maximum recombination frequency of 30%. Recombination frequencies were converted to genetic distances in centiMorgan using the Kosambi function. Marker order was estimated using the maximum-likelihood mapping algorithm with default settings.

QTL analysis

Quantitative trait loci (QTL) analysis was performed in the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations using the R/qtl software. QTL significance was estimated by 10,000 permutations of the data, each generating the maximum test statistic for a complete genome-wide scan using simple interval mapping (SIM) and composite interval mapping (CIM), with an experiment-wide type I error rate of p < 0.05. For the SIM genome scan, the multiple imputation method was applied using the sim.geno function, followed by the imp method. For the CIM genome scan, the two SNP markers with the most significant effects in the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations in SIM were selected as covariates to control for background variance using the cim function. Previous studies in oat (Sunstrum et al. 2019) and wheat (Wolde et al. 2019) were taken into account to select markers with the highest statistical significance. The QTL analysis was performed using the phenotypic variation in the number of days from emergence to heading observed in the F_4:5 self-pollinated generation in both populations and the phenotypic variation in the number of days from emergence to heading evaluated for early and late planting in the F_5:6 self-pollinated generation in both populations.

Comparative genomics

Newly developed SNP markers linked to QTLs affecting heading date in RIL lines from the oat populations FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett were submitted for a recent comparative analysis using a reference genome from GrainGenes, a database for Triticeae and Avena (https://wheat.pw.usda.gov/GG3/). The nucleotide sequence of each SNP marker was first subjected to a Basic Local Alignment Search Tool (BLAST; Altschul et al. 1990) using the collection of PepsiCo OT3098 Hexaploid Oat v2 pseudomolecules (2021) available from PepsiCo at https://wheat.pw.usda.gov/jb?data=/ggds/oat-ot3098v2-pepsico. SNP markers were mapped to oat chromosomes on the OT3098 v2 physical map. Only oat chromosomes with the highest BLAST query coverage and identity were further considered in this study. JBrowse was used to define the position of the SNP markers on the oat chromosomes. Using the annotation tools GBS Markers and Oat-2018-Consensus (2018 Mrg) map regions vs. OT3098 v2 from GrainGenes, the chromosomal position of each SNP marker was mapped to the corresponding 2018 Mrg linkage group and names from the high-density SNP consensus linkage map (Bekele et al. 2018). A genomic region of 40 Mbp on each Mrg group was scanned to locate annotated candidate genes and mapped QTLs controlling flowering time in the oat consensus linkage map. The nucleotide sequence of SNP markers was mapped to the specific positions of candidate genes using Gene Set v3 with annotations and to QTLs from previous studies using QTL/gene inventory: comparative mapping vs. OT3098 v2 from GrainGenes, according to Wight et al. (2024).

Phenotypes

The number of days from emergence to heading varied between oat populations and field trials. In the FL0206B-S-B-S1 × UFRGS 078030-1 population, days to heading varied from 59 to 79 days among the 460 F_4:5 RIL lines, with a variation of 20 days. The average number of days to heading for this population was 69 days. Phenotypic variation was also observed for the parents FL0206B-S-B-S1 and UFRGS 078030-1, with average days to heading of 78 and 65 days, respectively (Fig. 1a). When this population was evaluated for early and late planting, the number of days from emergence to heading ranged from 78 to 102 days for early planting and from 66 to 86 days for late planting. The average number of days to heading among the 460 F_5:6 RIL lines was 90 days for early planting and 74 days for late planting. Parents FL0206B-S-B-S1 and UFRGS 078030-1 showed average days to heading of 94 and 88 days for early planting and 79 and 72 days for late planting, respectively (Fig. 1c). Early and late transgressive phenotypes were observed in the FL0206B-S-B-S1 × UFRGS 078030-1 population. In the F_4:5 generation, 73 RIL lines showed a lower number of days to heading compared to the early-flowering parent UFRGS 078030-1, while 11 RIL lines showed a higher number of days to heading compared to the late-flowering parent FL0206B-S-B-S1 (Fig. 1a). For early planting, 95 F_5:6 RIL lines were earlier than UFRGS 078030-1 and 47 RIL lines were later than FL0206B-S-B-S1. For late planting, 153 F_5:6 RIL lines showed an earlier phenotype than UFRGS 078030-1 and 32 RIL lines showed a later phenotype than FL0206B-S-B-S1 (Fig. 1c).

The number of days from emergence to heading for the URS Taura × Leggett ranged from 64 to 109 days among the 326 F_4:5 RIL lines, with an average of 82 days. The average number of days to heading for the parents was 66 days for URS Taura and 114 days for Leggett, indicating significant phenotypic variation between them (Fig. 1b). Heading dates evaluated in the F_5:6 generation ranged from 87 to 127 days for early planting and 70 to 102 days for late planting. The average days to heading for the 326 F_5:6 RILs was 103 for early planting and 84 for late planting. The URS Taura and Leggett parents had average days to heading of 91 and 119 days for early planting and 71 and 100 days for late planting (Fig. 1d). RIL lines and parents from the URS Taura × Leggett population had a greater variation in the number of days to heading in all field trials than RIL lines and parents from the FL0206B-S-B-S1 × UFRGS 078030-1 population. However, transgressive segregation was only observed in a few RIL lines in the URS Taura × Leggett population. In the F_4:5 generation, only one RIL line headed earlier than URS Taura, but none headed later than Leggett (Fig. 1b). At early planting, six RIL lines headed earlier than URS Taura, and six RIL lines headed later than Leggett. At late planting, one RIL line headed earlier than URS Taura and two RIL lines headed later than Leggett (Fig. 1d). For the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations, the highest average number of days to heading was observed for the RIL lines and parents evaluated at early planting. This can be attributed to the lower mean daily temperature (around 16 ^oC) and photoperiod (around 11 hours) recorded during the field trial. Conversely, the lowest average number of days to heading for the RIL lines and parents in both populations occurred in the F_4:5 generation, where the highest mean daily temperature (around 19 ^oC) was recorded, exceeding the mean daily temperature (around 18 ^oC) recorded at late planting (Fig. S1). All RIL lines and parents in both populations had fewer days to heading when plants were grown in late planting than in early planting (Fig. 1c, 1d). This response was mainly due to environmental factors such as temperature and photoperiod. The photoperiod was longer and the temperature was higher in late planting, and these environmental factors can accelerate the heading date.

Marker analysis and map construction

A set of 666 newly developed SNP markers identified by genotyping-by-sequencing (GBS) was mapped in the FL0206B-S-S-S1 × UFRGS 078030-1 population, resulting in 28 linkage groups. Marker density varied between linkage groups, ranging from three in linkage groups 7 and 18 to 81 in linkage group 3, indicating an uneven distribution of markers between groups. The genetic length of the linkage groups varied from 4.5 cM in linkage group 18 to 203.5 cM in linkage group 3. The total map distance for this population covered a genetic distance of 1929.5 cM. In the URS Taura × Leggett population, 869 newly developed SNP markers were mapped, resulting in 27 linkage groups. Linkage groups 26 and 27 were the smallest with two SNP markers, while linkage group 1 was the largest with 98 SNP markers. The genetic length of the linkage groups varied from 1.8 cM in linkage group 26 to 176.8 cM in linkage group 4. The total map distance for this population covered a genetic distance of 2220.4 cM. The complete linkage maps for the oat populations FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett are shown in Supplementary Figs. S2 and S3.

QTL analysis

Several genomic regions on the linkage maps contained QTLs affecting heading date in the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett oat populations. In the FL0206B-S-B-S1 × UFRGS 078030-1 population, 21 SNP markers were associated with nine QTLs across all environments. Of these, 16 SNP markers contributed to an increase and five to a decrease in the number of days from emergence to heading. In the F_4:5 generation, the observed phenotypic variation for days to heading was associated with the markers avgbs1380849_19, avgbs2550895_20, avgbs2070084_58, avgbs227981_5, avgbs683618_13, avgbs2382938_73, avgbs60602_48 and avgbs1464089_47. The avgbs60602_48 and avgbs1464089_47 markers in linkage group 2, had the largest effects, increasing the heading date by five days (Table 1). For early planting, heading date was associated with the markers avgbs691688_27, avgbs915246_5, avgbs557366_70, avgbs1601155_57, avgbs447148_10, avgbs1261907_19, avgbs396242_32 and avgbs1464089_47. In this environment, smaller effects were estimated compared to the evaluation carried out in the F_4:5 generation, where alleles from the FL0206B-S-B-S1 parent for the markers avgbs915246_5, avgbs447148_10, avgbs1261907_19, avgbs396242_32 and avgbs1464089_47 increased the heading date by two days (Table 1). For late planting, heading date was associated with the markers avgbs809730_11, avgbs447152_34, avgbs447148_10, avgbs169487_61, avgbs1511138_28, avgbs1497003_26, avgbs1773912_44 and avgbs1464089_47, with alleles from the FL0206B-S-B-S1 parent for these markers increasing the heading date by one or two days (Table 1). The avgbs1464089_47 marker had a statistically significant effect in all environments. In addition, the avgbs447148_10 marker had statistically significant effects in early and late planting.

