Haplotypes And Evolution Analysis Of Barley Ear Trait Domestication Genes

Cultivated barley shows significant changes in spikelets compared with its wild progenitor Hordeum vulgare ssp.spontaneum,such as row-type,brittleness of the rachis,covered/naked caryopsis,which are the significant events in the progress of domestication.The non-brittle rachis is the most important standard to distinguish the domesticated barley from the wild.The six-rowed barley can greatly improve the yield and the naked barley is convenient to eat.In this study,the brittle-rachis genes HvBtr1 and HvBtr2,row-type genes Vrs1 and Int-c,the naked caryopsis gene Nud were re-sequenced among universal barley germplasm population originated from different countries especially from China to examine the genetic diversity of the domestication genes and association with the phenotype,phylogenetic analysis,ecologo-geographic distribution,genetic mechanism of domesticated ear trait gene alleles,and elucidate the origin and spread of Chinese barley.The main conclusions are as follows: 1.The brittle-rachis genes HvBtr1 and HvBtr2HvBtr1 and HvBtr2 existed many polymorphic sites,but the coding regions were more conservative.There were 41 and 22 nucleotide variations that were detected in Btr1 and Btr2 interval with 11 and 14 haplotypes,respectively.The two prevalent haplotypes of HvBtr1 and HvBtr2 made up the proportion at 70.9%(Btr1.1a)and 24.8%(btr1.4a),51.5%(Btr2.3a)and 36.7%(btr2.1a).All of the 54 wild relatives collected from the Qinghai-Tibet Plateau were brittle barley and Btr1Btr2 genotype.The cultivated barley accessions carried either 1-bp or 11-bp microdeletions in btr1 or btr2 and no double recessive and heterozygous alleles were found.The ecologo-geographic distribution in world and China,as well as the germplasm accessions distribution of HvBtr1 and HvBtr2 haplotypes were well identified in this study.Phylogenetic and geographical analysis of HvBtr1 and HvBtr2 haplotypes divided the 398 barley accessions into typical Western and Eastern groups.The most accessions from Central and East Asia were btr2-type.European and Southwest Asia barley accessions were almost typical occidental btr1-type.This result revealed that the brittle-rachis genes btr1 and btr2 resulted from two independent domestication events.Chinese wild barleys were divided into Western and Eastern groups.Most of Chinese wild barleys were genetically closer to Chinese cultivars.In view of the symbiosis situation of wild barley and cultivated barley in China,we considered that barley has gone through four stages during domestication and spread in China:(1)introduction and plantation of ancestral wild brittle rachis two-rowed barley H.vulgare ssp.spontaneum in the Tibetan Plateau and its vicinity,(2)mutation and selection of the wild brittle rachis six-rowed barley,H.vulgare ssp.Agriocrithon,(3)development of the first tough rachis six-rowed barley landrace via spontaneous mutation,(4)advent of the tough rachis two-rowed barley by natural pollination between the tough rachis six-rowed barley and H.vulgare ssp.spontaneum.There is a part of the so called Western btr1-type Chinese barley landraces in some special regions.Those btr1-type landraces were speculated to be introduced from Western countries in the early time by checking their names and original records.The btr1 gene was recently introduced into Chinese modern barley cultivars via cross breeding between Chinese landraces and foreign cultivars.Furthermore,a nearly 1:1 proportion of btr1-type to btr2-type genotypes in Chinese modern barley cultivars resulted from random selection of breeders,showing no difference existed in ecological adaptability of the two alleles btr1 and btr2.2.The row-type genes HvVrs1 and HvInt-cIn this study,15 HvVrs1 haplotypes and 7 HvInt-c haplotypes were identified by re-sequencing the two row-type genes in 445 Chinese landraces and 71 wild barleys tegether with three mutants.Of the 15 HvVrs1 haplotypes,six haplotypes Vrs1.1c,Vrs1.1d,vrs1.2d,Vrs1.2e,Vrs1.4b and vrs1.5b were novel