Complex Horticultural Quality Traits in Broccoli Are Illuminated by Evaluation of the Immortal BolTBDH Mapping Population.

Improving horticultural quality in regionally adapted broccoli (Brassica oleracea var. italica) and other B. oleracea crops is challenging due to complex genetic control of traits affecting morphology, development, and yield. Mapping horticultural quality traits to genomic loci is an essential step in these improvement efforts. Understanding the mechanisms underlying horticultural quality enables multi-trait marker-assisted selection for improved, resilient, and regionally adapted B. oleracea germplasm. The publicly-available biparental double-haploid BolTBDH mapping population (Chinese kale × broccoli; N = 175) was evaluated for 25 horticultural traits in six trait classes (architecture, biomass, phenology, leaf morphology, floral morphology, and head quality) by multiple quantitative trait loci mapping using 1,881 genotype-by-sequencing derived single nucleotide polymorphisms. The physical locations of 56 single and 41 epistatic quantitative trait locus (QTL) were identified. Four head quality QTL (OQ_C03@57.0, OQ_C04@33.3, OQ_CC08@25.5, and OQ_C09@49.7) explain a cumulative 81.9% of phenotypic variance in the broccoli heading phenotype, contain the FLOWERING LOCUS C (FLC) homologs Bo9g173400 and Bo9g173370, and exhibit epistatic effects. Three key genomic hotspots associated with pleiotropic control of the broccoli heading phenotype were identified. One phenology hotspot reduces days to flowering by 7.0 days and includes an additional FLC homolog Bo3g024250 that does not exhibit epistatic effects with the three horticultural quality hotspots. Strong candidates for other horticultural traits were identified: BoLMI1 (Bo3g002560) associated with serrated leaf margins and leaf apex shape, BoCCD4 (Bo3g158650) implicated in flower color, and BoAP2 (Bo1g004960) implicated in the hooked sepal horticultural trait. The BolTBDH population provides a framework for B. oleracea improvement by targeting key genomic loci contributing to high horticultural quality broccoli and enabling de novo mapping of currently unexplored traits.

Quantitative trait loci mapping of heat tolerance in broccoli (Brassica oleracea var. italica) using genotyping-by-sequencing.

KEY MESSAGE: Five quantitative trait loci and one epistatic interaction were associated with heat tolerance in a doubled haploid population of broccoli evaluated in three summer field trials. Predicted rising global temperatures due to climate change have generated a demand for crops that are resistant to yield and quality losses from heat stress. Broccoli (Brassica oleracea var. italica) is a cool weather crop with high temperatures during production decreasing both head quality and yield. Breeding for heat tolerance in broccoli has potential to both expand viable production areas and extend the growing season but breeding efficiency is constrained by limited genetic information. A doubled haploid (DH) broccoli population segregating for heat tolerance was evaluated for head quality in three summer fields in Charleston, SC, USA. Multiple quantitative trait loci (QTL) mapping of 1,423 single nucleotide polymorphisms developed through genotyping-by-sequencing identified five QTL and one positive epistatic interaction that explained 62.1% of variation in heat tolerance. The QTL identified here can be used to develop markers for marker-assisted selection and to increase our understanding of the molecular mechanisms underlying plant response to heat stress.

A GBS-SNP-based linkage map and quantitative trait loci (QTL) associated with resistance to Fusarium oxysporum f. sp. niveum race 2 identified in Citrullus lanatus var. citroides.

