Interactive effects of multiple vernalization (<Emphasis Type="Italic">Vrn-1</Emphasis>)- and photoperiod (<Emphasis Type="Italic">Ppd-1</Emphasis>)-related genes on the growth habit of bread wheat and their association with heading and flowering time

Background

The precise identification of Winterness/Springness (growth habit) for bread wheat, which is determined by genes involved in vernalization and photoperiod, will contribute to the effective utilization of bread wheat varieties. Here, 198 varieties from the Yellow and Huai wheat production region (YHW) in China were collected to identify their vernalization (Vrn-1) and photoperiod (Ppd-1) gene composition via a series of functional markers and their association with vernalization and photoperiod requirements at three locations during two years of experiments. The growth habits were measured during the spring sowing season.

Results

The results showed that the semi-winter varieties (grades1–4) were most prevalent in the population. The relative effects of single Vrn alleles on the growth period, such as heading date (HD) and/or flowering date (FD), were as follows: Vrn-B1b?>?Vrn-B1a?>?Vrn-D1b?>?Vrn-D1a?>?vrn-D1?=?vrn-B1. The interactive effects of Vrn-B1 and Vrn-D1 on HD and FD were identical to those of Vrn-B1b. Approximately 35.3% of the cultivars carried Ppd-B1a (photoperiod-insensitive) and exhibited the earliest HD and FD. The Ppd-D1a-insensitive allele (Hapl II) was carried by just 0.5% of the varieties; however, the other two sensitive alleles were present at a higher frequency, and their effects were slightly weaker than those of Ppd-B1a. In addition, strong interactive effects between Ppd-B1 and Ppd-D1 were detected. In terms of mean values among various genotypes, the effects followed the order of Vrn-1?>?Ppd-1.

Conclusions

According to the results of ANOVA and least significant range (LSR) tests, we can conclude that Vrn-1 rather than Ppd-1 played a major role in controlling vernalization and photoperiod responses in this region. This research will be helpful for precisely characterizing and evaluating the HD, FD and even growth habit of varieties in the YHW at molecular levels.

     

Detection of genetic determinants that define the difference in photoperiod sensitivity of Triticum aestivum L. near-isogenic lines

Identification of genetic determinants that define different degrees of line sensitivity to the photoperiod was acomplished using near-isogenic lines of the soft hexaploid wheat Triticum aestivum L. using SSR markers and markers specific to the Vrn and Ppd genes. It was established that the Ppd-s line contains a dominant Ppd-D1a allele located on chromosome 2D. This allele is characterized by a large deletion in the gene promoter region. For two other lines (Ppd-m and Ppd-w), introgression of the Ppd-B1 gene on chromosome 2B was detected from the parental Sonora variety, which is insensitive to the day length; however, the previously described Ppd-B1a.1 allele was not found. Another polymorphism that can cause weak photoperiodic sensitivity, an increased copy number of the Ppd-B1 gene, was detected for these lines.     

<Emphasis Type="Italic">CaVIL1</Emphasis>, a plant homeodomain gene that promotes flowering in pepper

Key message

CaVIL1 is a homolog of VIL1, a regulator of vernalization response in Arabidopsis and acts as a flowering promoter in pepper which does not respond to vernalization and photoperiod.

Abstract

As part of our goal to study the genetic and molecular basis of transition to flowering in pepper, we isolated the late-flowering mutant E-2698. Aside from late flowering, multiple pleiotropic alterations of the shoot structure, such as enlarged and distorted leaves, weak apical dominance, and reduced angle of the lateral branches were observed, indicating a broad role for the mutated gene in pepper development. Genetic mapping and sequence analyses revealed that the disrupted gene in E-2698 is the pepper homolog of VERNALIZATION INSENSITIVE 3-LIKE 1 (VIL1) that acts as a regulator of vernalization in Arabidopsis through chromatin modification. The pepper gene, CaVIL1, contains a plant homeodomain motif associated with chromatin modification and a VERNALIZATION INSENSITIVE 3-interacting domain that is truncated in E-2698 and in two other allelic mutants. Because pepper flowering does not respond to vernalization, we postulate that CaVIL1 regulates flowering time via chromatin modification of unknown targets. Expression analysis indicated that CaVIL1 activates the flowering promoter CaFLOWERING LOCUS T and represses the flowering repressor CaAPETALA2. Furthermore, CaVIL1 represses several genes from the FLOWERING LOCUS C (FLC)-LIKE clade that are clustered together in the pepper genome. This indicates their possible involvement in flowering regulation in this species. Our results show that CaVIL1 is a major regulator of flowering and interacts with other flowering promoters and repressors, as well as with FLC-LIKE genes whose function in flowering regulation is not yet known in pepper.