haplotypes.The haplotype vrs1.a2 with a single base insertion at the second exon of HvVrs1 was widely distributed in Mediterranean,but may not exist in China.A two-rowed deficient barley with rudimental lateral spikelets from Heilongjiang was identified,which carried the Vrs1.t allele and a SNP(A1333G)lead to the phenotype.In addition,the EMS-induced mutant from the two-rowed deficiens showed the six row-type phenotype.Sequence analysis revealed that a SNP(G1440A)of Vrs1 formed a stop codon,leading to early termination of translation and the absence of four amino acid residues in the mutant.In this study,the ecologo-geographic distribution and germplasm accessions distribution of HvVrs1 and HvInt-c haplotypes were clarified.There were three main Vrs1 haplotypes(Vrs1.1a,vrs1.2 and Vrs1.4a)and two main Int-c haplotypes(Int-c.1a and int-c.2a)in Chinese barley accessions.Among them,Vrs1.1a,Vrs1.4a and Int-c.1a had the highest frequency in wild and cultivated barley from Qinghai-Tibet Plateau with its vicinity.The vrs1.2 and int-c.2a were distributed in Northwest China,Huanghuai and the middle and lower reaches of the Yangtze River.Then the association of haplotype combinations HvVrs1 and HvInt-c with the row-type was analyzed.The haplotype combination Vrs1.1a/Int-c exhibited two-rowed phenotype,while the vrs1.2/Int-c showed six-rowed barley.The Vrs1.4a haplotype in landraces were all six-rowed,while the wild barley had three types: two-rowed,six-rowed and labile barley.We speculated that the six-rowed landraces originated from artificial selection,whereas the wild-type barley was results of the long-term continuous isolation and homozygosity after hybridization with different row-type barley.And there is another gene regulating the development of lateral spikelet except HvVrs1 and HvInt-c.Median-joining network of Vrs1 revealed that Vrs1.1a and vrs1.2 were derived directly from the common unknown ancestor Mv1.Vrs1.4a originated from another ancestor Mv2.If we assume that the unknown ancestor was the original wild barley introduced into Qinghai-Tibet Plateau with its vicinity,combined with the row-type of Vrs1.4a/Int-c wild barley,which is consistent with the deduction about the four stages during domestication and spread in China based on the evolutionary relationship of Btr1 and Btr2 haplotypes.3.The naked caryopsis gene HvNud genePrevious study revealed the naked barleys harbored a 17 kb deletion containg the entire HvNud gene.In our study,all hulled barley had the Nud gene and most naked barley possessed the 17 kb deletion fragment.However,seven naked barley from Tibet possessed the Nud gene without the 17 kb deletion.A missense mutation(T643A)of Nud might be the reason for the naked caryopsis phenotype.The result revealed a second domestication pathway of the naked barley.Sequence analysis of Nud gene showed that the 40 hulled barleys and the 7 naked barleys had 4 polymorphic sites with 5 haplotypes.Of which,H3,H4 and H5 were novel haplotypes.H3 had a mutation in the intron region;H5 also carried a SNP missense mutation in the left base of the mm domain;H4 had a 6 bp Indel between the mm and cm domains.Haplotype H1 was normal hulled barley.The seeds of haplotypes H4 and H5 showed semi-naked or incomplete naked,which the caryopsis was partially exposed.We speculated that the 6 bp deletion of H4 and the missense mutation of H5 were responsible for the phenotypic change.The mutations at both sites may reduce the activity of the NUD and affect the lipid biosynthesis.H2 was the seven naked barley cultivars identified in this study.The SNP missense mutation(V148D)occurs at the right of the mm domain in HvNud gene affected the lipid biosynthesis.Thus,different variations at mm domain have different effects on HvNud gene.In addition,the Indel variations in other sites also reduced the function of the Nud gene.The evolutionary relationship between the five haplotypes was deduced.H1 was the original haplotype,H2,H3 and H5 derived from the SNP mutation of H1,and then the H3 mutated to H4 by a 6 bp base deletion.