Genetic Mapping Of Genes Controlling Seed Shattering And Stem Epidermis Color In Lettuce

Lactuca serriola,the progenitor of cultivated lettuce（L.sativa）,plays an important role in genetic improvement of lettuce,but seed shattering is a bad agronomic trait for the utilization of wild lettuce.The domestication of lettuce showed that the trait of seed shattering changed into none seed shattering.In this study,we used the forward genetic method to map the gene controlling seed shattering.Our results can provide gene resources for the utilization of wild lettuce.The previous study showed that an F₂ segregating population derived from Wo111×PI491245 was used to map a gene controlling seed shattering.After scanning 3,962individuals,we mapped the gene controlling seed shattering to the region of 9.28-17.00Mb,approximately 7.72 Mb on chromosome 6.This result indicated that the F₂ population we used may have the suppression of chromosome recombination.Then three F₂segregating populations were constructed to overcome the recombination suppression and were used to narrow the mapping region.We screened 1,044 individuals from these three segregating populations.Ultimately,the candidate region was mapped between 10.75 Mb and 16.04 Mb on chromosome 6,approximately 5.29 Mb.In order to understand the mechanism of recombination suppression,we first aligned the candidate region between cultivated lettuce and wild lettuce.The alignment showed that an inversion between 11.22 Mb and 15.76 Mb was on chromosome 6.Moreover,a fragment of 297 kb at 14.77 Mb on chromosome 6 was lost.Further,we analyzed the sequence of the inversion breakpoint,there is no gene in and near the breakpoint.Annotated the 297 kb sequence of the wild genome,all the sequences are repetitive sequences.We predicted seven genes of 163 genes in the region.The candidate genes are LG6533864,LG6581063,LG6577075,LG6580918,LG6580935,LG6577149,LG6592781.The CRISPR/Cas9 gene-editing technology was used to verify the function of candidate genes.We obtained T₀generation transgenic plants of LG6533864,and T₁generation obtained homozygous editing plants,but the phenotype was still seed shattering,so this gene was excluded.We also obtain one T₀generation hybrid editing plant of LG6577149,and the phenotype was shattering.We need to wait for the T₁ phenotype and observe if the phenotype changes.Other genes didn’t obtain editing materials yet.The previous study predicated two shattering candidate genes which encoded an ethylene-responsive transcription factor（LG6581050）and a polygalacturonase（LG6580947）.T₁generation homozygous editing plants of LG6581050 and LG6580947were still shattering.The two genes may not be the candidate gene to regulate stem color in lettuce.It showed that a locus controlling the red stem and green stem in the F₂segregating population derived from Wo111（non-shattering/green stem）×CGN10978（shattering/red stem）.The previous study cloned five genes that controlled the anthocyanin biosynthesis in the leaf,but they can’t explain the anthocyanin biosynthesis in the stem.So there will be other loci to control the stem color.Using the F₂ and F_2:3 segregating populations with 1,790individuals,the candidate region was mapped to the region of 47.23-47.66 Mb on chromosome 1,approximately 0.43 Mb.We analyzed the gene expression in two pools and SNPs in two parents of fifteen genes in this region.A gene LG1145654 was possible the candidate gene,which encodes the Phytochrome B.A nonsense mutation on the third exon abolished the gene function.The CRISPR/Cas9 gene-editing technology was used to verify the function of the candidate gene.Ten T₀ transgenic plants were obtained,but colors of stems in these plants are still red.We need to wait for the T₁ phenotype and then see if the phenotype changes.