Allopolyploid Formation In Trapa,and The Origin And Domestication Of Water Caltrop

Plant domestication altered the course of human history,causing a shift from hunter-gatherer to agricultural societies and greatly advancing human civilization.Thereby,it is essential to elucidate the genetic basis of domestication traits and history of domestication,which not only facilitates our understanding of early human society,but also strengthen the improvement of modern crops.Trapa L.(Lythraceae)is an annual herbaceous,floating-leaf aquatic genus containing two morphological species,i.e.T.incisa and T.natans.Notably,the fruit of Trapa is an important source of food in early Chinese agricultural societies due to its high starch content.However,the evolution and domestication history of Trapa remain unclear.In this study,we utilized de novo sequencing,population resequencing and transcriptome sequencing data,and performed comparative genomics,phylogenomics,and population genomics analyses,as well as phenotype detection,cytological analysis to investigate the species differentiation,allopolyploid formation,and domestication origins in Trapa.The main findings obtained are as follows:(1)Cytological and phenotypic analysis of Trapa and de novo genome assembly of tetraploid T.natansKaryotype analyses and flow cytometry were performed on individuals covering all species and cultivars in Trapa.Results showed that T.incisa was diploid with 2n =2x = 48,while T.natans was a complex containing diploid(2n = 2x = 48)and tetraploid(2n = 4x = 96).Phenotypic statistics result suggested that the morphology traits of tetraploid T.natans are intermediate between the two diploids(i.e.diploid T.natans and T.incsia).We assembled and annotated the genome of tetraploid T.natans.The assembled genome of the tetraploid T.natans was 1005.46 Mb with Contig N50 of3.19 Mb and Scaffold N50 of 20.8Mb.Approximately 90% of the assembled scaffolds were anchored onto 48 pseudo-chromosomes using high-throughput chromosome conformation capture(Hi-C)technology.We identified 62.31% of the genome sequence as repetitive elements and annotated 68,946 protein-coding genes,96% of which had functional annotations.By using method of mapping resequencing data to the reference genome and Hi-C interaction,we found that the tetraploid genome was composed of two sets of subgenomes(A-subgenome and B-subgenome),indicating AA(diploid T.natans),BB(T.incisa),and AABB(tetraploid T.natans)three genome types in Trapa.(2)Population genetic structure and genetic diversity of TrapaA total of 57 individuals from China were selected for resequencing,and a total of9,133,926 SNPs were obtained for analysis.The neighbor-joining(NJ)tree revealed that the A and B(sub)genomes of Trapa clustered into two monophyletic branches with highly supported(bootstrap = 100%).ADMIXTURE analyses and principal components analysis(PCA)further divided 57 accessions into five genetic groups:Group I comprised cultivated and wild T.nantans(2x,AA)from the Yellow River,Yangtze River,and Pearl River regions(PYY);Group II comprised T.nantans(2x,AA)from the Heilongjiang River basin(HLR);Group III contained the A subgenome of tetraploid T.nantans(4x,AA);Group IV contained the B subgenome of tetraploid T.nantans(4x,BB)and T.incisa(2x,BB)from the HLR;Group V consists of T.incisa from PYY.Genetic diversity and pairwise linkage disequilibrium analyses indicated that the genetic diversity of cultivated T.natans was lower than that of wild populations(0.68 vs.1.51 × 10-3),and higher level of linkage disequilibrium in the cultivated T.natans genome.(3)Origin and domestication history of TrapaThe best-supported demographic model supported that diploid T.natans(AA)and T.incisa(BB)diverged approximately one million years ago,and may be related to allopatric speciation resulting from Late Quaternary refugial isolation.During the early-to-mid Pleistocene(0.23-0.31 Ma),changes in the connectivity of the presently discontinuous aquatic habitat systems induced by the Quaternary climatic fluctuations likely promoted population expansions and secondary contacts between diploid T.?natans and T.?incisa,thus leading to the formation of tetraploid T.?natans via hybridization and polyploidization.The phylogenetic analysis based on the complete chloroplast genome indicated that diploid T.natans was the maternal progenitor and T.incisa was the paternal progenitor of tetraploid T.natans.Based on resequencing data covering diploid T.natans,the phylogenetic analysis confirmed that all cultivated T.natnas(AA)were derived from the diploid T.natans(AA)in the Yangtze River.Water caltrop was first domesticated in the Neolithic period(c.6,300 yr BP),and underwent a second improvement during the ‘Nansong’ Dynasties(c.800 yr BP),giving rise to the most commonly cultivated modern variety named ‘Wuling’.(4)Genetic basis of domestication and improvement of cultivated T.natansCombined with XP-CLR score,πwild/πcult? and Tajima’s D,a total of 126 genomic regions were identified to be under selective sweeps during domestication,of which 66(52%)were distributed on five chromosomes(i.e.Chr4 A,Chr8A,Chr17 A,Chr19A and Chr21A).Within those 126 regions,we identified 205 protein-coding genes.Four genes(PME7,G9,At1g02816 and At4g02250)involved in the metabolism of pectin(a major cell wall component)were located in a particular region(5.79–5.86 Mb)of Chr19 A.Based on RNA-seq of five samples of flower/fruit tissues that represented five developmental stages and one sample of leaf tissue,we found 137 of 205 selected genes exhibited significant expression differentiations between cultivars and wild T.?natans.By integrating selective sweep and transcriptome profiling analyses,we identified a number of genes potentially selected and/or differentially expressed during domestication,some of which likely contributed not only to larger fruit sizes(SUS3,XTH8,CRA1 etc.)but also to a more vigorous root system(SAC7,RPD1,OCT1 etc.),facilitating nutrient uptake,environmental stress response and underwater photosynthesis.These gene resources will provide a valuable resource for genomicassisted breeding of Trapa.In conclusion,our study has provided insights into the differentiation of diploid species and the formation of allopolyploids in Trapa.Furthermore,it has shed light on the historical process of ‘two step domestication’ of cultivated water caltrop,which was likely first domesticated in the Yangtze River Valley as early as 6300?yr BP,and experienced a second improvement c.800?years ago,and we also uncovered the genetic basis for the formation of key agronomic traits during domestication.