KEY MESSAGE: A major QTL for resistance to Fusarium oxysporum f. sp. niveum race 2 was mapped to a narrow 1.2 Mb interval using a high-density GBS-SNP linkage map, the first map of Citrullus lanatus var. citroides. Fusarium wilt, a fungal disease caused by Fusarium oxysporum f. sp. niveum (Fon), devastates watermelon crop production worldwide. Several races, which are differentiated by host range, of the pathogen exist. Resistance to Fon race 2, a particularly virulent strain prevalent in the United States, does not exist in edible cultivars of the sweet cultivated watermelon Citrullus lanatus var. lanatus (Cll) and has been well described in a few plant introductions of the wild subspecies of watermelon, C. lanatus var. citroides (Clc). Clc provides a vital source of genetic diversity, as well as resistance to numerous diseases. Unfortunately, both genetic diversity and disease resistance are lacking in Cll due to the narrow genetic base. Despite the importance of Clc to continued watermelon improvement, intra-variety genetic studies are lacking. Here, we present the first Clc genetic linkage map, generated with 2495 single nucleotide polymorphisms developed through genotyping-by-sequencing, and use it to identify quantitative trait loci associated with Fon race 2 resistance. Multiple QTL mapping in a Clc F2:3 population (N = 173) identified one major and four minor QTL. The major QTL explained 43% of the variation in Fon race 2 resistance and was delimited to a 1.2-Mb interval on chromosome 9, a region spanning 44 genes.

Suppression of Bacterial Blight on Mustard Greens with Host Plant Resistance and Acibenzolar-S-Methyl.

Bacterial blight, caused by Pseudomonas cannabina pv. alisalensis, attacks the leaves of most brassica vegetables, including mustard greens (Brassica juncea). 'Carolina Broadleaf,' a new mustard cultivar, is resistant to bacterial blight, whereas 'Florida Broadleaf,' a commonly grown cultivar, is susceptible. Acibenzolar-S-methyl (trade name Actigard) has been used to manage bacterial diseases caused by P. syringae on a variety of crops. The objective of this study was to evaluate host plant resistance and acibenzolar-S-methyl alone and in combination to manage bacterial blight. Three field experiments were done in spring and fall 2011 and fall 2014. In each experiment, acibenzolar-S-methyl was applied twice as a foliar spray, once before and once after plants were inoculated. Severity of bacterial blight was 81% less on nontreated Carolina Broadleaf than on nontreated Florida Broadleaf (P ≤ 0.0003). Acibenzolar-S-methyl applications reduced severity of bacterial blight by 55% compared with the water control treatment on susceptible Florida Broadleaf. Mean weight of diseased leaves, averaged across acibenzolar-S-methyl treatments, was 53% less with Carolina Broadleaf than with Florida Broadleaf (P < 0.0001). However, acibenzolar-S-methyl applied at the recommended rate (14.2 g/ha) significantly injured leaves of Carolina Broadleaf in two experiments and injured leaves of Florida Broadleaf in one experiment. Overall, host plant resistance was more effective than acibenzolar-S-methyl for managing bacterial blight on mustard greens.

Genetic Diversity and Population Structure of Collard Landraces and their Relationship to Other Brassica oleracea Crops.

Landraces have the potential to provide a reservoir of genetic diversity for crop improvement to combat the genetic erosion of the food supply. A landrace collection of the vitamin-rich specialty crop collard (Brassica oleracea L. var. viridis) was genetically characterized to assess its potential for improving the diverse crop varieties of B. oleracea. We used the Illumina 60K Brassica SNP BeadChip array with 52,157 single nucleotide polymorphisms (SNPs) to (i) clarify the relationship of collard to the most economically important B. oleracea crop types, (ii) evaluate genetic diversity and population structure of 75 collard landraces, and (iii) assess the potential of the collection for genome-wide association studies (GWAS) through characterization of genomic patterns of linkage disequilibrium. Confirming the collection as a valuable genetic resource, the collard landraces had twice the polymorphic markers (11,322 SNPs) and 10 times the variety-specific alleles (521 alleles) of the remaining crop types examined in this study. On average, linkage disequilibrium decayed to background levels within 600 kilobase (kb), allowing for sufficient coverage of the genome for GWAS using the physical positions of the 8273 SNPs polymorphic among the landraces. Although other relationships varied, the previous placement of collard with the cabbage family was confirmed through phylogenetic analysis and principal coordinates analysis (PCoA).

Impact of thermal processing on sulforaphane yield from broccoli ( Brassica oleracea L. ssp. italica).