     

Taxonomic implications of the morphological and genetic variation of cultivated and domesticated populations of the <Emphasis Type="Italic">Agave angustifolia</Emphasis> complex (Agavoideae,Asparagaceae) in Oaxaca,Mexico

The Agave angustifolia complex, distributed from Mexico to Costa Rica, comprises four species and five varieties, including three species used for mescal production. The complex is represented in the Mexican state of Oaxaca by two wild taxa, A. angustifolia var. angustifolia and A. angustifolia var. rubescens, the cultivated form A. angustifolia “Espadín” and the partially cultivated species A. rodacantha. The aims of this study were to investigate the morphological and genetic variation of the A. angustifolia complex in the state of Oaxaca and to identify traits useful for taxonomic delimitation. Four wild and three cultivated populations of A. angustifolia from Oaxaca, one population of A. tequilana from Guanajuato and one population of A. angustifolia from Sonora were sampled for morphological, genetic and cytometric analyses. We showed that cultivated populations of A. angustifolia “Espadin,” A. rhodacantha and A. tequilana could be clearly differentiated from wild populations. Furthermore, the domesticated populations of A. angustifolia, known locally as “Espadin,” had a higher ploidy level and lower genetic variation than their related wild populations. The population of A. angustifolia from Sonora could be recognized as a different entity. Populations of A. rhodacantha need to be studied throughout their entire distribution area to further evaluate their taxonomic delimitation.     

Mutant Alleles of Photoperiod-1 in Wheat (Triticum aestivum L.) That Confer a Late Flowering Phenotype in Long Days

Flowering time in wheat and barley is known to be modified by mutations in the Photoperiod-1 (Ppd-1) gene. Semi-dominant Ppd-1a mutations conferring an early flowering phenotype are well documented in wheat but gene sequencing has also identified candidate loss of function mutations for Ppd-A1 and Ppd-D1. By analogy to the recessive ppd-H1 mutation in barley, loss of function mutations in wheat are predicted to delay flowering under long day conditions. To test this experimentally, introgression lines were developed in the spring wheat variety ‘Paragon’. Plants lacking a Ppd-B1 gene were identified from a gamma irradiated ‘Paragon’ population. These were crossed with the other introgression lines to generate plants with candidate loss of function mutations on one, two or three genomes.Lines lacking Ppd-B1 flowered 10 to 15 days later than controls under long days. Candidate loss of function Ppd-A1 alleles delayed flowering by 1 to 5 days while candidate loss of function Ppd-D1 alleles did not affect flowering time. Loss of Ppd-A1 gave an enhanced effect, and loss of Ppd-D1 became detectable in lines where Ppd-B1 was absent, indicating effects may be buffered by functional Ppd-1 alleles on other genomes. Expression analysis revealed that delayed flowering was associated with reduced expression of the TaFT1 gene and increased expression of TaCO1.A survey of the GEDIFLUX wheat collection grown in the UK and North Western Europe between the 1940s and 1980s and the A.E. Watkins global collection of landraces from the 1920s and 1930s showed that the identified candidate loss of function mutations for Ppd-D1 were common and widespread, while the identified candidate Ppd-A1 loss of function mutation was rare in countries around the Mediterranean and in the Far East but was common in North Western Europe. This may reflect a possible benefit of the latter in northern locations.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

High-throughput sequencing of <Emphasis Type="Italic">Prunus ferganensis</Emphasis> indicates that it is a geographical population of <Emphasis Type="Italic">P. persica</Emphasis>

