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A utotetraploid alfalfa (2n = 4x = 32) is the third largest crop by acreage in the United States with an estimated 28 million kg of seed produced per year (NASS, 2009). Although alfalfa tends to outcross (i.e., xenogamous), it is a self-compatible species meaning that self-pollination or "selfing" is possible. Selfing in alfalfa is more likely to occur in the absence of pollen from another genotype, suggesting that, when present, outcross pollen may outcompete self pollen (Viands et al., 1988). Selfing has been shown to occur in insect-pollinated alfalfa seed production fields. Reported field-wide selfing rate estimates range from 9 to 53% (Burkart, 1937, 15% selfing;Johansen, 1963, multiple studies 9 to 26% selfing; Bradner and Frakes, 1964, 34% selfing; Pedersen, 1968, 53% selfing; Knapp and Teuber, 1993, 24% selfing; and Brown and Bingham, 1994, 28% selfing). Most of the pre-1990s studies used white flowered "sentinel" plants in fields of purple flowered alfalfa plants to estimate selfing rates. The pollinators used in these studies varied widely or were not mentioned and ABSTRACT Alfalfa (Medicago sativa L.) self-pollination (i.e., selfing) causes inbreeding depression. Determining factors influencing alfalfa seed production selfing rates could inform potential mitigation strategies to reduce selfing. We measured in situ selfing rates from seed sampled from random plants in a commercial alfalfa seed production field pollinated by leafcutter bees (Megachile rotundata F.). Alfalfa selfing rates were estimated by genotyping ~24 progeny from each of 38 maternal plants. Maternal plant distance to pollinator domicile, pod position on racemes, raceme position on stems, and seeds per pod were noted during seed and tissue collection. Selfing rates averaged 11.8% with individual selfing rates ranging from 0% to 52.2%. Seed from pods collected from upper parts of racemes had lower selfing rates (9.1%) compared to pods from lower parts of racemes (15.1%). When "low" self-compatible (<15% selfing rate in 3+ seeded pods) and "high" self-compatible (≥15% selfing rate in 3+ seeded pods) plants were examined separately, however, this pattern remained significant only for low self-compatible plants (upper raceme selfing rates 3.1% vs. lower raceme 8.3%). Low self-compatible plants had higher selfing rates in 1-2 seeded pods (12.9%) compared to 3+ seeded pods (3.8%) while high self-compatible plants showed no differences in selfing rates based on seed number per pod. Genetic differences for self-pollen's ability to outcompete outcross pollen when growing down the style best explained observed differences between low and high self-compatible plants. Best management practices and selection could help reduce but not eliminate selfing in alfalfa seed production fields.

Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

The introduction of genetically engineered (GE) alfalfa requires a mechanism for producers to successfully grow and market alfalfa (Medicago sativa L.) hay destined for GE-sensitive markets such as organic and export. A process of coexistence includes elements of respect for diverse agricultural systems, improved communication, scientific knowledge, and market clarity. A definition for "non-GE alfalfa forage" is proposed, along with suggested production protocols. These protocols include securing non-GE-detect seed, steps to reduce the probability of gene flow in hay fields, equipment sanitation, hay-lot identification, and hay testing for low-level presence. The largest risk for low-level presence in hay is likely to originate from unwanted GE presence in the planting seed. Secondary risks include accidental mixing of hays during harvest or storage, followed by gene flow between forage fields. The tolerance for low-level presence in non-GE hay must meet specific market sensitivities. Promoting absolute zero GE hay (e.g., GMO free) is a practical and analytical impossibility, creates difficulties for farmers, and makes no sense for a nontoxic, unwanted market factor. Regulatory-based tolerances, driven largely by countries that do not permit a GE trait, may require non-GE determination to a limit of detection of approximately 0.1%. Market-based tolerance thresholds may differ greatly depending on the sensitivity of markets. For market purposes, a definition of non-GE alfalfa as having a low-level presence of less than 0.9% of dry matter is suggested. Coexistence strategies for alfalfa forage require an understanding of the sources of low-level presence, market tolerances of diverse markets, and market assurance processes.