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Zooming into the genomic vicinity of the major locus for vicine and convicine in faba bean (Vicia faba L.)

The versatility of faba bean (Vicia faba L.) seed as valuable protein feed is hampered by its relatively high level of the compounds vicine and convicine (VC), which are antinutritive factors in poultry and further non-ruminant feed. The objective here was to develop the first-ever genetically low-VC winter faba bean. Hence, the low VC allele vc-, that should be the basis of a known, major locus for VC, need verification and molecular identification and be based on appropriately developed DNA-markers; the low VC feature awaited its transfer into the high-performing winter faba bean germplams. Based on bi-parental F2-families and isogenic lines, we thus developed highly useful SNP markers exploiting transcriptomic data. Furthermore, we fine-mapped and, based on synteny to Medicago truncatula and Cicer arietinum, we identified a candidate gene for the VC locus. A novel, genetically low VC winter faba bean population was bred. The path is now well-prepared for further marker-based breeding progress.


Introduction
Faba bean (Vicia faba L.; V.f.) is a large-seeded, annual grain legume which is grown for food and feed. It is appreciated for its high protein content of approximately 30% (Crépon et al. 2010;Link et al. 1994), as a regional vegetal protein source and as an alternative crop to improve soil fertility and break rotation circles of pests and diseases (Köpke and Nemecek 2010;Kulak et al. 2013). Genomic tools are still underdeveloped for faba bean (Annicchiarico et al. 2017); a sequence of the huge genome of faba bean (13 Gb) is not yet available. Therefore, the high syntenic correspondence of Vicia faba to the sequenced genomes of Medicago truncatula (M.t.), Cicer arietinum (C.a.) and further legumes of the subfamily Faboideaei is of high importance for genomic analyses. Faba beans are cultivated in Germany on only approximately 50.000 ha (Statistisches Bundesamt 2020). One limiting factor is the presence of the antinutritive seed compounds vicine and convicine (VC), two pyrimidine glycosides which occur in faba bean seed with approx. 0.3% up to 1.5% in dry matter. This quantita-tive variation has a clear genetic component (Duc 1997;Frauen et al. 1984;Khamassi et al. 2013).
Vicine and convicine occur in an approximate 2:1 ratio in faba bean seeds (Goyoaga et al. 2008). Faba beans contain VC in all parts of the plant (Goyoaga et al. 2008). These compounds are nearly unique to the genus Vicia; Momordica charantia (M.c.) is the only species outside this genus containing vicine (Gauttam and Kalia 2013;Khazaei et al. 2019).
More than 400 million people will, upon ingestion of vicine and convicine, suffer from a hemolytic anemia, called favism, caused by a human X-chromosomal inherited genetic deficiency of glucose-6phosphate-dehygrogenase (G6PD). This human genetic condition has a delicate epidemiological connection to malaria (Arese 2006;Arese et al. 2012;Luzzatto and Arese 2018).
Vicine and convicine can also have a negative effect e.g. on laying hen and broiler performances (Guillaume and Bellec 1977;Halle 2006;Larbier and Leclercq, 1994;Marquardt et al. 1981;Munduuli et al. 1981;Muduuli et al. 1982;Naber et al. 1988;Olaboro et al. 1981a;Olaboro et al. 1981b). Duc et al. (1989) reported the presumably monogenetically inherited low vicine and convicine (LVC) content of the genebank accession 1268(4)(1) from Radzikov (Poland), which has since been used in breeding and research. This accession showed a seed content of 0,046% of VC, this is about 1/10 to 1/20 of the wild type seed content. That trait is controlled by one locus, designated as VC locus, with alleles VC+ (wild type) and vc-(from that genebank accession 1268(4)(1)). The low VC level in homozygous vc-/vc-faba bean genotypes prevents favism in G6PD-deficient humans (Gallo et al. 2018) and prevents dietary disadvantages if such faba beans are used as compounds of feed (Crépon et al. 2010 Based on scattered hints in classical literature, vicine and convicine are assumed to be formed in the faba bean seed coat and transported into the embryo (Duc et al. 1989;Brown and Roberts 1972).
