Bee species visiting Medicago sativa differ in pollen deposition curves with consequences for gene flow.

PREMISE: Pollinator foraging behavior can influence pollen dispersal and gene flow. In many plant species a pollinator trips a flower by applying pressure to release its sexual organs. We propose that differences in tripping rate among grooming pollinators could generate distinct pollen deposition curves, the pattern of pollen deposition over successive flowers visited. This study compares the pollen deposition curves of two grooming pollinators, a social bumble bee and a solitary leafcutting bee, with distinct tripping rates on Medicago sativa flowers. We predict a steeper deposition curve for pollen moved by leafcutting bees, the pollinator with the higher tripping rate. METHODS: Medicago sativa plants carrying a gene (GUS) whose product is easily detected by staining, were used as pollen donors. After visiting the GUS plants, a bee was released on a linear array of conventional M. sativa plants. The number of GUS pollen grains deposited over successive flowers visited or over cumulative distances was examined. Distinct mixed effect Poisson regression models, illustrating different rates of decay in pollen deposition, were fitted to the pollen data for each bee species. RESULTS: Pollen decay was steeper for leafcutting bees relative to bumble bees for both models of flowers visited and cumulative distance, as predicted by their higher tripping rate. CONCLUSIONS: This is the first report of a difference in pollen deposition curves between two bee species, both grooming pollinators. Such differences could lead to distinct impacts of bee species on gene flow, genetic differentiation, introgression, and ultimately speciation.

A simple model for pollen-parent fecundity distributions in bee-pollinated forage legume polycrosses.

A simple Weibull distribution based empirical model that predicts pollen-parent fecundity distributions based on polycross size alone has been developed in outbred forage legume species for incorporation into quantitative genetic theory. Random mating or panmixis is a fundamental assumption in quantitative genetic theory. Random mating is sometimes thought to occur in actual fact, although a large body of empirical work shows that this is often not the case in nature. Models have been developed to explain many non-random mating phenomena. This paper measured pollen-parent fecundity distributions among outbred perennial forage legume species [autotetraploid alfalfa (Medicago sativa L.), autohexaploid kura clover (Trifolium ambiguum M. Bieb.), and diploid red clover (Trifolium pratense L.)] in ten polycrosses ranging in size (N) from 9 to 94 pollinated with bee pollinators [Bumble Bees (Bombus impatiens Cr.) and leafcutter bees (Megachile rotundata F.)]. A Weibull distribution best fit the observed pollen-parent fecundity distributions. After standardizing data among the 10 polycrosses, a single Weibull distribution-based model was obtained with an R (2) of 0.978. The model is able to predict pollen-parent fecundity distributions based on polycross size alone. The model predicts that the effective polycross size will be approximately 9 % smaller than under random mating (i.e., N e/N ~ 0.91). The model is simple and can easily be incorporated into other models or simulations requiring a pollen-parent fecundity distribution. Further work is needed to determine how widely applicable the model is.

Pooled DNA sequencing in hairy vetch (Vicia villosa Roth) reveals QTL for seed dormancy but not pod dehiscence.

Introduction: Hairy vetch (Vicia villosa Roth) is a promising legume cover crop, but its use is limited by high rates of pod dehiscence and seed dormancy. Methods: We used phenotypically contrasting pooled DNA samples (n=24 with 29-74 individuals per sample) from an ongoing cover crop breeding program across four environments (site-year combinations: Maryland 2020, Maryland 2022, Wisconsin 2021, Wisconsin 2022) to find genetic associations and genomic prediction accuracies for pod dehiscence and seed dormancy. We also combined pooled DNA sample genetic association results with the results of a prior genome-wide association study. Results and discussion: Genomic prediction resulted in positive predictive abilities for both traits between environments and with an independent dataset (0.34-0.50), but reduced predictive ability for DNA pools with divergent seed dormancy in the Maryland environments (0.07-0.15). The pooled DNA samples found six significant (false discovery rate q-value<0.01) quantitative trait loci (QTL) for seed dormancy and four significant QTL for pod dehiscence. Unfortunately, the minor alleles of the pod dehiscence QTL increased the rate of pod dehiscence and are not useful for marker-assisted selection. When combined with a prior association study, sixteen seed dormancy QTL and zero pod dehiscence QTL were significant. Combining the association studies did not increase the detection of useful QTL.

