Mapping QTL for vernalization requirement identified adaptive divergence of the candidate gene Flowering Locus C in polyploid Camelina sativa.

Vernalization requirement is an integral component of flowering in winter-type plants. The availability of winter ecotypes among Camelina species facilitated the mapping of quantitative trait loci (QTL) for vernalization requirement in Camelina sativa. An inter and intraspecific crossing scheme between related Camelina species, where one spring and two different sources of winter-type habit were used, resulted in the development of two segregating populations. Linkage maps generated with sequence-based markers identified three QTLs associated with vernalization requirement in C. sativa; two from the interspecific (chromosomes 13 and 20) and one from the intraspecific cross (chromosome 8). Notably, the three loci were mapped to different homologous regions of the hexaploid C. sativa genome. All three QTLs were found in proximity to Flowering Locus C (FLC), variants of which have been reported to affect the vernalization requirement in plants. Temporal transcriptome analysis for winter-type Camelina alyssum demonstrated reduction in expression of FLC on chromosomes 13 and 20 during cold treatment, which would trigger flowering, since FLC would be expected to suppress floral initiation. FLC on chromosome 8 also showed reduced expression in the C. sativa ssp. pilosa winter parent upon cold treatment, but was expressed at very high levels across all time points in the spring-type C. sativa. The chromosome 8 copy carried a deletion in the spring-type line, which could impact its functionality. Contrary to previous reports, all three FLC loci can contribute to controlling the vernalization response in C. sativa and provide opportunities for manipulating this requirement in the crop.

Deep neural networks for genomic prediction do not estimate marker effects.

Genomic prediction is a promising technology for advancing both plant and animal breeding, with many different prediction models evaluated in the literature. It has been suggested that the ability of powerful nonlinear models, such as deep neural networks, to capture complex epistatic effects between markers offers advantages for genomic prediction. However, these methods tend not to outperform classical linear methods, leaving it an open question why this capacity to model nonlinear effects does not seem to result in better predictive capability. In this work, we propose the theory that, because of a previously described principle called shortcut learning, deep neural networks tend to base their predictions on overall genetic relatedness rather than on the effects of particular markers such as epistatic effects. Using several datasets of crop plants [lentil (Lens culinaris Medik.), wheat (Triticum aestivum L.), and Brassica carinata A. Braun], we demonstrate the network's indifference to the values of the markers by showing that the same network, provided with only the locations of matches between markers for two individuals, is able to perform prediction to the same level of accuracy.

Development of B. carinata with super-high erucic acid content through interspecific hybridization.

KEY MESSAGE: Disomic alien chromosome addition Brassica carinata lines with super-high erucic acid content were developed through interspecific hybridization with B. juncea and characterized using molecular, cytological and biochemical techniques. Brassica carinata [A.] Braun (BBCC, 2n = 34) is a climate-resilient oilseed. Its seed oil is high in erucic acid (> 40%), rendering it well suited for the production of biofuel and other bio-based applications. To enhance the competitiveness of B. carinata with high erucic B. napus (HEAR), lines with super-high erucic acid content were developed through interspecific hybridization. To this end, a fad2B null allele from Brassica juncea (AABB, 2n = 36) was introgressed into B. carinata, resulting in a B. carinata fad2B mutant with erucic acid levels of over 50%. Subsequently, the FAE allele from B. rapa spp. yellow sarson (AA, 2n = 20) was transferred to the fad2B B. carinata line, yielding lines with erucic acid contents of up to 57.9%. Molecular analysis using the Brassica 90 K Illumina Infinium™ SNP genotyping array identified these lines as disomic alien chromosome addition lines, with two extra A08 chromosomes containing the BrFAE gene. The alien chromosomes from B. rapa were clearly distinguished by molecular cytogenetics in one of the addition lines. Analysis of microspore-derived offspring and hybrids from crosses with a CMS B. carinata line showed that the transfer rate of the A08 chromosome into male gametes was over 98%, resulting in almost completely stable transmission of an A08 chromosome copy into the progeny. The increase in erucic acid levels was accompanied by changes in the proportions of other fatty acids depending on the genetic changes that were introduced in the interspecific hybrids, providing valuable insights into erucic acid metabolism in Brassica.

Protocol for the Production of Doubled Haploid Plants of Brassica carinata.

Brassica carinata, also known as Ethiopian or Abyssinian mustard, is a drought- and heat-tolerant oilseed with great potential as a dedicated industrial feedstock crop for use in biofuel and other bio-based applications. Doubled haploid technology, a system that allows for the rapid development of doubled haploid, completely homozygous plants through microspore embryogenesis, has been applied routinely in both B. carinata breeding and basic research. Here, we present a comprehensive isolated microspore culture protocol detailing the various steps involved in doubled haploid plant production for this species, from growing donor plants over harvesting flower buds and isolating, culturing and inducing microspores to regenerating doubled haploid embryos and plantlets.

Camelina as a sustainable oilseed crop: contributions of plant breeding and genetic engineering.

Camelina is an underutilized Brassicaceae oilseed plant with a considerable agronomic potential for biofuel and vegetable oil production in temperate regions. In contrast to most Brassicaceae, camelina is resistant to alternaria black spot and other diseases and pests. Sequencing of the camelina genome revealed an undifferentiated allohexaploid genome with a comparatively large number of genes and low percentage of repetitive DNA. As there is a close relationship between camelina and the genetic model plant Arabidopsis, this review aims at exploring the potential of translating basic Arabidopsis results into a camelina oilseed crop for food and non-food applications. Recently, Arabidopsis genes for drought resistance or increased photosynthesis and overall productivity have successfully been expressed in camelina. In addition, gene constructs affecting lipid metabolism pathways have been engineered into camelina for synthesizing either long-chain polyunsaturated fatty acids, hydroxy fatty acids or high-oleic oils in particular camelina strains, which is of great interest in human food, industrial or biofuel applications, respectively. These results confirm the potential of camelina to serve as a biotechnology platform in biorefinery applications thus justifying further investment in breeding and genetic research for combining agronomic potential, unique oil quality features and biosafety into an agricultural production system.

Single-nucleotide polymorphism identification and genotyping in Camelina sativa.

Camelina sativa, a largely relict crop, has recently returned to interest due to its potential as an industrial oilseed. Molecular markers are key tools that will allow C. sativa to benefit from modern breeding approaches. Two complementary methodologies, capture of 3' cDNA tags and genomic reduced-representation libraries, both of which exploited second generation sequencing platforms, were used to develop a low density (768) Illumina GoldenGate single nucleotide polymorphism (SNP) array. The array allowed 533 SNP loci to be genetically mapped in a recombinant inbred population of C. sativa. Alignment of the SNP loci to the C. sativa genome identified the underlying sequenced regions that would delimit potential candidate genes in any mapping project. In addition, the SNP array was used to assess genetic variation among a collection of 175 accessions of C. sativa, identifying two sub-populations, yet low overall gene diversity. The SNP loci will provide useful tools for future crop improvement of C. sativa.

Monolignol biosynthesis is associated with resistance to Sclerotinia sclerotiorum in Camelina sativa.

The ascomycete Sclerotinia sclerotiorum is a necrotrophic plant pathogen with an extremely broad host range. It causes stem rot in Camelina sativa, a crucifer with great potential as an alternative oilseed crop. Lignification is a common phenomenon in the expression of resistance against necrotrophs, but the molecular mechanisms underlying this defence response are poorly understood. We present histochemical, gene expression and biochemical data investigating the role of monolignols in the resistance of C. sativa to S. sclerotiorum. Comparative studies with resistant and susceptible lines of C. sativa revealed substantial differences in constitutive transcript levels and gene regulation patterns for members of the gene family encoding cinnamoyl-CoA reductase (CCR), the first enzyme specifically committed to the synthesis of lignin monomers. These differences were associated with anatomical and metabolic factors. While the induction of CsCCR2 expression after inoculation with S. sclerotiorum was associated with the deposition of lignin mainly derived from guaiacyl monomers, high constitutive levels of CsCCR4 paralleled a high syringyl lignin content in healthy stems of resistant plants. The results provide evidence that plant cell wall strengthening plays a role in the resistance of C. sativa to S. sclerotiorum, and that both constitutive and inducible defence mechanisms contribute to reduced symptom development in resistant germplasm. This study provides the first characterization of quantitative resistance in C. sativa to S. sclerotiorum.