Table 1

Summary of QTL affecting heading date in the FL0206B-S-B-S1 × UFRGS 078030-1 oat population and comparative analysis
Loci	Linkage group	Map position (cM)	Additive main effect^a	Oat chromosome^b	Identity (%)^c	Consensus map^d	Position (cM)^e
Days to heading at F_4:5
avgbs1380849_19	28	0.0	1	4A	98	Mrg20	241
avgbs2550895_20	9	26.2	-1	5C	100	Mrg03	18.6
avgbs2070084_58	24	0.0	1	5C	100	Mrg03	~ 100
avgbs227981_5	14	95.7	2	6A	100	Mrg05	~ 130.9
avgbs683618_13	14	41.7	2	6D	98.5	Mrg04	~ 40.1
avgbs2382938_73	3	134.6	1	7C	99.5	Mrg09	49.2
avgbs60602_48	2	66.0	5	7D	100	Mrg02	~ 73.3
avgbs1464089_47	2	76.4	5	7D	100	Mrg02	~ 86.5
Days to heading in the early planting
avgbs691688_27	6	16.3	-1	2C	99	Mrg13	~ 64
avgbs915246_5	6	0.0	2	2C	100	Mrg13	~ 69.5
avgbs557366_70	12	86.6	-1	2D	99	Mrg08	~ 63.5
avgbs1601155_57	1	74.5	-2	5D	90	Mrg06	72.1
avgbs447148_10	14	100.8	2	6A	100	Mrg05	~ 124.2
avgbs1261907_19	14	103.0	2	6A	99.5	Mrg05	135.5
avgbs396242_32	2	84.1	2	7D	99	Mrg02	72.7
avgbs1464089_47	2	76.4	2	7D	100	Mrg02	~ 86.5
Days to heading in the late planting
avgbs809730_11	1	84.3	-1	5D	100	Mrg06	66.1
avgbs447152_34	14	100.6	2	6A	100	Mrg05	124.1
avgbs447148_10	14	100.8	2	6A	100	Mrg05	~ 124.2
avgbs169487_61	14	114.9	1	6A	99	Mrg05	~ 140
avgbs1511138_28	3	203.5	1	7C	100	Mrg09	25.2
avgbs1497003_26	2	63.5	2	7D	100	Mrg02	~ 34.1
avgbs1773912_44	2	45.3	1	7D	100	Mrg02	~ 80
avgbs1464089_47	2	76.4	2	7D	100	Mrg02	~ 86.5
^aAdditive main effect (number of days increase caused by parent 1 (FL0206B-S-B-S1) relative to parent 2 (UFRGS 078030-1))
^bSNP markers assigned to oat chromosomes through BLAST analysis using the OT3098 v2 physical map
^cThe extent to which nucleotide sequences had the same residues at the same positions in BLAST analysis from GrainGenes
^d,eCorresponding 2018 Mrg linkage group names and cM distances

In the URS Taura × Leggett population, 10 SNP markers were associated with seven QTLs controlling heading date in all environments. Of these, eight markers contributed to a reduction in the number of days from emergence to heading, while two contributed to an increase. In the F_4:5 generation, the phenotypic variation for days to heading was associated with the avgbs292292_44, avgbs292351_17, avgbs1491480_21, and avgbs390936_44 markers. Alleles from the URS Taura parent for avgbs292292_44 and avgbs1491480_21 reduced the heading date by five days, while avgbs292351_17 reduced it by seven days (Table 2). For early planting, the heading date was influenced by the markers avgbs798773_8, avgbs1040030_69, avgbs1491480_21, avgbs342711_16, avgbs390936_44, and avgbs674946_34. Marker avgbs1040030_69 had the greatest effect, reducing the heading date by nine days, while avgbs798773_8 and avgbs674946_34 each reduced it by eight days (Table 2). For late planting, heading date was associated with the markers avgbs1452471_63, avgbs605397_30, and avgbs390936_44. Marker avgbs605397_30 had the most significant effect, reducing the heading date by seven days. Marker avgbs390936_44 increased heading by three days and had a statistically significant and consistent additive main effect across all environments (Table 2). In addition, the URS Taura × Leggett population showed greater effects than the FL0206B-S-B-S1 × UFRGS 078030-1 population in all environments. The short-read sequence of the newly developed SNP markers associated with QTLs affecting heading date in the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett oat populations is shown in Supplementary Table S1.