In broccoli, sulforaphane forms when the glucosinolate glucoraphanin is hydrolyzed by the endogenous plant thiohydrolase myrosinase. A myrosinase cofactor directs hydrolysis away from the formation of bioactive sulforaphane and toward an inactive product, sulforaphane nitrile. The cofactor is more heat sensitive than myrosinase, presenting an opportunity to preferentially direct hydrolysis toward sulforaphane formation through regulation of thermal processing. Four broccoli cultivars were microwave heated, boiled, or steamed for various lengths of time. Production of nitrile during hydrolysis of unheated broccoli varied among cultivars from 91 to 52% of hydrolysis products (Pinnacle > Marathon > Patriot > Brigadier). Boiling and microwave heating caused an initial loss of nitrile, with a concomitant increase in sulforaphane, followed by loss of sulforaphane, all within 1 min. In contrast, steaming enhanced sulforaphane yield between 1.0 and 3.0 min in all but Brigadier. These data are proof of concept that steaming for 1.0-3.0 min provides less nitrile and more sulforaphane yield from a broccoli meal.

Development of public immortal mapping populations, molecular markers and linkage maps for rapid cycling Brassica rapa and B. oleracea.

Publicly available genomic tools help researchers integrate information and make new discoveries. In this paper, we describe the development of immortal mapping populations of rapid cycling, self-compatible lines, molecular markers, and linkage maps for Brassica rapa and B. oleracea and make the data and germplasm available to the Brassica research community. The B. rapa population consists of 160 recombinant inbred (RI) lines derived from the cross of highly inbred lines of rapid cycling and yellow sarson B. rapa. The B. oleracea population consists of 155 double haploid (DH) lines derived from an F1 cross between two DH lines, rapid cycling and broccoli. A total of 120 RFLP probes, 146 SSR markers, and one phenotypic trait (flower color) were used to construct genetic linkage maps for both species. The B. rapa map consists of 224 molecular markers distributed along 10 linkage groups (A1-A10) with a total distance of 1125.3 cM and a marker density of 5.7 cM/marker. The B. oleracea genetic map consists of 279 molecular markers and one phenotypic marker distributed along nine linkage groups (C1-C9) with a total distance of 891.4 cM and a marker density of 3.2 cM/marker. A syntenic analysis with Arabidopsis thaliana identified collinear genomic blocks that are in agreement with previous studies, reinforcing the idea of conserved chromosomal regions across the Brassicaceae.

High-density Brassica oleracea linkage map: identification of useful new linkages.

We constructed a 1,257-marker, high-density genetic map of Brassica oleracea spanning 703 cM in nine linkage groups, designated LG1-LG9. It was developed in an F2 segregating population of 143 individuals obtained by crossing double haploid plants of broccoli "Early-Big" and cauliflower "An-Nan Early". These markers are randomly distributed throughout the map, which includes a total of 1,062 genomic SRAP markers, 155 cDNA SRAP markers, 26 SSR markers, 3 broccoli BAC end sequences and 11 known Brassica genes: BoGSL-ALK, BoGSL-ELONG, BoGSL-PROa, BoGSL-PROb, BoCS-lyase, BoGS-OH, BoCYP79F1, BoS-GT (glucosinolate pathway), BoDM1 (resistance to downy mildew), BoCALa, BoAP1a (inflorescence architecture). BoDM1 and BoGSL-ELONG are linked on LG 2 at 0.8 cM, making it possible to use the glucosinolate gene as a marker for the disease resistance gene. By QTL analysis, we found three segments involved in curd formation in cauliflower. The map was aligned to the C genome linkage groups and chromosomes of B. oleracea and B. napus, and anchored to the physical map of A. thaliana. This map adds over 1,000 new markers to Brassica molecular tools.

Heterosis for horticultural traits in broccoli.

Over the last three decades, broccoli (Brassica oleracea L., Italica Group) hybrids made by crossing two inbred lines replaced open-pollinated populations to become the predominant type of cultivar. The change to hybrids evolved with little or no understanding of heterosis or hybrid vigor in this crop. Therefore, the purpose of the present study was to determine levels of heterosis expressed by a set of hybrids derived by crossing relatively elite, modern inbreds (n = 9). An additional objective was to determine if PCR-based marker derived genetic similarities among the parents can be useful to predict heterosis in this crop. Thirty-six hybrids derived from a diallel mating design involving nine parents were evaluated for five horticultural characters including the head characteristics of head weight, head stem diameter, and maturity (e.g., days from transplant to harvest), and the plant vigor characteristics of plant height, and plant width in four environments. A total of 409 polymorphic markers were generated by 24 AFLP, 23 SRAP and 17 SSR primer combinations. Euclidean distances between parents were determined based on phenotypic traits. About half of the hybrids exhibited highparent heterosis for head weight (1-30 g) and stem diameter (0.2-3.5 cm) when averaged across environments. Almost all hybrids showed highparent heterosis for plant height (1-10 cm) and width (2-13 cm). Unlike other traits, there was negative heterosis for maturity, indicating that heterosis for this character in hybrids is expressed as earliness. Genetic similarity estimates among the nine parental lines ranged from 0.43 to 0.71 and were significantly and negatively correlated with highparent heterosis for all traits except for stem diameter and days from transplant to harvest. Euclidean distances were not correlated with heterosis. With modern broccoli inbreds, less heterosis was observed for head characteristics than for traits that measured plant vigor. In addition, genetic similarity based on molecular markers was more highly correlated with plant vigor characteristics than head traits. Unlike with molecular marker-based estimates of genetic similarity, euclidean distance determined using phenotypic trait data was not predictive of heterosis. In conclusion, this study has documented heterosis in Brassica oleracea L., and the ability to predict heterosis in this crop using molecular marker-based estimates of genetic similarity among parents used in producing the hybrid.

AFLP analysis of a worldwide collection of Didymella bryoniae.

Didymella bryoniae (anamorph Phoma cucurbitacearum) is an ascomycete that causes gummy stem blight, a foliar disease that occurs on cucurbits in greenhouses and fields throughout the world. In a previous study using RAPD analysis, little genetic diversity was found among isolates of D. bryoniae from New York and South Carolina, USA. Here we report the use of amplified fragment length polymorphism (AFLP) analysis to assess the genetic variation within a worldwide collection of D. bryoniae, 102 field and greenhouse isolates from ten states in the USA (California, Delaware, Florida, Georgia, Indiana, Maryland, Michigan, Oklahoma, South Carolina, and Texas) and seven other countries (Australia, Canada, China, Greece, Israel, Sweden, and The Netherlands) were examined. Seven different AFLP primer-pair combinations generated 450 bands, of which 134 were polymorphic (30%). Using cluster analysis, two groups and a total of seven subgroups were delineated. Representative isolates varied in their virulence on muskmelon and watermelon seedlings, but the degree of virulence was not strongly associated with AFLP groupings. However, isolates from the northern USA grouped separately from isolates originating from the southern USA.

Internal Transcribed Spacer Regions 1 and 2 and Random Amplified Polymorphic DNA Analysis of Didymella bryoniae and Related Phoma Species Isolated from Cucurbits.

ABSTRACT Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.

Effect of Wirestem Severity on Survival and Head Production of Transplanted Broccoli and Cabbage.

Field experiments were conducted with transplants of Brassica oleracea with known severity levels of wirestem caused by Rhizoctonia solani anastomosis group 4. Seedlings of broccoli and cabbage were grown in steamed soil infested with R. solani at 5 to 25 sclerotia/kg. Two weeks after inoculation, plants were separated into five severity classes based on wirestem symptoms, then transplanted into fumigated field plots in the spring and fall of 1995. The percentage of plants with and without aboveground symptoms was assessed at 14 and 42 days after transplanting. Marketable-sized heads were harvested eight times. In both seasons, percentages of symptomless plants, surviving (symptomless plus symptomatic) plants, and plants producing a marketable-sized head decreased as wirestem severity increased. Only 33 and 29% of cabbage transplants with >75% of the stem circumference girdled survived and produced a marketable head, respectively, compared with 95 and 83% of healthy transplants, respectively. For broccoli, only 15% of transplants with girdled stems survived and produced heads in spring but, in the fall, 74 and 72% of transplants with girdled stems survived and produced heads, respectively. Percentage of plants producing a marketable-sized head was highly correlated (P = 0.0001) with percentage of symptomless plants at 14 days after transplanting and percentage of surviving plants at 42 days after transplanting.