The biosynthetic pathway to vicine and convicine was unknown until recently, when Björnsdotter et al. (bioRxiv preprint, 2020) presented new, pioneering findings, which provided convincing experimental evidence that a bi-functional RIBA1 protein, which catalyzes the first step of the riboflavin biosynthetic pathway, also catalyzes the key step of the so far unresolved vicine and convicine pathway. Starting with the GTP cyclohydrolase II function of this protein, the two pyrimidine glucosides are synthesized from GTP as demonstrated by feeding isotopically labelled GTP precursor into roots of V. faba. However, the additional enzymatic steps, which finally lead to the compounds vicine and convicine, remain to be elucidated, although the authors propose that VC are synthesized in three additional steps from the first two intermediates in the riboflavin pathway, respectively, the first of which is probably catalyzed by an N-glycosidase described by Frelin et al. (2015). Björnsdotter et al. (2020) could also show that there is a copy of the gene encoding the RIBA1 protein at the VC locus, whose GTP cyclohydrolase II function is destroyed by a frameshift mutation in the vc-allele. This gene was consequently named VC1.
A major objective of research of VC characteristics is to facilitate breeding of faba beans which are genetically low in vicine and convicine seed content. The black and white hilum colours of faba bean seeds, a monogenic trait (Sirks 1931), can be used as morphological marker to select for LVC status.
The accession 1268(4)(1), source of the LVC feature, shows white seed hilum. The linkage between the VC locus and the hilum colour locus is relatively tight (5-10cM; Duc et al. 2004;Khazaei et al. 2017). Yet, white versus black hilum is only visible late in the life of plants, when their seeds are mature. In an early attempt to overcome this limitation, Gutierrez et al. (2006) developed two CAPS markers by bulked segregant analysis, which were linked to the VC locus. Later, Khazaei et al. (2015) were able to map the VC locus as a major QTL on the large first chromosome of faba bean. The QTL mapped in an interval of 3.6 cM, flanked by two pairs of cosegregating SNP markers. Unfortunately, the two markers closest to this mapped QTL (0.8 cM) proved to be not diagnostic for the trait in a larger set of diverse genotypes.
The genetic map used for the mapping of the VC locus had been constructed with SNP markers (Khazaei et al. 2014) that were a subset of a larger set of 687 SNP markers which were used to construct a comprehensive consensus map for faba bean (Webb et al. 2016). The majority of these SNP markers had been developed based on selected mRNAseq contigs of faba bean that could be unambiguously assigned to a specific gene of M.t. by sequence comparisons. These markers therefore allow comparative mapping between faba bean and M. truncatula. Khazaei et al. (2015) used this approach to identify a region on chromosome 2 of M. t. with strong collinearity to the region in faba bean carrying the VC locus. A total of 340 genes were found in M. t. in the interval delimited by the markers flanking the VC locus. However, due to lacking knowledge about the biosynthetic pathway for vicine and convicine, it was not possible to identifiy a candidate gene for the VC locus. In an early approach to identify candiate genes for the VC locus, Ray et al. (2015) identified six RNAseq contigs that were differentially expressed between high and low VC genotypes.
One of these contigs, contig 4518, was later located by Khazaei et al. (2017) in M. t. in the target region for the VC gene on chromosome 2. The contig 4518 corresponds to the M. t. gene Medtr2g009270, which is annotated as 3,4-dihydroxy-2-butanone 4-phosphate synthase. In soybean and chickpea, the same gene is annotated as RIBA 1, a bifunctional riboflavin biosynthesis protein (Khazaei et al. 2017) that has two catalytic domains, one with a 3,4-dihydroxy-2-butanone 4phosphate synthase activity and a second one with a GTP cyclohydrolase activity. Based on contig 4518, Khazaei et al. (2017) developed an SNP marker whose segregation completely matched the segregation of VC content in a RIL population from a cross between a high and low VC genotype and in a set of 51 diverse genotypes. A comprehensive and detailed review on the current classical and molecular genetic background of the VC feature of faba bean was very recently presented by Khazaei et al. (2019).
Here, the main objectives were to demonstrate and facilitate the breeding of faba beans with low VC content. Therefore, we developed new SNP markers which were closely linked to the VC locus via fine-mapping and, if possible, locate and identify the VC gene.
Finally, we aimed to employ all our findings to develop a novel, LVC winter faba bean germplam.

Plant material for genetic analyses
We used two pairs of near-isogenic lines (NILs) for mRNA analysis and as crossing parents to develop two F 2 mapping populations (Cross1, Cross2). These two NIL pairs were themselves generated from two crosses of a high vicine and convicine (HVC) and a LVC parental inbred line, respectively. The first NIL pair originated from the cross between the inbred line Mélodie/2 and ILB938/2. Mélodie/2 was bred via single-seed descent (SSD) from the French LVC cultivar Mélodie, whereas ILB938/2 was bred via SSD from the HVC gene bank accession ILB938 (Khamassi et al. 2013;Khazaei et al. 2015). The second NIL pair was accordingly generated from a cross facilitated by NPZ (Norddeutsche Pflanzenzucht Hans-Georg Lembke KG). This cross included the cultivar Fabelle as the LVC parent. From both crosses, recombinant inbred lines (RILs) were bred until generation F5 (Khazaei et al. 2015). Here, one rare F5-individual per cross was identified which gave F6 offspring that still segregated for VC and included a homozygous HVC and homozygous LVC individual. These individuals were maintained by self-fertilization and became one pair of NILs. Hence, one such pair of NILs was derived from each of the two initial crosses. From Mendelian expectation, the two lines of each NIL pair should be isogenic at about 15/16 of their genome.

Creation of mapping populations
To create the two F2-mapping populations, the F 1 of Cross 1 and of Cross 2 were self-fertilized under insect-proof conditions. These two mapping populations contained N=751 (Cross 1) and N=899 (Cross 2) F 2 -individuals. The two F 2 populations where subsequently used to generate linkage map frag-ments and to conduct fine-mapping analyses while seed coat material from their parental NIL pairs was further used for mRNA analyses.
Until 2015 and generation BC 2 F 2 , the selection was carried out via hilum colour among F 2 , BC 1 F 2 and BC 2 F 2 individuals. For actual backcrossing, corresponding F 3 , BC 1 F 3 and BC 2 F 3 individuals were used. Starting in BC 3 F 2 , selection was based on high liquid pressure chromatography (HPLC) analyses (service provided by National Institute of Agricultural Botany in England, NIAB 2021). Further on, we employed marker assisted selection for the next backcrossing program. mRNA sampling, RNA-Isolation, transcriptome, and SNP analysis For mRNA analyses, we collected seed coats of immature seeds (Figure 1), in their developmental stages four, five and six (Borisjuk et al. 1995).
The frozen tissue was ground to a fine powder under liquid nitrogen conditions using a mixer mill (MM200, Retsch, Haan, settings 30 Hz for 2 min). Total RNA was isolated with the InviTrap® Spin were pooled on a ratio of 1:1 and subsequently converted into RNASeq libraries and paired-end sequenced at 2x75 b on a Hiseq2000 device yielding 64-77 million quality filtered reads per sample.
Combined high-quality reads from all samples were used for a de novo assembly and annotation of a duplicate free testa-specific reference transcriptome as described in Santos et al. (2018). The software package JointSNVMix was used to detect single nucleotide variants (Roth et al. 2012). For SNP candidates, a locus must have had at least five reads in both samples where one of two possible bases might occur (either allele A or B). A maximum of one percent false allele reads was tolerated.
NGS template preparation, sequencing and bioinformatics took place at GenXPro GmbH (Frankfurt am Main). Transcriptome data evaluation for the identification of SNPs that correlated with the VC phenotype was performed stepwise: for the RNASeq and MACE libraries separately and for each of the two NIL pairs (parents of Cross 1 and Cross 2). For each genotype, MACE-data sets derived from developmental stages 4, 5, and 6 were pooled to increase sensitivity for SNP-detection. Subsequently, those SNP candidates which were common to both NIL pairs were identified.

SNP marker development
KASP marker development, DNA extraction and KASP analysis were conducted at TraitGenetics (Gatersleben, Germany). KASP assay designs were based on app. 50 bps to the left and right side of SNPs found via the mRNA analysis.

Marker analysis and map construction
Throughout, the genetic linkage maps were constructed using R (R Development Core Team 2011; R version 3.5.3) and the R package ASMap (Taylor and Butler 2017). For all linkage maps, the Kosambi function was used to calculate the map distances in cM (Kosambi 1943). The functions of the package were used as described by Taylor and Butler (2017).
Missing marker data and marker segregation ratios were identified with the R Package ASMap.
Markers that were monomorphic in a cross and markers showing strongly skewed segregations (log(P) of 2.0 or higher) were excluded from further analyses. The selected SNP markers were used for the linkage map fragment construction.
An initial marker set, which contains markers from Webb et al. 2016, and the group of Donal O`Sullivan, yielded the so-called draft maps. These were used to define the putative region of the VC locus for future analyses, especially for the transcription analysis.
The set of newly developed markers from our transcription analyses and certain markers from the initial marker set (termed new marker set) yielded the final linkage map fragments, which were used for the fine-mapping.

Phenotyping of VC content for fine-mapping and breeding
Selected F 2 individuals from Cross 1 and Cross 2 as well as the backcross generation BC 3 F 2 were phenotyped (HPLC; via NIAB 2021). As checks and benchmarks for the HPLC results, F 2 individuals with marker-deduced homozygous and heterozygous VC genotypes were identified (see Table 1).

Fine mapping
For the manual fine-mapping of the sought-for VC-locus, phenotypic VC results were displayed along with the SNP genotypes of the F 2 individuals of Cross 1 and Cross 2. The SNP markers were ordered according to their linkage map positions. The correspondences between genotype and phenotype were thoroughly studied.

BLASTing of SNPs from finemapping
To determine candidate genes, specific KASP marker sequences which are found within the fine

SNP marker selection
For the marker-assisted selection, mapping and fine-mapping, markers within the region around the putative VC locus (Khazaei et al. 2015) were chosen and tested. The markers Vf_Mt2g005900_001 and Vf_Mt2g015010_001 from Khazaei et al. (2015) were used to serve as initial boundary markers of the chromosomal to define the initial interval-of-interest and encompass all other chosen markers.
In total, six markers from Khazaei et al. (2015), including the initial boundary markers, were chosen.
Ten markers from Webb et al. 2016 were added since they were determined to be in the interval-ofinterest according to the physical placement of the initial boundary markers in M. t. genome and the genetic map of Webb et al. 2016. Additionally, the marker SNP384 from Song (2017) was included.
These 16 markers make up our initial marker set (Table 2) which was used for our first marker assisted selection in the backcross and the construction of draft maps for Cross 1 and Cross 2.
--X Vf_Mt2g015010_001 * ---"*": Marker from Khazaei et al. 2015; "-" : not polymorphic; "#": removed from the analysis due to skewed segregation patterns; "X" used in draft maps and final genetic maps and for marker assisted selection Only subsets of the 16 markers (initial marker set) were polymorphic in the respective genetic backgrounds ( Table 2). Most notably, the marker Vf_Mt2g015010_001, which served as initial boundary marker, was monomorphic. The polymorphic marker Vf_Mt2g013690_001 was hence chosen as substitute (see Table 2). So, markers Vf_Mt2g005900_001 and Vf_Mt2g013690_001 were subsequently used to define the search frame (Table 3). All markers polymorphic in Cross 1 and Cross 2 were used for the construction of the draft maps. Only three markers were polymorphic in all genetic backgrounds (Table 2, markers SNP384, Vf_Mt2g009320_001 and Vf_Mt2g010740_001) and their chromosomal vicinity was deemed as the most likely region to contain the VC gene, based on the data of Song (2017) and Khazaei et al. (2017), and was therefore the focus for our search for new SNP markers and the VC gene in the transcription analysis. In addition, the markers polymorphic in the backcross were used for marker assisted selection in the backcrossing program.
Analysis of RNAseq and MACE data sets revealed, in the intersection of Cross 1 and Cross 2, a total of 448 unique SNPs within 123 contigs (Table 3). These contigs could be assigned to chromosome 2 of M. truncatula. Of these, 135 unique SNPs from 35 contigs were found within the new interval-ofinterst. A total of 73 SNPs were found with both analyses, RNAseq and MACE.

Table 3. Comparison of LVC vs. HVC data from transcription analysis of Cross 1 and Cross 2.
Given are the numbers of detected SNPs and contigs which were found with different transcription analysis methods and search frames. chr. 2; 390,001-3,620,000 bp, see Table 3).

Table 4. Fifty-eight SNP markers from mRNA analyses RNAseq and MACE. Their origin as well as their function in the three families (Cross 1 and 2 and backcross) is noted. Usability in families Marker
Filtering process Cross 1 Cross 2 Back-cross VFS18002.A03 Taken from the 73 SNP markers found with both techniques, RNAseq and MACE (Table 3) From RNAseq only, mapped to M.t.
X X X "-": not polymorphic; "#": removed from the analysis due to skewed segregation patterns; "X" used in final genetic maps and for marker assisted selection Of the 58 newly developed markers, 42 markers were used for the construction of the final linkage map fragment and fine-mapping of Cross 1 and 38 markers were used for Cross 2 (see Table 1), since these were polymorphic and did not show skewed segregation patterns. For the backcross, 34 markers were polymorphic and were tested for their suitability for our further winter bean breeding.
Those of the initial set markers which were polymorphic for the genetic backgrounds of Cross 1 and 2 plus the above described newly developed markers form the new marker set.

Linkage maps
For the construction of the draft linkage maps, all F 2 genotypes present in the F 2 families were used, i.e., 751 individuals for Cross 1, and 899 individuals for Cross 2. The initial marker set that was employed for draft maps is depicted in Table 2. The marker order in the draft maps followed nearly always the order of their physical placement in the genome sequence of M. truncatula. The new marker set gave the final linkage map fragments for Cross 1 and Cross 2.
Only informative F 2 genotypes were genotyped with the new marker set. Such informative F 2 genotypes had one or more putative recombination events as determined by their marker genotypes and the draft mapping. A total of 70 individuals for Cross 1 and 74 individuals for Cross 2 were thus reanalyzed with the new marker set. In addition, and as controls, the parental genotypes (10 plants each) of the respective cross were genotyped with the new marker set. The marker data of the remaining F 2 individuals were inferred based on results from the draft maps.
SNP results of the informative and control individuals were checked for missing data points and for plausibility of their numbers of single and double crossovers. Five F 2 -individuals of Cross 1 and nine of Cross 2 were excluded based on more than 10 missing marker data per individual and on extremely unlikely recombination patterns, such as the apparent occurrence of two or more crossovers in both gametes within 3 cM. This cleansing combined with exclusion of markers with strongly skewed seg-regations (see "SNP marker selection", Table 4) reduced the length of the map fragments from app. 20 cM in a first, uncleansed approach to between 3 and 6 cM for the final linkage map fragments.

Fine mapping of the VC locus
For the manual fine-mapping of the sought-for VC-locus, the informative F 2 plants of Cross 1 and Cross 2 were phenotyped for their VC content.
The resulting VC phenotypes were matched with the genotypes. For this purpose, markers were arranged according to the final linkage map fragments s (Figure 2). The F 2 individuals were grouped as homozygous HVC, heterozygous and homozygous LVC. Crossovers were thus made visible (see Tables   5A and 5B).
It was hence determined which crossovers were closest to the VC locus (see Tables 5A and 5B). The smallest possible SNP interval bearing the sought-for VC locus was determined for both crosses. This interval is referred to as the core region (depicted by orange background of marker names in Tables   5A and 5B Table 5A. Fine-mapping of the vicinity of the VC locus for Cross 1. F 2 genotypes of the respective individuals are sorted according to their haplotypes, with homozygous for SNP-allele (green, A) as detected in LVC parent on the left to heterozygous (yellow, H) in the middle to homozygous for SNPalleles (red, B) as detected in HVC parent on the right. The respective HPLC VC contents are given above with the individual numbers of genotypes. Marker names as well as their positions on the genetic linkage map fragments (see Figure 2) are displayed, thus crossovers are visualized. Orange colored marker names define outer boundaries of the interval which, according to this fine-mapping, contains the VC locus. The light green colored marker name depicts the SNP384 which was defined by Khazaei et al. (2017)  The region between markers VFS18002.A51 and VFS18002.A54 (Cross 1) was 0.14cM smaller than the region between markers VFS18002.A49 and VFS18002.A53 (Cross 2; Table 5A, 5B). All markers in this Cross 1 region were included in the region of Cross 2, and in same order. VFS18002.A51 and VFS18002.A54 of Cross 1 (0.13cM) are consequently the final boundary markers which define the core region where the VC locus must be located.

