Extensive allele mining discovers novel genetic diversity in the loci controlling frost tolerance in barley

Key message Exome sequencing-based allele mining for frost tolerance suggests HvCBF14 rather than CNV at Fr-H2 locus is the main responsible of frost tolerance in barley. Abstract Wild relatives, landraces and old cultivars of barley represent a reservoir of untapped and potentially important genes for crop improvement, and the recent sequencing technologies provide the opportunity to mine the existing genetic diversity and to identify new genes/alleles for the traits of interest. In the present study, we use frost tolerance and vernalization requirement as case studies to demonstrate the power of allele mining carried out on exome sequencing data generated from > 400 barley accessions. New deletions in the first intron of VRN-H1 were identified and linked to a reduced vernalization requirement, while the allelic diversity of HvCBF2a, HvCBF4b and HvCBF14 was investigated by combining the analysis of SNPs and read counts. This approach has proven very effective to identify gene paralogs and copy number variants of HvCBF2 and the HvCBF4b-HvCBF2a segment. A multiple linear regression model which considers allelic variation at these genes suggests a major involvement of HvCBF14, rather than copy number variation of HvCBF4b-HvCBF2a, in controlling frost tolerance in barley. Overall, the present study provides powerful resource and tools to discover novel alleles at relevant genes in barley.

Non-vernalization requirement in Chinese kale caused by loss of BoFLC and low expressions of its paralogs

Key message We identified the loss ofBoFLC gene as the cause of non-vernalization requirement inB. oleracea. Our developed codominant marker ofBoFLCgene can be used for breeding program ofB. oleraceacrops. Abstract Many species of the Brassicaceae family, including some Brassica crops, require vernalization to avoid pre-winter flowering. Vernalization is an unfavorable trait for Chinese kale (Brassica oleracea var. chinensis Lei), a stem vegetable, and therefore it has been lost during its domestication/breeding process. To reveal the genetics of vernalization variation, we constructed an F2 population through crossing a Chinese kale (a non-vernalization crop) with a kale (a vernalization crop). Using bulked segregant analysis (BSA) and RNA-seq, we identified one major quantitative trait locus (QTL) controlling vernalization and fine-mapped it to a region spanning 80 kb. Synteny analysis and PCR-based sequencing results revealed that compared to that of the kale parent, the candidate region of the Chinese kale parent lost a 9,325-bp fragment containing FLC homolog (BoFLC). In addition to the BoFLC gene, there are four other FLC homologs in the genome of B. oleracea, including Bo3g005470, Bo3g024250, Bo9g173370, and Bo9g173400. The qPCR analysis showed that the BoFLC had the highest expression among the five members of the FLC family. Considering the low expression levels of the four paralogs of BoFLC, we speculate that its paralogs cannot compensate the function of the lost BoFLC, therefore the presence/absence (PA) polymorphism of BoFLC determines the vernalization variation. Based on the PA polymorphism of BoFLC, we designed a codominant marker for the vernalization trait, which can be used for breeding programs of B. oleracea crops.

Initial developmental rate delay in modern highly productive cultivars of winter bread wheat from West Europe

Aim. To evaluate the vernalization requirement duration and sensitivity to photoperiod in the set of modern high yielding winter bread wheat cultivars from West Europe which are proposed for production in Ukraine. Methods. Planting in natural and shortened photoperiods followed by artificial vernalizing variants of 5-days green seedlings have been used for heading dates registration. Results. Many foreign cultivars revealed the durable vernalization requirement and strong photosensitivity. Few stocks were characterized by the week analogous physiological reactions, which are typical in most modern Ukrainian cultivars and breeding lines. Conclusions. Reveled data confirmed the adaptive value of discussed properties for specific environment which are similar to Ukrainian one in many zones of winter wheat cultivation. And they refute the assertion that strong photosensitivity and durable vernalization requirement limit the improvement of yield potential by selection. Keywords: winter bread wheat, vernalization requirement, photosensitivity, modern cultivars from West Europe.