Characterization of Fusarium Yellows Resistance in Collard.

The yellows disease of cole crops, caused by Fusarium oxysporum f. sp. conglutinans, can be very damaging to collard. Growers in the southeastern United States frequently produce collard in hot, summer months when conditions for yellows development are favorable, and thus, incidence of this disease is increasing. A collection of essentially all U.S. commercial cultivars of collard, various landraces of collard, and other representative cole crops was evaluated for response to artificial inoculation with F. oxysporum f. sp. conglutinans under controlled-temperature conditions. In addition, the same collection was evaluated following transplanting for response to naturally infested soil in the field during summer 1997 and 1998. In all trials, genotype had the most significant effect on percentage of diseased plants, and genotype responses ranged from resistant (0 to 20% diseased) to susceptible (61 to 100% diseased). There was a significant temperature effect on percentage of diseased plants in one growth chamber experiment with five genotypes that resulted primarily from an increase in disease incidence for the cultivar Blue Max at 30°C compared with 25°C. Temperature was not significant in a second experiment with 20 genotypes. In the field, although significant differences were observed among genotypes and between years, a significant genotype × year interaction was not detected for percentage of diseased plants, indicating a similar ranking of genotypes for resistance between years. There was a significant correlation between results from controlled-environment studies and the field. A resistant response to F. oxysporum f. sp. conglutinans was expressed in certain cultivars of collard, including Flash, Heavicrop, and Morris Heading, and also in specific landraces. This resistance was stable in relatively high temperature environments used in evaluations. Results of this research indicate that choice of cultivar is a critical factor in producing collard where conditions favor infection by F. oxysporum f. sp. conglutinans. This information will aid in development of new yellows-resistant cultivars.

Differential Cultivars and Criteria for Evaluating Resistance to Rhizoctonia solani in Seedling Brassica oleracea.

Growth-room and field experiments were conducted to develop methods of studying resistance in Brassica oleracea crops to Rhizoctonia solani anastomosis groups (AG) 2-1 and 4, causal agents of wirestem. Seedlings of 12 cultivars (3 each of broccoli, cauliflower, cabbage, and collard) at the four- to five-leaf stage were transplanted to trays in a growth room and covered with steamed soil infested with cornmeal-sand cultures or sclerotia of R. solani or to fumigated field plots infested with sclerotia. The percent healthy, diseased, and dead plants was assessed every 3 to 5 days for 2 weeks in the growth room and for 3 weeks in field trials. At harvest, plants were dug out with roots intact and rated for wirestem severity. In most experiments, wirestem incidence (percent diseased and dead plants) stabilized within 10 to 14 days after inoculation. Inoculation with cornmeal-sand cultures of both AGs and sclerotia of AG-4 resulted in severe wirestem in all experiments, whereas sclerotia of AG-2-1 were less effective in the growth room and not effective in the field. Percent healthy and surviving (healthy plus diseased) plants, area under the disease progress curve (AUDPC), and wirestem severity all separated the most susceptible from the partially resistant cultivars more consistently than fresh weight of inoculated plants expressed as a percentage of noninoculated plant weight. Wirestem severity and AUDPC were always negatively and significantly (P ≤ 0.01) correlated with percent healthy plants. Although genotype by environment interactions were observed, the cauliflower cvs. Snowcone and Snow Crown were severely diseased in all experiments, whereas collard cv. Blue Max was consistently and significantly (P ≤ 0.05) less diseased.