SNP marker validation for breeding novel, winterhardy, vc-faba bean lines
Those nine markers which are denominated by the outer boundaries of the intervals in Cross 1 and Cross 2 (see Tables 5A and 5B) were tested in our backcross material to confirm markers which might be good candidates for marker assisted selection. The usability of these markers for the backcross material and the accuracy of the respective marker prediction for the VC content was verified with BC 3 F 2 genotypes and their respective HPLC results.
Eight of these promising nine markers (see Tables 5A and 5B) proved to be precise selection tools for our backcrossing program, namely markers VFS18002.A49 to VFS18002.A13 (see Table 6) while one marker was not polymorphic here (VFS18002.A53). Two markers did not perfectly match; they were indeed not included in this group of promising markers (markers VFS18002.A14 and VFS18002.A55; see Table 6). Putative candidate genes from synteny studies and from transcriptome analyses The core region which was identified via fine-mapping of Cross 1 and Cross 2 was inspected for possible candidate genes. The sequences of the markers covering the core region were BLASTed against the genomes of M.t. and C.a. (see Table 7) and genes within the core region were noted (see Table   8). A total of 30 genes were thus found in M.t., while a subset of 17 of these could also be found in the corresponding region of C.a. ( Table 8).
In 2020, we arrived at generation BC 4 F 4 . From this material, four LVC components (see Table 9) were sown in October 2019 as generation Syn 0 and in October 2020 as Syn 1, to initiate a novel LVC synthetic variety of winter faba beans as experimental cultivar and as breeding germplasm pool.  (Khazaei et al. 2015). Gutiérrez et al. (2016) reported corresponding findings from their cross Vf6 * 1268. The putative region of the respective locus in their approach amounts also to app. 4cM. We employed two crosses with two different genetic backgrounds containing 751 and 899 individuals, respectively. Sixteen markers from Webb et al. (2016) and Song (2017)  The relatively small area of this core region yielded a set of putative candidates. Additionally, our approach towards a candidate gene via expression analysis also yielded a set of candidates, which were differentially expressed in immature seed coat tissue of our LVC and HVC NIL pairs.
The overlap between the two approaches clearly indicated that the most likely VC candidate gene was 3,4-dihydroxy-2-butanone 4 -phosphate synthase, or bifunctional riboflavin biosynthesis protein RIBA 1. Recently, Björnsdotter et al. (2020) named the gene RIBA 1 as the putative candidate gene for the vc-phenotype. Their findings strongly suggest that vicine and convicine are side products of the riboflavin biosynthesis from the purine GTP. Their hypothesis for the cause of the LVC phenotype is a frame shift insertion in the RIBA1 enzyme. They accordingly named the gene for the RIBA1 enzyme VC1. Our findings seem to verify the conclusion of Björnsdotter et al. (2020).
Additionally, the mRNA analyses lead to a set of new markers in the VC core region, ready for use for marker assisted selection. We employed them and bred new, LVC winter faba bean lines, aiming at highly versatile feed stuff from highly productive winter beans. Syn 1 of the first, novel LVC winter faba bean variety is currently (2021 season) in the field. This introduction of the LVC feature will make winter faba beans more attractive for farmers and feed producers.