Genome-wide association mapping in hairy vetch (Vicia villosa) discovers a large effect locus controlling seed dormancy.

Hairy vetch (Vicia villosa Roth), a winter-hardy annual legume, is a promising cover crop. To fully leverage its potential, seed production and field performance of V. villosa must be improved to facilitate producer adoption. Two classic domestication traits, seed dormancy (hard seed) and dehiscence (pod shatter), are selection targets in an ongoing breeding program. This study reports a genome-wide association study of 1,019 V. villosa individuals evaluated at two sites (Knox City, Texas and Corvallis, Oregon) for the proportion of dormant seed, visual pod dehiscence scores, and two dehiscence surrogate measures (force to dehiscence and pod spiraling score). Trait performance varied between sites, but reliability (related to heritability) across sites was strong (dormant seed proportion: 0.68; dehiscence score: 0.61; spiraling score: 0.42; force to dehiscence: 0.41). A major locus controlling seed dormancy was found (q-value: 1.29 × 10-5; chromosome 1: position: 63611165), which can be used by breeding programs to rapidly reduce dormancy in breeding populations. No significant dehiscence score QTL was found, primarily due to the high dehiscence rates in Corvallis, Oregon. Since Oregon is a potentially major V. villosa seed production region, further dehiscence resistance screening is necessary.

Transcript profiling of hairy vetch (Vicia villosa Roth) identified interesting genes for seed dormancy.

Hairy vetch, a diploid annual legume species, has a robust growth habit, high biomass yield, and winter hardy characteristics. Seed hardness is a major constraint for growing hairy vetch commercially. Hard seeded cultivars are valuable as forages, whereas soft seeded and shatter resistant cultivars have advantages for their use as a cover crop. Transcript analysis of hairy vetch was performed to understand the genetic mechanisms associated with important hairy vetch traits. RNA was extracted from leaves, flowers, immature pods, seed coats, and cotyledons of contrasting soft and hard seeded "AU Merit" plants. A range of 31.22-79.18 Gb RNA sequence data per tissue sample were generated with estimated coverage of 1040-2639×. RNA sequence assembly and mapping of the contigs against the Medicago truncatula (V4.0) genome identified 76,422 gene transcripts. A total of 24,254 transcripts were constitutively expressed in hairy vetch tissues. Key genes, such as KNOX4 (a class II KNOTTED-like homeobox KNOXII gene), qHs1 (endo-1,4-ß-glucanase), GmHs1-1 (calcineurin-like metallophosphoesterase), chitinase, shatterproof 1 and 2 (SHP1, SHP2), shatter resistant 1-5 (SHAT1-5)(NAC transcription factor), PDH1 (prephenate dehydrogenase 1), and pectin methylesterases with a potential role in seed hardness and pod shattering, were further explored based on genes involved in seed hardness from other species to query the hairy vetch transcriptome data. Identification of interesting candidate genes in hairy vetch can facilitate the development of improved cultivars with desirable seed characteristics for use as a forage and as a cover crop.

A reference assembly for the legume cover crop hairy vetch (Vicia villosa).

Vicia villosa is an incompletely domesticated annual legume of the Fabaceae family native to Europe and Western Asia. V. villosa is widely used as a cover crop and forage due to its ability to withstand harsh winters. Here, we generated a reference-quality genome assembly (Vvill1.0) from low error-rate long-sequence reads to improve the genetic-based trait selection of this species. Our Vvill1.0 assembly includes seven scaffolds corresponding to the seven estimated linkage groups and comprising approximately 68% of the total genome size of 2.03 Gbp. This assembly is expected to be a useful resource for genetically improving this emerging cover crop species and provide useful insights into legume genomics and plant genome evolution.

Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species.

Relative to other crops, red clover (Trifolium pratense L.) has various favorable traits making it an ideal forage crop. Conventional breeding has improved varieties, but modern genomic methods could accelerate progress and facilitate gene discovery. Existing short-read-based genome assemblies of the ∼420 megabase pair (Mbp) genome are fragmented into >135,000 contigs, with numerous order and orientation errors within scaffolds, probably associated with the plant's biology, which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. Here, we present a high-quality long-read-based assembly of red clover with a more than 500-fold reduction in contigs, improved per-base quality, and increased contig N50 by three orders of magnitude. The 413.5 Mbp assembly is nearly 20% longer than the 350 Mbp short-read assembly, closer to the predicted genome size. We also present quality measures and full-length isoform RNA transcript sequences for assessing accuracy and future genome annotation. The assembly accurately represents the seven main linkage groups in an allogamous (outcrossing), highly heterozygous plant genome.

Self-Fertilization, Inbreeding, and Yield in Alfalfa Seed Production.

Selfing (self-pollination) is the ultimate form of inbreeding, or mating among close relatives. Selfing can create yield loss when inbreeding depression, defined as a lower survival and reproduction of inbred relative to outbred progeny, is present. To determine the impact of selfing in alfalfa (Medicago sativa L.), we quantified the selfing rate of 32 alfalfa seed production fields located in three regions, namely, the Pacific Northwest (PNW), the Central Valley of California (CEV), and the Imperial Valley of California (IMP). Selfing rates (the proportion of selfed seeds) varied between 5.3 and 30% with an average of 12.2% over the 32 seed production fields. In both the parents and their progeny, we observed an excess of heterozygotes relative to Hardy-Weinberg expectations. We detected notable levels of inbreeding in parents (0.231 ± 0.007 parental inbreeding coefficient) and progeny (0.229 ± 0.005). There were a 15% decrease in the number of seeds per stem (seed set) and a 13% decline in the number of seeds per pod in selfed relative to outcrossed stems, but negligible inbreeding depression for pods per raceme and seed weight. The number of racemes on selfed stems increased significantly in fields with greater selfing rates, supporting the presence of geitonogamous or among flower selfing. Despite the significant level of inbreeding depression, seed set did not decrease in fields with higher selfing rates, where the greater number of racemes on the selfed stems increased the seed set. The effects of the field selfing rate on the seed yield metrics were mostly indirect with direct effects of the number of racemes per stem. Available data indicate that the majority of selfing in alfalfa is pollinator-mediated, and thus, eliminating selfing in alfalfa seed production would require the selection of self-incompatible varieties, which, by eliminating inbreeding depression, would provide a 15% potential increase in seed yield and an increase in future hay yield.

Genetic map-based location of the red clover (Trifolium pratense L.) gametophytic self-incompatibility locus.

Red clover is a hermaphroditic allogamous diploid (2n = 2x = 14) with a homomorphic gametophytic self-incompatibility (GSI) system (Trifolium pratense L.). Red clover GSI has long been studied, and it is thought that the genetic control of GSI constitutes a single locus. Although GSI genes have been identified in other species, the genomic location of the red clover GSI-locus remains unknown. The objective of this study was to use a mapping-based approach to identify simple sequence repeats (SSR) that were closely linked to the GSI-locus. Previously published SSR markers were used in this effort (Sato et al. in DNA Res 12:301-364, 2005). A bi-parental cross was initiated in which the parents were known to have one self-incompatibility allele (S-allele) in common. S-allele genotypes of 100 progeny were determined through test crosses and pollen compatibility. Pseudo F(1) linkage analysis isolated the GSI-locus on red clover linkage-group one within 2.5 cM of markers RCS5615, RCS0810, and RCS3161. A second 256 progeny mapping testcross population of a heterozygous self-compatible mutant revealed that this specific self-compatible mutant mapped to the same location as the GSI-locus. Finally, 82 genotypes were identified whose parents putatively shared one S-allele in common from maternal halfsib families derived from two random mating populations in which paternal identity was determined using molecular markers. Unique S-allele identity in the two random mating populations was tentatively inferred based on haplotypes of two highly allelic linkage-group one SSR (RCS0810 and RCS4956), which were closely linked to each other and the GSI-locus. Paternally derived pollen haplotype linkage analysis of RCS0810 and RCS4956 SSR and the GSI-locus again revealed tight linkage at 2.5 and 4.7 cM between the GSI-locus and RCS0810 and RCS4956, respectively. The map-based location of the GSI-locus in red clover has many immediate applications to red clover plant breeding and could be useful in helping to sequence the GSI-locus.

Pod Dehiscence in Hairy Vetch (Vicia villosa Roth).

Hairy vetch, Vicia villosa (Roth), is a cover crop that does not exhibit a typical domestication syndrome. Pod dehiscence reduces seed yield and creates weed problems for subsequent crops. Breeding efforts aim to reduce pod dehiscence in hairy vetch. To characterize pod dehiscence in the species, we quantified visual dehiscence and force required to cause dehiscence among 606 genotypes grown among seven environments of the United States. To identify potential secondary selection traits, we correlated pod dehiscence with various morphological pod characteristics and field measurements. Genotypes of hairy vetch exhibited wide variation in pod dehiscence, from completely indehiscent to completely dehiscent ratings. Mean force to dehiscence also varied widely, from 0.279 to 8.97 N among genotypes. No morphological traits were consistently correlated with pod dehiscence among environments where plants were grown. Results indicated that visual ratings of dehiscence would efficiently screen against genotypes with high pod dehiscence early in the breeding process. Force to dehiscence may be necessary to identify the indehiscent genotypes during advanced stages of selection.

Identification of Single Nucleotide Polymorphism in Red Clover (Trifolium pratense L.) Using Targeted Genomic Amplicon Sequencing and RNA-seq.

Red clover (Trifolium pratense L.) is a diploid, naturally cross-pollinated, cool-season species. As a perennial forage legume, red clover is mostly cultivated in temperate regions worldwide. Being a non-model crop species, genomic resources for red clover have been underdeveloped. Thus far, genomic analysis used in red clover has mainly relied on simple sequence repeat (SSR) markers. However, SSR markers are sparse in the genome and it is often difficult to unambiguously map them using short reads generated by next generation sequencing technology. Single nucleotide polymorphisms (SNPs) have been successfully applied in genomics assisted breeding in several agriculturally important species. Due to increasing importance of legumes in forage production, there is a clear need to develop SNP based markers for red clover that can be applied in breeding applications. In this study, we first developed an analytical pipeline that can confidently identify SNPs in a set of 72 different red clover genotypes using sequences generated by targeted amplicon sequencing. Then, with the same filtering stringency used in this pipeline, we used sequences from publicly available RNA-seq data to identify confident SNPs in different red clover varieties. Using this strategy, we have identified a total of 69,975 SNPs across red clover varieties. Among these, 28% (19,116) of them are missense mutations. Using Medicago truncatula as the reference, we annotated the regions affected by these missense mutations. We identified 2,909 protein coding regions with missense mutations. Pathway analysis of these coding regions indicated several biological processes impacted by these mutations. Specifically, three domains (homeobox domain, pentatricopeptide repeat containing plant-like, and regulator of Vps4 activity) were identified with five or more missense SNPs. These domain might also be a functional contributor in the molecular mechanisms of self-incompatibility in red clover. Future in-depth sequence diversity analysis of these three genes may yield valuable insights into the molecular mechanism involved in self-incompatibility in red clover.