Table 2

Summary of QTL affecting heading date in the URS Taura × Leggett oat population and comparative analysis
Loci	Linkage group	Map position (cM)	Additive main effect^a	Oat chromosome^b	Identity (%)^c	Mrg linkage group^d	Distance (cM)^e
Days to heading at F_4:5
avgbs292292_44	11	98.1	5	1D	100	Mrg01	~ 45.1
avgbs292351_17	11	96.4	7	1D	100	Mrg01	~ 52.6
avgbs1491480_21	2	45.8	5	3C	99	Mrg15	50.3
avgbs390936_44	12	55.8	-3	5A	97	Mrg24	76.4
Days to heading in early planting
avgbs798773_8	9	123.7	8	2C	98	Mrg13	21.2
avgbs1040030_69	2	51.0	9	3C	100	Mrg15	~ 35.1
avgbs1491480_21	2	45.8	5	3C	99	Mrg15	50.3
avgbs342711_16	1	50.9	-5	4C	100	Mrg11	52.8
avgbs390936_44	12	55.8	-3	5A	97	Mrg24	76.4
avgbs674946_34	20	2.6	8	7A	98	Mrg12	29.5
Days to heading in late planting
avgbs1452471_63	14	28.3	4	4D	99	Mrg21	~ 28.3
avgbs605397_30	18	48.4	7	4D	98	Mrg21	180
avgbs390936_44	12	55.8	-3	5A	97	Mrg24	76.4
^aAdditive main effect (number of days decrease caused by parent 1 (URS Taura) relative to parent 2 (Leggett))
^bSNP markers assigned to oat chromosomes through BLAST analysis using the OT3098 v2 physical map
^cThe extent to which nucleotide sequences had the same residues at the same positions in BLAST analysis from GrainGenes
^d,eCorresponding 2018 Mrg linkage group names and cM distances

Comparative genomics

The SNP markers associated with QTLs affecting heading date in all field trials were assigned to oat chromosomes using BLAST analysis with the OT3098 v2 physical map from GrainGenes. They were then assigned to the corresponding 2018 Mrg linkage groups and cM distances from the oat consensus linkage map. For the RIL lines from the FL0206B-S-B-S1 × UFRGS 078030-1 population, the SNP markers were placed on chromosomes 2C, 2D, 4A, 5C, 5D, 6A, 6D, 7C and 7D, corresponding to linkage groups Mrg13, Mrg08, Mrg20, Mrg03, Mrg06, Mrg05, Mrg04, Mrg09 and Mrg02 (Table 1). For the RIL lines from the URS Taura × Leggett population, the SNP markers were placed on chromosomes 1D, 2C, 3C, 4C, 4D, 5A and 7A, corresponding to linkage groups Mrg01, Mrg13, Mrg15, Mrg11, Mrg21, Mrg24 and Mrg12 (Table 2). The start and end positions in base pairs of the corresponding chromosomal regions relative to the newly developed SNP markers in the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations are shown in Supplementary Table S2.

Comparative analyses showed that the chromosomal locations of most loci associated with QTLs affecting heading date in both populations of this study were linked to annotated candidate genes and heading-related QTLs from previous studies when a genomic region of 40 Mbp on each Mrg group was analysed in GrainGenes. In the FL0206B-S-B-S1 × UFRGS 078030-1 population, avgbs1464089_47 was the only SNP marker to show significant effects in all field trials. This marker, together with the markers avgbs60602_48, avgbs396242_32, avgbs1497003_26, and avgbs1773912_44, was located on Mrg02 (chr7D), which contains several candidate genes involved in flowering time control. These genes include COL16 (CONSTANS-like 16), COL9 (CONSTANS-like 9), MFT (Mother of FT and TFL1 homolog 1), Ehd3 (Early heading date 3), Hd3a (Heading date 3a), VIL2 (VIN-like 2), COL5 (CONSTANS-like 5), PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1) and RVE6 (REVEILLE 6). Previous molecular mapping and genome-wide association studies have also identified QTLs controlling flowering time on Mrg02 (De Koyer et al. 2001; Holland et al. 2002; Locatelli et al. 2006; McNish et al. 2020; Siripoonwiwat et al. 1996; Sunstrum et al. 2019; Tumino et al. 2016; Winkler et al. 2016). SNP markers on Mrg05 (chr6A) were also consistent across different environments. This genomic region contains the PRR37 (pseudo response regulator-like PRR37) and FPFL5 (flowering-promoting factor 1-like 5) genes. QTLs for heading date on Mrg05 have been identified in previous studies (Holland et al. 2002; Nava et al. 2012; Siripoonwiwat et al. 1996).

The markers avgbs1601155_57 and avgbs809730_11 showed statistically significant effects for early and late planting, respectively, and were located on Mrg06 (chr5D). The candidate genes FPA (Flowering time control FPA), FT (Flowering locus T) and SUF (SUPPRESSOR OF FRI 4) with multiple QTLs for heading date have been mapped in this genomic region (Holland et al. 1997; Holland et al. 2002; Siripoonwiwat et al. 1996). Marker avgbs683618_13 showed a significant effect in the F_4:5 generation and was associated with Mrg04 (chr6D) by comparative analysis. Candidate genes PRR1 (pseudo response regulator-like PRR1) and HDR1 (HEADING DATE REPRESSOR 1) and previously mapped QTLs (Hizbai et al. 2012; He et al. 2013) are located in this region. Markers avgbs691688_27 and avgbs915246_5 were significant only at early planting and were associated with Mrg13 (chr2C). Comparative analysis showed that this linkage group contained the candidate genes FTIP1 (FT-INTERACTING PROTEIN 1), COL5 (CONSTANS-like 5) and RVE1 (REVEILLE 1), in addition to previously mapped QTLs near these markers (Tumino et al. 2016). Markers avgbs557366_70 and avgbs1380849_19, located on Mr08 (chr2D) and Mrg20 (chr4A), were associated with the FTIP3 (FT-INTERACTING PROTEIN 3) and FTIP7 (FT-INTERACTING PROTEIN 7) genes, respectively, but not with QTLs affecting flowering time in oat. SNP markers located on Mrg03 (chr5C) and Mrg09 (chr7C) showed associations with previously mapped QTLs (Bekele et al. 2018; Holland et al. 1997; Holland et al. 2002; Siripoonwiwat et al. 1996), but not with candidate genes (Fig. 2a, Supplementary Tables S2 and S3).

In the URS Taura × Leggett population, the avgbs390936_44 marker showed significant effects in all environments. Unlike most of the other SNP markers, avgbs390936_44 contributed to a three-day delay in heading (Table 2). The sequence of avgbs390936_44 was located at position 76.4 cM on Mrg24 (chr5A) from the oat consensus map. The candidate genes Hd3a, FT, FPA, PRR95 (pseudo response regulator-like 95) and SUF4 (SUPPRESSION OF FRIGIDA 4) and the QTL 'QHD.64Lines_23' (Beer et al. 1997) were identified in this genomic region by comparative analysis. The avgbs342711_16 marker on Mrg11 (chr4C) also delayed heading date and was associated with the GI (GIGANTEA) and COL13 (CONSTANS-like 13) genes, together with the QTLS 'QHD-Abe11h.2018-CORE_Mrg11' and 'QEFLD.2002-OxT_7' (Bekele et al. 2018; Holland et al. 2002). Marker avgbs1040030_69 showed the largest effect in the URS Taura × Leggett population. The significant association of this marker was estimated at early planting, which reduced the heading date by nine days (Table 2). This marker was linked to Mrg15 (chr3C) at position 35.1 cM on the oat consensus map. The avgbs1491480_21 marker was also linked to Mrg15 and showed the same effect in the F_4:5 generation and early planting. The candidate genes Hd3a, FRL4a (FRIGIDA-like 4a), GI-like (GIGANTEA-like) and FPL1 (flowering-promoting factor 1-like 1) were identified, together with the previously mapped flowering time QTLs (Holland et al. 1997; Holland et al. 2002).

