Genetic Mapping Reveals Broader Role of Vrn-H3 Gene in Root and Shoot Development beyond Heading in Barley

The aim of the present study was to dissect the genetic inheritance and interplay of root, shoot and heading attributes for a better understanding of these traits in crop production. For this, we utilized quantitative trait loci (QTL) and candidate gene analysis approach using a second filial (F2) population originated from a cross between spring cultivar Cheri and wild barley accession ICB181160. The F2 population comprising 182 plants was phenotyped for root dry weight (RDW), root volume (RV), root length (RL) and shoot dry weight (SDW), tiller number per plant (TIL) and days to heading (HEA). In parallel, this population was genotyped using polymerase chain reaction (PCR) based cleaved amplified polymorphic sequence (CAPS) markers distributed across the whole genome. Marker by trait analysis revealed 16 QTL for root and shoot traits localized on chromosomes 1H, 3H, 4H, 5H and 7H. The strongest and a common QTL effect for root, shoot and heading traits was identified on chromosome 7H at the putative region of Vrn-H3 gene. Later, we have established PCR based gene specific marker HvVrnH3 revealing polymorphism for early heading Vrn-H3 allele in Cheri and late heading allele vrn-H3 in ICB181160. Genotyping of these alleles revealed a clear co-segregation of early heading Vrn-H3 allele with lower root and shoot attributes, while late heading vrn-H3 allele with more TIL and higher root biomass suggesting a primary insight on the function of Vrn-H3 gene beyond flowering. Genetic interactions of vernalization genes Vrn-H3 with Vrn-H2 and Vrn-H1 also suggested the major role of Vrn-H3 alleles in determining root and shoot trait variations in barley. We believe, these data provide an opportunity for further research to test a precise significance of early heading on yield components and root associated sustainability in crops like barley and wheat.


PPDH1
This study This study a Detection of the described SNPs in the exon 8 with sequencing.
The dominant PPDH1 allele is prevalent in wild progenitor H. vulgare spp. spontaneum and accelerates flowering under long days in short growing seasons. The recessive ppdh1 allele causes reduced response to long days and was selected for adaptation to long growing seasons [3]. Previous studies described polymorphisms between dominant and recessive PPDH1 alleles. In this study, primers were designed spanning from exon 7 to end of exon 8for detection of previously described mutations(SNP22 and SNP23) [4]. Sequencing of PPDH1 was showed that wild ICB181160 parent carrying a dominant PPDH1 allele while Cheri carrying a recessive ppdh1 allele. The dominant PPDH2 allele is functional and providing early flowering under short days whereas the recessive allele is truncated gene having only part of exon 4 and delay flowering under short days [5]. Previously shown that most of the spring cultivars carrying dominant allele of PPDH2 gene whereas the majority of winter cultivars possessed recessive null ppdh2 [6]. Sequencing of the PPDH2 was carried out in Cheri and ICB181160. PCR results were showed that both of the parents carrying a dominant PPDH2 allele. Two SNPs were detected in intron 3 which is previously described also by Casao et al. [7].
b Letters "in" and "del" indicate a 6 bp indel (TGCATT) c Letters "in" and "del" indicate a 3 bp indel (AAG) d Detection of large deletion and described mutations in exon 2 with sequencing Transcript based cloning of the EAM8 gene was showed that EAM8 is the orthologue of the Arabidopsis ELF3 and eam8 mutation has a dramatic effect on the expression level of circadian clock and photoperiod pathway genes [8]. First mutant line (mat-a.8) Mari was released commercially in 1961 as an early barley mutant [9]. In field trials under long-day and short-day conditions, it flowered earlier than the mother cultivar Bonus [9]. Primers were designed spanning from exon 2 to exon 3 for detection of previously described mutations [

HvCCA1
A partially redundant Arabidopsis thaliana MYB-transcription factor AtCCA1(CIRCADIAN CLOCK ASSOCIATED) 1 plays a role in the formation of morning active genes [13].
Previous study experimentally proofed that HvCCA1 functionality similar to AtCCA1 [11].Sequencing of the 556 bp genomic region spanning from exon1 to exon3 of HvCCA1 sequenced from Cheri and ICB181160.Alignment of the sequences with available HvCCA1 genes from other cultivars Morex, Triumph and Igri also were not showed any polymorphism.
Sequencing results were showed that both of the parents having same allele of the HvCCA1 gene.

HvLUX1
HvLUX1 is a candidate gene underlying the early maturity 10 (eam10) locus in barley.
HvLUX1 characterized as a transcription factor which encodes an MYB domain-containing SHAQKYF-type GARP family protein. The mutation identified in eam10 changed the first amino acid residue in this conserved motif and this described as hvlux1 allele [3]. Sequencing of the complete gene was carried out and results were showed that Cheri and ICB181160 both carrying haplotype 1 allele of HvLUX1 gene. TFL1(TERMINAL   FLOWER1), which regulates inflorescence architecture and flowering time [15]. Three major haplotypes (I, II and III) identified at higher frequencies in domesticated barleys compare to wild progenitors. Major haplotypes I and III (which are later flowering than haplotype II in domesticated barleys) both harbor the mutation encoding the p.Pro135Ala alteration. The haplotype I is most frequently found in wild winter barleys from Fertile Crescent while haplotype III has been selected from wild barley and is completely fixed in northern European spring barley cultivars [15] . In this study HvCEN sequences of Cheri and ICB181160 were sequenced and aligned with previously described haplotypes, results were showed that Cheri is carrying a haplotype III allele of HvCEN gene while ICB181160 carrying a haplotype I.