Peach belongs to the genus Prunus, which includes Prunus persica and its relative species, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. Of these, P. ferganensis have been classified as a species, subspecies, or geographical population of P. persica. To explore the genetic difference between P. ferganensis and P. persica, high-throughput sequencing was used in different peach accessions belonging to different species. First, low-depth sequencing data of peach accessions belonging to four categories revealed that similarity between P. ferganensis and P. persica was similar to that between P. persica accessions from different geographical populations. Then, to further detect the genomic variation in P. ferganensis, the P. ferganensis accession “Xinjiang Pan Tao 1” and the P. persica accession “Xia Miao 1” were sequenced with high depth, and sequence reads were assembled. The results showed that the collinearity of “Xinjiang Pan Tao 1” with the reference genome “Lovell” was higher than that of “Xia Miao 1” and “Lovell” peach. Additionally, the number of genetic variants, including single nucleotide polymorphisms (SNPs), structural variations (SVs), and the specific genes annotated from unmapped sequence in “Xia Miao 1” was higher than that in “Xinjiang Pan Tao 1” peach. The data showed that there was a close distance between “Xinjiang Pan Tao 1” (P. ferganensis) and reference genome which belong to P. persica, comparing “Xia Miao 1” (P. persica) and reference ones. The results accompany with phylogenetic tree and structure analysis confirmed that P. ferganensis should be considered as a geographic population of P. persica rather than a subspecies or a distinct species. Furthermore, gene ontology analysis was performed using the gene comprising large-effect variation to understand the phenotypic difference between two accessions. The result revealed that the pathways of gene function affected by SVs but SNPs and insertion-deletions markedly differed between the two peach accessions.     

Genetic structure of diploid-polyploid complex of spined loach genus <Emphasis Type="Italic">Cobitis</Emphasis> (<Emphasis Type="Italic">Cypriniformes,Cobitidae</Emphasis>) in the Lower Danube

The extremely high diversity of spined loach biotypes in the Lower Danube has been detected by biochemical genetic investigation and cytometric analysis of 358 specimens collected in the riverbed and shallow channels. Along with two diploid species (C. elongatoides and C. “tanaitica”), six hybrid forms were revealed, namely, diploid C. elongatoides-“tanaitica”; triploid C. 2 elongatoides-“tanaitica,” C. elongatoides-2 “tanaitica,” and C. 2 elongatoides-species-1; and tetraploid C. 3 elongatoides-“tanaitica” and C. elongatoides-species-2-2 “tanaitica.” In addition, specimens with recombinant genotypes were also found. In spite of the apomictic mode of reproduction, the polyploids did not possess clonal structure, but according to the level of polymorphism and the genotype distribution, they were isomorphous to parental diploid species. Thus, in contrast to the polyploidy in Cobitids of the Dnieper, which have appeared in the basin due to the expansion, the polyploids of the Lower Danube are autochthonous and were derived by crossing with local diploid species. The process is apparently proceeds without any limitations.     

Developmental transcriptome analysis of floral transition in <Emphasis Type="Italic">Rosa odorata</Emphasis> var. <Emphasis Type="Italic">gigantea</Emphasis>

Key message

Expression analyses revealed that floral transition of Rosa odorata var. gigantea is mainly regulated by VRN1, COLs, DELLA and KSN, with contributions by the effects of phytohormone and starch metabolism.

Abstract

Seasonal plants utilize changing environmental and developmental cues to control the transition from vegetative growth to flowering at the correct time of year. This study investigated global gene expression profiles at different developmental stages of Rosa odorata var. gigantea by RNA-sequencing, combined with phenotypic characterization and physiological changes. Gene ontology enrichment analysis of the differentially expressed genes (DEGs) between four different developmental stages (vegetative meristem, pre-floral meristem, floral meristem and secondary axillary buds) indicated that DNA methylation and the light reaction played a large role in inducing the rose floral transition. The expression of SUF and FLC, which are known to play a role in delaying flowering until vernalization, was down-regulated from the vegetative to the pre-floral meristem stage. In contrast, the expression of VRN1, which promotes flowering by repressing FLC expression, increased. The expression of DELLA proteins, which function as central nodes in hormone signaling pathways, and probably involve interactions between GA, auxin, and ABA to promote the floral transition, was well correlated with the expression of floral integrators, such as AGL24, COL4. We also identified DEGs associated with starch metabolism correlated with SOC1, AGL15, SPL3, AGL24, respectively. Taken together, our results suggest that vernalization and photoperiod are prominent cues to induce the rose floral transition, and that DELLA proteins also act as key regulators. The results summarized in the study on the floral transition of the seasonal rose lay a foundation for further functional demonstration, and have profound economic and ornamental values.

     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.     

QTL-seq for rapid identification of candidate genes for flowering time in broccoli × cabbage

Key message

A major QTL controlling early flowering in broccoli × cabbage was identified by marker analysis and next-generation sequencing, corresponding to GRF6 gene conditioning flowering time in Arabidopsis.

