Artificially improving traits of cultivated alfalfa (Medicago sativaL.), one of the most important forage crops, is challenging due to the lack of a reference genome and an efficient genome editing protocol, which mainly result from its autotetraploidy and self-incompatibility. Here, we generate an allele-aware chromosome-level genome assembly for the cultivated alfalfa consisting of 32 allelic chromosomes by integrating high-fidelity single-molecule sequencing and Hi-C data. We further establish an efficient CRISPR/Cas9-based genome editing protocol on the basis of this genome assembly and precisely introduce tetra-allelic mutations into null mutants that display obvious phenotype changes. The mutated alleles and phenotypes of null mutants can be stably inherited in generations in a transgene-free manner by cross pollination, which may help in bypassing the debate about transgenic plants. The presented genome and CRISPR/Cas9-based transgene-free genome editing protocol provide key foundations for accelerating research and molecular breeding of this important forage crop.

The rice orthologue of maize domestication geneTeosinte branched 1(Tb1) affects tillering. But, unlike maizeTb1gene, it was not selected during domestication. Here, we report that anOsTb1duplicate gene (OsTb2) has been artificially selected during upland rice adaptation and that natural variation inOsTb2is associated with tiller number. Interestingly, transgenic rice overexpressing this gene shows increased rather than decreased tillering, suggesting thatOsTb2gains a regulatory effect opposite to that ofOsTb1following duplication. Functional analyses suggest that the OsTb2 protein positively regulates tillering by interacting with the homologous OsTb1 protein and counteracts the inhibitory effect of OsTb1 on tillering. We further characterize two functional variations withinOsTb2that regulate protein function and gene expression, respectively. These results not only present an example of neo-functionalization that generates an opposite function following duplication but also suggest that theTb1homologue has been selected in upland rice.

Spermidine (SPD), as a signal molecule, participates in the response of plants to adversity stress. In the biosynthesis of SPD, spermidine synthase (SPDS) is the key enzyme that catalyzes the synthesis of putrescine into SPD. TWO cDNA sequence coding spermidine synthases have been cloned from mulberry (MnSPDS) in the present report. Sequence analysis revealed that their ORF were 1005 and 1188bp in length, encoding 334 and 395 amino acid protein, respectively. MnSPDS genes belonged to S-adenosyl-L-methionone, AdoMet-MTase, Class I superfamily. Phylogenetic analysis of the amino acid sequences encoded by the spermidine synthase gene from different species reveals that Morus notabilis C.K. Schneid. was closely connected to Arabidopsis thaliana. MnSPDS genes expression patterns treated under drought, pathogen treatment were investigated using quantitative PCR (RT-qPCR) in real-time. The level of mRNA showed a significant change in drought and pathogen treatment compared to the standard growth environment. Especially, the peak expression of MnSPDS2 gene was 3.2 times that of MnSPDS1gene under drought stress, however, for the treatment of Botrytis cinerea, the peak of transcript level of MnSPDS1 gene was 4.2 times that of MnSPDS2 gene. These findings provide useful reference information for the molecular basis of signal transduction in mulberry trees during stress responses.