Features of modern winter wheat varieties in terms of winter hardiness components under conditions of Ukrainian Forest-Steppe

In recent years, there has been a significant change in climatic conditions affecting the cultivation and yield of winter wheat. Therefore, the creation of wheat varieties with high adaptive potential is one of the main tasks of modern breeding. A significant component of the overall adaptive potential of winter wheat is winter hardiness, which is determined by a set of characters enabling plants to overwinter. To a large extent, winter hardiness is determined by gene systems that control vernalization requirement duration, photoperiod reaction, and frost resistance. The research is aimed at determining the features of modern winter wheat varieties developed at the V. M. Remeslo Myronivka Institute of Wheat of the National Academy of Agrarian Sciences of Ukraine in terms of winter hardiness components and adaptive potential in the environment of the Central part of the Ukrainian Forest-Steppe. Winter bread wheat varieties Estafeta myronivska, Hratsiia myronivska, MIP Assol, and Balada myronivska were studied. They also were crossed on incomplete diallele scheme with three near-isogenic lines derived from Erythrospermum 604 with different alleles of Vrd genes 1) Vrd1Vrd1vrd2vrd2, 2) vrd1vrd1Vrd2Vrd2, and 3) vrd1vrd1vrd2vrd2. It was established that vernalization requirement duration in the varieties Estafeta myronivska and Balada myronivska was short whereas in the varieties Hratsiia myronivska and MIP Assol it was medium. All the varieties studied have medium photoperiod sensitivity. The results of the hybridological analysis indicate the absence of the Vrd1 and Vrd2 genes in the varieties. Frost tolerance of these varieties is at the same level and higher than in the highly tolerant to the low temperatures variety Myronivska 808. Thus, the results indicate the possibility of recombining different levels of expression of these traits in genotypes by breeding efforts. This has great practical importance in farming, because in recent years the areas of crops harvested late (corn, sunflower, etc.) in the production conditions has significantly increased. It causes a shift in sowing dates of winter wheat to a later period. In this case, varieties Estafeta myronivska, Hratsiia myronivska, MIP Assol, and Balada myronivska are able to undergo sufficient hardening, to satisfy the vernalization requirement, and to form a high level of winter hardiness. Their relatively medium photoperiod sensitivity allows vegetation to be restored a little earlier in the spring and winter reserves of moisture to be used more effectively.

MiR396 is involved in plant response to vernalization and flower development in Agrostis stolonifera

MicroRNA396 (miR396) has been demonstrated to regulate flower development by targeting growth-regulating factors (GRFs) in annual species. However, its role in perennial grasses and its potential involvement in flowering time control remain unexplored. Here we report that overexpression of miR396 in a perennial species, creeping bentgrass (Agrostis stolonifera L.), alters flower development. Most significantly, transgenic (TG) plants bypass the vernalization requirement for flowering. Gene expression analysis reveals that miR396 is induced by long-day (LD) photoperiod and vernalization. Further study identifies VRN1, VRN2, and VRN3 homologs whose expression patterns in wild-type (WT) plants are similar to those observed in wheat and barley during transition from short-day (SD) to LD, and SD to cold conditions. However, compared to WT controls, TG plants overexpressing miR396 exhibit significantly enhanced VRN1 and VRN3 expression, but repressed VRN2 expression under SD to LD conditions without vernalization, which might be associated with modified expression of methyltransferase genes. Collectively, our results unveil a potentially novel mechanism by which miR396 suppresses the vernalization requirement for flowering which might be related to the epigenetic regulation of VRN genes and provide important new insight into critical roles of a miRNA in regulating vernalization-mediated transition from vegetative to reproductive growth in monocots.

The role of FRIGIDA and FLOWERING LOCUS C genes in flowering time of Brassica rapa leafy vegetables

There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower.