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.

Tracing Heterozygosity in the Vvlexp1 Locus in Grapevine by Sequencing and High-resolution Melt Analysis

Multiple loci in a continuously asexually reproducing genome such as vegetatively propagated grapevine (Vitis vinifera) can be heterozygote. The methodology to analyze heterozygous loci is manifold ranging from traditional breeding and studying segregating offspring, codominant marker analyses to whole sequence analysis. Results of heterozygosity studies on challenging loci need to be carefully confirmed to ensure accuracy and avoid misinterpretation. One of these methods is high-resolution melt (HRM) analysis in combination with sequencing and segregation analysis. We present first the adoption of HRM analyses for grapevine and its potential to confirm heterozygotic markers with low or no sequence size differences.

Development of Molecular Markers Linked to Cladosporium fulvum Resistant Gene Cf-6 in Tomato by RAPD and SSR Methods

Leaf mold, caused by the fungus Cladosporium fulvum, is a serious disease of tomato. In the current study, the main physiological races of C. fulvum collected from three northeastern provinces of China were identified using a set of identification hosts. The results showed that the prevalent pathogenic physiological races were 1.2.3, 1.3, 3, 1.2.3.4, and 1.2.4. F1, F2, and BC1 tomato plants were obtained by crossing C. fulvum-resistant cultivar 03748 carrying the Cf-6 gene and susceptible cultivar 03036. Three 10-mer oligonucleotide random amplified polymorphic DNA (RAPD) primers and two simple sequence repeat (SSR) primers were selected for the further molecular marking analysis after 210 RAPD primers and 50 SSR primers were screened using the bulked segregate analysis method. The polymorphic DNA bands were amplified among parents, 10 F1 plants, 184 F2 plants including 145 resistant plants and 39 sensitive plants using three RAPD primers and two SSR primers so that three RAPD molecular markers and two SSR molecular markers linked to the Cf-6 loci were identified. Three RAPD markers were linked to the Cf-6 resistant locus separated with 8.7 cM, 20.3 cM, and 33.4 cM. Also, one RAPD codominant marker S374619/559 was found. The locations of the two SSR markers were 12.6 cM and 9.7 cM away from the Cf-6 locus. After cloning and sequencing two specific DNA fragments closely connected to the Cf-6 resistant and susceptible alleles respectively, in the RAPD codominant marker S374619/559 and one codominant sequence characterized amplified region marker S674619/559 was converted from RAPD marker S374619/559. In the RAPD marker S374619/559, the length difference of two specific fragments, 619-bp fragment and 559-bp fragment, is the result of one insertion (60 bp) in the 619-bp fragment. These markers will facilitate the selection of resistant tomato germplasm containing the Cf-6 gene and cloning of Cf-6 to breed new C. fulvum resistant tomato cultivars.

A Codominant Randomly Amplified Polymorphic DNA (RAPD) Marker Useful for Indirect Selection of Bean Golden Mosaic Virus Resistance in Common Bean

Bean golden mosaic virus (BGMV) is a devastating disease of common bean (Phaseolus vulgaris L.) in tropical America. The disease is effectively controlled by combinations of genetic resistances. The most widely deployed source of resistance to BGMV is a recessive gene (bgm-1) derived from the dry bean landrace cultivar Garrapato (Mexico) that conditions a nonmosaic partial resistance response to the pathogen. To expedite introgression of partial resistance into snap bean for southern Florida and other susceptible dry bean market classes for the Caribbean and Central American regions, a RAPD marker tightly linked to bgm-1 has been identified. Two contrasting DNA bulks, one consisting of five BGMV-resistant and the other five susceptible F6 recombinant inbred lines, were used to screen for polymorphic fragments amplified by 300 decamer primers in the polymerase chain reaction. RAPDs generated between the bulks were analyzed across F2 populations segregating for the marker and the gene. One codominant RAPD marker (R2570/530) tightly linked to the recessive resistance gene bgm-1 was found. The 530-base pair (bp) fragment was linked in repulsion with bgm-1 and the other 570-bp fragment was linked in coupling. No recombinants between R2570/530 and bgm-1 were observed among 91 F2 progeny from one dry bean population, and there were two recombinants (4.2 cM) observed among 48 F2 progeny combined across four snap bean populations. Assays of R2570/530 across susceptible germplasm and lines likely to have the `Garrapato'-derived partial resistance to BGMV have revealed that the codominant marker is gene-pool nonspecific and maintains its original linkage orientation with the recessive bgm-1 gene through numerous meioses. The codominant marker is useful for rapidly introgressing partial resistance to BGMV into susceptible germplasm.