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Abstract

16Background: Sainfoin (Onobrychis viciifolia) is a highly nutritious, tannin-containing, and 17 tetraploid forage legume. Due to the lack of detailed transcriptomic and genomic information on 18 this species, genetic and breeding projects for sainfoin improvement have been significantly 19 hindered. 20Methods: In this study, a total of 24,630,711 clean reads were generated from 14 different 21 sainfoin tissues using Illumina paired-end sequencing technology and deposited in the NCBI 22 SRA database (SRX3763386). From these clean reads, 77,764 unigene sequences were obtained 23 and 6,752 EST-SSRs were identified using denovo assembly. A total of 2,469 primer pairs were 24 designed, and 200 primer pairs were randomly selected to analyze the polymorphism in five 25 sainfoin wild accessions. 26Results: Further analysis of 40 sainfoin individuals from the five wild populations using 61 27 EST-SSR loci showed that the number of alleles per locus ranged from 4 to 15, and the expected 28 heterozygosity varied from 0.55 to 0.91. Additionally, by counting the EST-SSR band number 29 and sequencing the three or four bands in one sainfoin individual, sainfoin was confirmed to be 30 autotetraploid. This finding provides a high level of information about this plant. 31Discussion: Through this study, 61 EST-SSR markers were successfully developed and shown to 32 be useful for genetic studies and investigations of population genetic structures and variabilities 33 among different sainfoin accessions. 34 Sainfoin (Onobrychis viciaefolia) is a cross-pollinated, autotetraploid and perennial legume 38 (2n=4x=28) that is commonly used as a silage. The nutritional value of sainfoin is universally 39 recognized, and it is known to be rich in proteins and secondary metabolites. Sainfoin can also fix 40 atmospheric nitrogen through its symbiotic relationship with rhizobia. The origin center of 41 sainfoin is known in the Middle East and Central Asia. It was introduced into Europe in the 42 fifteenth century, and it was rapidly adopted by that region due to its high fodder value 43 (Mora-Ortiz et al. 2016). In China, sainfoin is mainly grown in the northeast, north and northwest 44 regions, including Gansu province. Sainfoin contains high levels of condensed tannins that 45 provide many potential nutritional and health benefits to animals: preventing bloating, reducing 46 fecundity of nematodes, improving nitrogen utilization, and reducing greenhouse gas emissions. 47In recent years, there was a renewed interest in sainfoin for its use in animal diets. Several studies 48 indicated that the voluntary intake of sainfoin by grazing heifers is higher than alfalfa (Medicago 49 sativa) (Parker and Moss, 1981;Kempf et al., 2016). Scharenberg and colleagues reported that 50 sainfoin was more palatable than birdsfoot trefoil when given to sheep (Scharenberg et al., 2007). 51Therefore, exploitation and conservation of sainfoin germplasms became important. Also, 52 knowledge of sainfoin genetic diversity and structures has become a prerequi...