Cloning Of A Yellow-leaf Controlling Gene GmYLD1 In Soybean And Its Role In Resistance To Phytophthora Infestans

Mutation breeding is one of the important ways of genetic improvement of crops.In the early stage of this laboratory,a soybean EMS mutation mutant library was established,and a large number of mutants for important functional gene mining and molecular breeding were obtained.In the mutant library,a weak yellowing mutant Gmyld1 was screened.In the early stage,genetic analysis of Gmyld1 has been conducted to determine that they are recessive homozygous mutations controlled by a single gene,and the mutation site has been initially located.On this basis,fine positioning of mutant genes,allele construction,gene function identification research and RNA-seq analysis were carried out on Gmyld1,which was to further reveal the regulatory mechanism of the gene function and cultivate high-yield,disease-resistant new varieties with excellent traits Lay the foundation.The main research process and experimental results include:1、Phenotype identification of soybean yellowing mutant Gmyld1The analysis of agronomic traits and test data showed that the mutant Gmyld1 exhibited a leaf yellowing phenotype compared with wild-type soybean Hedou 12(H12).Its young leaves are golden yellow,and as the leaves mature,the color of the leaves gradually changes to light green leaves.The height of Gmyld1 is shorter,and the number of nodes,number of pods,number of seeds per plant are reduced,but there is no significant difference in seed size and 100-kernel weight.Further analysis revealed that the pigment content of Gmyld1 leaves decreased,the chloroplast thylakoid arrangement was disordered,and the number of basal granules was less.Comparison of paraffin sections revealed that the turning point of Gmyld1 stem tip growth point from vegetative growth to reproductive growth was later than that of wild type.2、Fine mapping and cloning of GmYLD1Preliminary genetic analysis in the laboratory confirmed that the mutant trait was controlled by a single recessive nuclear gene,and the F2 generation population of Gmyld1 and Williams82 was used to determine the mutation gene in the 0.90Mb interval on chromosome 20,and the F2 population of Gmyld1 and H12 was used to BSA sequencing.In this study,we further expanded the F2:3 generation population,using 1564 mutants to finely locate the mutant gene in the 68kb interval on chromosome 20,which contains 6 annotated genes.Sequencing and comparison of 6 candidate genes in Gmyldl and wild type revealed that one candidate gene GmYLD1 gene had a base C-T substitution at the CDS+1629 base position,its encoding amino acid from P(proline)-S(Serine).BSA-seq data analysis obtained a candidate interval with a total length of 2.27Mb with 3 non-synonymous SNP sites.The CDS+1629 base site of GmYLD1 gene is one of the points.It is speculated GmYLD1 is a candidate gene for regulating leaf color.Sequence analysis found that GmYLD1 belongs to the R family resistance gene.Subcellular localization found that the protein encoded by GmYLD1 is located in the cytoplasm,and the homologous genes of Gm YLD1 are mainly located in soybean,kidney bean and Medicago truncatula legumes.3.Functional verification of candidate gene GmYLD1Screening and identification of the Gmyldl allele:In order to further verify the accuracy of GmYLD1 isolated by map-based cloning,this study screened 8 mutants with weak yellowing from the M3 generation(yellow leaf:green leaf=1:3)strain of the EMS mutagenic mutant library,GmYLD1 sequence alignment was performed on these mutants,and it was found that except for 1 strain with the same mutation site as GmYLD1,the other 7 strains had different nucleotide mutations at different positions in the GmYLD1 CDS sequence:666 TC,816 TA,906 TC,930 AT,1164 AT,1209 GA,1288 CT,1392 CT,indicating that they are Gmyld1 alleles.Construction and molecular identification of GmYLD1 editing plants:The CRISPR-Cas9 vector pGmYLD1-Pscl of GmYLD1 was transformed into soybean by Agrobacterium transformation method,and 4 transgenic T1 generation lines were obtained by Basta screening and gene fragment sequencing.The GmYLD1 fragment sequence has double peaks,and some of the sites are mutated,indicating that the GmYLD1-Pscl editing plant has a yellowing mutant phenotype.Construction and molecular identification of Gmyld1 restorer line:The overexpression vector p35S::GmYLD1 was transformed into soybean mutant Gmyld1 by Agrobacterium transformation method.After Basta screening and PCR identification,four p35S::GmYLD1 in Gmyld1 T1 soybean lines were obtained,a total of 36 plants,and the GmYLD1 fragment sequence showed double peaks.The GmYLD1 gene was integrated into the Gmyld1 genome,indicating that GmYLD1 overexpression restored the yellowing phenotype of Gmyld1.4、The role of GmYLD1 gene in Phytophthora infestansGmYLD1 is a disease resistance gene of the R family.Therefore,Gmyldl and wild-type Phytophthora infestation experiments were carried out.The leaves of H12 and Gmyld1 were injected with Phytophthora spore suspension,and it was found that compared with wild type H12,the area of water-like stains on the cotyledons of Gmyld1 was smaller,and the distribution of Phytophthora white hyphae was less.Trypan blue staining found that compared with H12,the Gmyld1 staining area was significantly reduced,and the mortality rate was smaller than that of the control,indicating that the mutant Gmyld1 had strong resistance to Phytophthora infestans.In order to reveal the molecular mechanism of GmYLD1 regulates Phytophthora infestans,transcriptome sequencing was performed on the mutants Gmyldl and H12.The results showed that compared with the wild type,Gmyld1 had 511 up-regulated genes and 265 down-regulated genes.Among them,there are 39 genes related to disease resistance,31 are up-regulated and 8 are down-regulated.In addition,there are differences expression of multiple genes in the alpha-linolenic acid anabolic pathway,MAPK signaling pathway,phytohormone signaling pathway,and plant-pathogen interaction pathway.The above results lay the foundation for further analysis of the molecular mechanism of Gmyldl regulating Phytophthora infestans Foundation.