Other important genomic regions with significant effects in the URS Taura × Leggett population were linked to Mrg01 (chr1D), Mrg12 (chr7A), Mrg13 (chr2C) and Mrg21 (chr4D). The markers avgbs292292_44 and avgbs292351_17 were linked to Mrg01. The nucleotide sequence of these markers is located close to the candidate gene FRL3 (FRIGIDA-like 3) and the QTLs 'QHDR.MedSet_Mrg01' and 'Days to heading, KxO-5-a' (Canales et al. 2021; Siripoonwiwat et al. 1996). Marker avgbs674946_34 showed a high association with heading date in early planting and mapped to position 29.5 cM of the Mrg12 linkage group. The search for candidate genes revealed that VIL3 (VIN3-like1), RVE6 and Hd3a are located in this genomic region. In addition, QTLs affecting flowering time have previously been mapped on Mrg 12 (De Koyer et al. 2001; Rispail et al. 2018; Siripoonwiwat et al. 1996; Sunstrum et al. 2019). The marker avgbs798773_8 was associated with Mrg13 and linked to the genes HULK3 (HUA2-like 3) and PIF3 (PHYTOCROME INTERACTING FACTOR 3). No QTLs affecting heading date were detected in this region by comparative analysis. The markers avgbs1452471_63 and avgbs605397_30 were associated with Mrg21 (chr4D) at positions 199730068 pb and 414225527 pb, respectively. The candidate genes Hd16 (Heading date-16), FY (Flowering time control FY) and SPL2 (Squamosa promoter binding-like 2) were found in the vicinity of these markers. The QTLs 'QHDEF.U8xU605_6' and 'QHD-A.TxM-5' were also mapped near the marker avgbs605397_30 (Nava et al. 2012; De Koyer et al. 2004) (Fig. 2b, Supplementary Tables S2 and S3).

Here, we present a phenotypic evaluation of heading date in two large populations of RIL lines and identify newly developed SNP markers associated with QTLs affecting heading date in hexaploid oat. In addition, we performed a comparative analysis using a reference genome to improve information on candidate genes and heading-related QTLs in oat. Heading date was collected in RIL lines from the oat populations FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett under field conditions in a subtropical environment in southern Brazil, where weather conditions vary considerably from year to year. These weather variations can make it difficult to interpret flowering time data and to identify the phenotypic and genetic patterns associated with this important adaptive trait in oats. In addition to environmental factors, the complexity and size of the allohexaploid oat genome and the quantitative inheritance of flowering time have further limited the study and effectiveness of phenotype-based selection for this trait in oat breeding programs, especially in subtropical environments.

Oat genotypes differ in their growth and developmental patterns in response to photoperiod, vernalization, ambient temperature, and the combination of these environmental cues (Trevaskis et al., 2022). In subtropical regions such as southern Brazil, consistent natural vernalization conditions may not occur during the early stages of the oat plant's vegetative cycle. Consequently, growing oat cultivars with specific vernalization requirements in these environments could be challenging in years when temperatures do not meet these requirements. To address this issue, the parents FL0206B-S-B-S1, UFRGS 078030-1, URS Taura and Leggett were selected for their lack of vernalization response before crossing, to eliminate the influence of vernalization on the phenotypic evaluation of heading date in this study. Therefore, the variation in days to heading and the identified QTLs in both populations, evaluated across different years and sowing dates within the same year, are attributed to photoperiod and temperature responses, and the interaction between these environmental factors.

The phenotypic difference between the parents of the FL0206B-S-B-S1 × UFRGS 078030-1 population and the different trials was not very large. However, the FL0206B-S-B-S1 parent consistently had the highest number of days to heading in all field trials compared to the UFRGS 078030-1 parent. Based on the photoperiod data collected during the trials, both parents were sensitive to day length. The difference in heading date between the parents may be related to the ability of UFRGS 078030-1 to flower at shorter day lengths and lower temperatures (Fig. 1a, 1c, and S1). Many RIL lines from the FL0206B-S-B-S1 × UFRGS 078030-1 population showed transgressive segregation for heading date (Fig. 1a, 1c). The presence of RIL lines with earlier heading date than UFRGS 078030-1 and later than FL0206B-S-B-S1 indicates natural allelic differences in the parental genome, segregation of a relatively large number of flowering time genes in the progeny, recombination of alleles from the parents, complex gene interactions in key pathways controlling heading date in response to photoperiod and temperature, and environmental influences on gene expression. Similarly, transgressive segregation in heading date has been observed in RIL lines from Brazilian oat populations (Locatelli et al. 2006; Nava et al. 2012) and from a southern oat population with parental complementary genes for earliness (Sunstrum et al. 2019).

The parents of the URS Taura × Leggett population showed significant differences in days to heading. Leggett consistently had the highest number of days to heading in all field trials. While both parents in this population showed some degree of photoperiod sensitivity, Leggett tended to flower under long day length conditions. In addition, the number of days to heading was dependent on higher temperatures in Leggett compared to URS Taura (Fig. 1b, 1d, and S1). It is important to note that Leggett is an oat cultivar adapted to the environmental conditions of western Canada, where plants experience long photoperiods and moderate to high temperatures from the beginning of the vegetative cycle. This is in contrast to the growing seasons of southern Brazil. In western Canada, Leggett typically heads early, with an average of 57 days to heading (Fetch et al. 2007). However, the field trials conducted for this study showed a very late phenotype with an average of 100 to 119 days to heading. On the other hand, URS Taura is well adapted to the growing conditions of southern Brazil, except for its susceptibility to crown rust. URS Taura flowers early and has been the dominant oat cultivar grown in Brazil for many years. Despite the large phenotypic variation in days from emergence to heading among the 326 RIL lines in the URS Taura × Leggett population, the variation was mainly concentrated between the phenotypes observed for the URS Taura and Leggett parents, with a few RIL lines showing transgressive segregation. These results suggest that URS Taura and Leggett carry different alleles for integrating genes such as Vrn3, CO, and GI, which are involved in interpreting environmental signals and controlling the final steps of the flowering induction pathways.

The field data showed that the URS Taura × Leggett population had a higher level of contrast adaptation than the FL0206B-S-B-S1 × UFRGS 078030-1 population (Fig. 1). This increased contrast adaptation, combined with the high marker density and genome coverage, contributed to the identification of SNP markers associated with QTLs affecting heading date, with a more significant effect observed in the URS Taura × Leggett population. These factors probably compensated for the lower number of RIL lines evaluated for heading date in all trials within the URS Taura × Leggett compared to the FL0206B-S-B-S1 × UFRGS 078030-1. In the FL0206B-S-B-S1 × UFRGS 078030-1 population, the average range of heading dates in the RIL lines across all trials was 21 days, while the sum of the average QTL effects at the nine identified QTLs was nine days. In the URS Taura × Leggett population, the average range of heading dates in the RIL lines across all trials was 39 days, and the sum of the average QTL effects at the seven identified QTLs was 18 days. These results suggest the presence of additional unidentified QTLs in both populations. Despite the reasonable size of the populations evaluated and the QTLs identified in this study, the relatively low marker density limited the detection of additional oat loci closely linked to the heading date. In addition, evaluation of heading date in a wider range of environments, including growth chamber tests at different photoperiods and temperatures, may have revealed new QTLs. These limitations probably also prevented the identification of similar genomic regions controlling heading date between the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations.

Comparative analyses showed that the QTLs affecting heading date in the FL0206B-S-B-S1 × UFRGS 078030-1 and URS Taura × Leggett populations were similarly distributed across the A, C, and D oat genomes. In addition, most of the SNP markers associated with QTLs affecting heading date in our study were supported by candidate genes and numerous other QTLs documented in the literature (Fig. 2, Supplementary Tables S2 and S3). In the FL0206B-S-B-S1 × UFRGS 078030-1 population, the genomic region on Mrg02 (chr7D) had the strongest additive effect on the heading date trait in all environments. Several candidate genes were identified in this region, including Hd3a, an ortholog of the Vrn3 gene in small grains, and the FT gene in Arabidopsis. The FT candidate gene was linked to our newly developed SNP markers avgbs1601155_57 and avgbs809730_11 on Mrg06 (chr5D). A Vrn3 locus has recently been mapped on oat chromosome 7D, corresponding to the Mrg02 linkage group of the oat consensus map (Tinker et al. 2022). Hd3a, Vrn3, and FT integrate the vernalization and photoperiod response pathways and encode a transferable signaling molecule that is produced in leaves and transported to function in the shoot apical meristem to promote flowering (Maple et al. 2024; Yan et al. 2006). Several historical QTLs have been reported in this region, including a large QTL for photoperiod response in the UFRGS 881971//Pc68/5*Starter population. This QTL was associated with the Di1 (daylength insensitivity 1) gene, an ortholog of the HD1 (heading date 1) gene in rice, and the CO gene in Arabidopsis (Locatelli et al. 2006). The oat loci on Mrg05 (chr6A) were also consistent across environments and were associated with the PRR37 gene, which acts as a pseudo-response regulator and belongs to a class of genes involved in circadian clock function. The PRR37 gene is an ortholog of Ppd1, the main determinant of photoperiod sensitivity in wheat and barley (Turner et al. 2005). Several historical QTLs have also been reported in the Mrg05 genomic region, and most of these QTLs were photoperiod dependent.

