Population Genetics And Functional Genomics Of Lactococcus Lactis Subsp.lactis In Naturally Fermented Milk

As a model species,Lactococcus genus,Lactococcus lactis subsp.lactis(L.lactis subsp.lactis)is one of the most common lactic acid bacteria in naturally fermented milk,which are commonly used as starter cultures for the production of fermented dairy products such as cheese and low-temperature fermented milk.It is well accepted that L.lactis subsp.lactis has highly economic value because of its extensive application prospects.Systematic analyses of the genetic background,evolution and functional characteristics of L.lactis subsp.lactis are of great significance for the development and utilization of this species.However,at present,few population genomic studies in this filed are carried out.In this present study,genomes of 227 different L.lactis subsp.lactis strains isolated from naturally fermented dairy products were sequenced using an Illumina Hiseq platform.Combined with genomes of another 90 strains available from NCBI,the genetic background,phylogenetic relationship,population structure and functional characteristics of L.lactis subsp.lactis were investigated using a series of population genetic techniques.Furthermore,the genome-wide association analysis(GWAS)was performed to locate the key phenotype in the genome.The main results of the research are as follows:(1)A total of 227 draft genomes from different L.lactis subsp.lactis strains were completed.According to the whole genome analyses and the ANI or TNI values,these L.lactis subsp.lactis strains were considered to have abundant genetic diversity,which was higher than that of the subspecies level defined in the traditional bacterial taxonomy.(2)Single nucleotide polymoiphisms(SNP)were analyzed by using L.lactis subsp.lactis IL1403 as the reference sequence,and a total of 78,624 SNP sites and 4,517 InDel were identified.The driving force of population evolution was analyzed at the whole gene level.The population of L.lactis subsp.lactis displayed various genomic characteristics,including fewer mutations SNPs,more recombination SNPs,larger effective population size and strong purification selection pressure.(3)Combined with another 90 strains published in NCBI,435 core genes and 18,719 pan-gene sets were constructed.Further phylogenetic and population structure analyses showed that L.lactis subsp.lactis population could be divided into four branches,including one environmental lineage and three domestication lineages.There were at least two separated domestication events between the four lineages.The environmental strains appeared earlier than domestication lineages and could be the provider of genetic diversity of the whole population.(4)Functional genomic studies found that the copy numbers of functional genes,such as carbohydrate active enzyme and cold/hot emergency protein coding genes,were significantly varied among the strains from different genetic lineages.Most of these variations were caused by movable genetic elements,such as insertion sequence and plasmid.Therefore,it could be indicated that mobile genetic elements might help strains from different lineages to adapt to the environment.(5)There were differences in fermentation ability between strains of different domestication lineages.Genome-wide association analysis(GWAS)was carried out to analyze the associations between the bacterial genomes and the fermentation phenotype data,which could locate the gene loci affecting the key phenotypes.As a result,a total of 76 loci and 152 genes were identified to be associated with 34 phenotypes.Among these genes,the most related genes were proteolytic enzyme pepF,pepO,oligopeptide transporter oppC,oppD and competing protein CoiA.The function of these genes might be related to the growth of strains so that they could affect most of the phenotypes.Based on the above sites,a random deep-forest model was established to distinguish the fermentation speed of bacterial strains.The AUC of the model was as high as 82.19%.In this study,the population genetic background and functional characteristics of L.lactis subsp.lactis were analyzed based on population genetics and functional genomics,and the key functional gene loci which affect phenotypic variations were located.This information will provide scientific guidance for the discovery of strains with excellent production characteristics and can be of great significance to practical productions.