Abstract

Flowering is an important agronomic trait for hybrid production in broccoli and cabbage, but the genetic mechanism underlying this process is unknown. In this study, segregation analysis with BC₁P1, BC₁P2, F₂, and F_2:3 populations derived from a cross between two inbred lines “195” (late-flowering) and “93219” (early flowering) suggested that flowering time is a quantitative trait. Next, employing a next-generation sequencing-based whole-genome QTL-seq strategy, we identified a major genomic region harboring a robust flowering time QTL using an F₂ mapping population, designated Ef2.1 on cabbage chromosome 2 for early flowering. Ef2.1 was further validated by indel (insertion or deletion) marker-based classical QTL mapping, explaining 51.5% (LOD = 37.67) and 54.0% (LOD = 40.5) of the phenotypic variation in F₂ and F_2:3 populations, respectively. Combined QTL-seq and classical QTL analysis narrowed down Ef1.1 to a 228-kb genomic region containing 29 genes. A cabbage gene, Bol024659, was identified in this region, which is a homolog of GRF6, a major gene regulating flowering in Arabidopsis, and was designated BolGRF6. qRT-PCR study of the expression level of BolGRF6 revealed significantly higher expression in the early flowering genotypes. Taken together, our results provide support for BolGRF6 as a possible candidate gene for early flowering in the broccoli line 93219. The identified candidate genomic regions and genes may be useful for molecular breeding to improve broccoli and cabbage flowering times.

     

Identification and fine mapping of a stay-green gene (<Emphasis Type="Italic">Brnye1</Emphasis>) in pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> L. ssp. <Emphasis Type="Italic">chinensis</Emphasis>)

Key message

Using bulked segregant analysis combined with next-generation sequencing, we delimited the Brnye1 gene responsible for the stay-green trait of nye in pakchoi. Sequence analysis identified Bra019346 as the candidate gene.

Abstract

“Stay-green” refers to a plant trait whereby leaves remain green during senescence. This trait is useful in the cultivation of pakchoi (Brassica campestris L. ssp. chinensis), which is marketed as a green leaf product. This study aimed to identify the gene responsible for the stay-green trait in pakchoi. We identified a stay-green mutant in pakchoi, which we termed “nye”. Genetic analysis revealed that the stay-green trait is controlled by a single recessive gene, Brnye1. Using the BSA-seq method, a 3.0-Mb candidate region was mapped on chromosome A03, which helped us localize Brnye1 to an 81.01-kb interval between SSR markers SSRWN27 and SSRWN30 via linkage analysis in an F₂ population. We identified 12 genes in this region, 11 of which were annotated based on the Brassica rapa annotation database, and one was a functionally unknown gene. An orthologous gene of the Arabidopsis gene AtNYE1, Bra019346, was identified as the potential candidate for Brnye1. Sequence analysis revealed a 40-bp insertion in the second exon of Bra019346 in nye, which generated the TAA stop codon. A candidate gene-specific Indel marker in 1561 F₂ individuals showed perfect cosegregation with Brnye1 in the nye mutant. These results provide a foundation for uncovering the molecular mechanism of the stay-green trait in pakchoi.

     

<Emphasis Type="Italic">SOC1</Emphasis> and <Emphasis Type="Italic">AGL24</Emphasis> interact with AGL18-1, not the other family members AGL18-2 and AGL18-3 in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Many MCM1-AGAMOUS-DEFICIENS-SRF (MADS) genes have been proved to play an important role in the flowering time regulation of plants. The flowering-inhibiting factor AGAMOUS-LIKE 18 (AGL18) integrates into the two flowering-activating factors SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24), which play an important role during the plant developmental stages of the flowering pathway. However, it remains unknown whether and how the AGL18 protein directly interacts with SOC1 and/or AGL24 genes to regulate flowering time in Brassica juncea. In this study, three members (AGL18-1 in florescence, AGL18-2 and AGL18-3 in young seedlings) of the AGL18 family, and SOC1 and AGL24 in florescence were cloned in Brassica juncea. Yeast One-Hybrid assays and Dual-Glo^® Luciferase assays showed that the SOC1 and AGL24 promoters interacted only with AGL18-1 protein, not AGL18-2 and AGL18-3. The typical conserved structure of the M-domain of AGL18-1 was the key region that mediated the interaction between the AGL18-1 protein and SOC1 promoter, and the I-domain, K-domain and C-domain did not regulate the interaction of AGL18-1/SOC1. In contrast, the K-domain and M-domain in AGL18-1 could mediate the interaction between the AGL18-1 protein and AGL24 promoter. This indicated that the AGL18-1 protein must have its unique functions that differed from AGL18-2 and AGL18-3. This work provides valuable information for in-depth studies into the molecular mechanisms of the AGL18 protein with SOC1 and AGL24 for flowering time control of Brassica juncea.