Major loci associated with QTLs affecting heading date in the URS Taura × Leggett population were identified in all environments. The locus associated with Mrg12 (chr7A) significantly reduced the heading date in early planting and was linked to the candidate genes VIL3 and Hd3a (= Vrn3). Similarly, a Vrn3 locus has recently been mapped on oat chromosome 7A, corresponding to the Mrg12 linkage group (Tinker et al. 2022). Furthermore, several QTLs related to photoperiod and vernalization responses have previously been detected in this genomic region. In a study reported by Sunstrum et al. (2019), a QTL identified on Mrg12 showed strong and significant effects in all trials, and was also associated with QTLs identified in a genome-wide association study (GWAS) consisting mainly of spring germplasm (Klos et al. 2016). The QTL on Mrg13 (chr2C) also had a strong additive effect and was associated with the candidate gene PIF3, a phytochrome interacting factor required for normal photoinduced signaling. In Arabidopsis, leaf expression of the FT gene (= Vrn3) is activated by a molecular network involving phytochromes (Trevaskis et al. 2022). However, this region was not associated with any annotated QTL on GrainGenes. Interestingly, the QTL on Mrg11 (chr4C) was linked to the candidate genes GI and COL13, which play an important role in the regulation of flowering by photoperiod. In our study, this QTL delayed the heading date in the URS Taura × Leggett population when evaluated in the early planting. This result is in agreement with a previous study in rice, where overexpression of the GI gene results in a delay in flowering time, with a more pronounced effect under short days (Brandoli et al. 2020). An oat QTL affecting photoperiod-dependent heading date was also associated with Mrg11 in a GWAS study performed by Bekele et al. (2018).

The heading date QTL on Mrg02 (near consensus position 87) identified in the FL0206B-S-B-S1 × UFRGS 078030-1 population, and the QTL on Mrg12 (near consensus position 30) identified in the URS Taura × Leggett population, are likely to represent identical genomic regions carrying important genes controlling flowering time in oat. These QTLs may also correspond to those identified on Mrg02 (near consensus position 87) and Mrg12 (near consensus position 41) from the TX07CS-1948 × Hidalgo cross, which was evaluated for crown rust resistance, days to heading, and plant height in different years and treatments in Canada (Sunstrum et al. 2019). It is noteworthy that Mrg02 and Mrg12 are likely to be homeologous chromosomes carrying the Vrn3 gene in oat (Klos et al. 2016). Oat loci corresponding to the Mrg15 (chr3C) and Mrg24 (chr5C) linkage groups were also linked to the Hd3a candidate gene in the URS Taura × Leggett population. This is intriguing as both genomic regions differ from where the three Vrn3 loci were mapped in the study reported by Tinker et al. (2022). In addition, the Mrg06 (chr5D) linkage group was associated with the FT candidate gene, and Hd3a was mapped twice at relatively long positions in the Mrg02 (chr7D) linkage group in the FL0206B-S-B-S1 × UFRGS 078030-1 population. Combining the results of this study with the role of the Vrn3 gene in integrating different flowering time pathways raises the question of whether there may be more than three copies of this gene in hexaploid oat. Further research is needed to determine the copy of Vrn3 in the oat genome and the adaptive consequences associated with possible variations in the copy number.

Heading date is the most important phenological stage in the growth and development of oats and serves as a key factor for adaptation and productivity as a crop species. The adaptive contrast of the parents based on their geographical origin contributed to the identification of genomic regions with strong additive effects on heading date in the two genetic populations. Most of these QTLs have already been reported in various studies available in the literature, increasing confidence for use by the scientific community and oat breeding programs. Advances in the sequencing and annotation of the oat genome have allowed reliable association of candidate genes with these genomic regions. Although this study is limited in its scope and depth of functional gene analysis, it is interesting to note that candidate genes associated with the positions of the oat loci affecting heading date in both populations perform a variety of biological functions as flowering regulators, mainly in the perception and response to photoperiodic pathways. No QTLs were associated with major genes involved in the vernalization response pathway, except for Vrn3, which is involved in both the vernalization and photoperiod pathways. Allelic variability for photoperiod response genes is essential for the adaptation of oat to different latitudes and the expansion of cultivation to new regions of the world. The validation of these markers linked to central flowering genes will be valuable for molecular selection in oat breeding programs aimed at developing new and improved oat cultivars adapted to specific growing conditions.

Author Contribution

Both authors, Ubert IP and Nava IC, contributed equally to all stages of the preparation of the manuscript.

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No competing interests reported.

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Genetic mapping and comparative analysis of heading date in hexaploid oat

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Abstract

Figures

Introduction

Material and methods

Population development

Phenotyping

Marker analysis

Map construction

QTL analysis

Comparative genomics

Results

Phenotypes

Marker analysis and map construction

QTL analysis

Comparative genomics

Discussion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Version 1