Genetics and Molecular Mapping of Black Rot Resistance Locus Xca1bc on Chromosome B-7 in Ethiopian Mustard (Brassica carinata A. Braun)

Black rot caused by Xanthomonas campestris pv. campestris (Pam.) Dowson is the most destructive disease of cauliflower causing huge loss to the farmers throughout the world. Since there are limited sources of resistance to black rot in B. oleracea (C genome Brassica), exploration of A and B genomes of Brassica was planned as these were thought to be potential reservoirs of black rot resistance gene(s). In our search for new gene(s) for black rot resistance, F2 mapping population was developed in Brassica carinata (BBCC) by crossing NPC-17, a susceptible genotype with NPC-9, a resistant genotype. Out of 364 Intron length polymorphic markers and microsatellite primers used in this study, 41 distinguished the parental lines. However, resistant and susceptible bulks could be distinguished by three markers At1g70610, SSR Na14-G02 and At1g71865 which were used for genotyping of F2 mapping population. These markers were placed along the resistance gene, according to order, covering a distance of 36.30 cM. Intron length polymorphic markers At1g70610 and At1g71865 were found to be linked to black rot resistance locus (Xca1bc) at 6.2 and 12.8 cM distance, respectively. This is the first report of identification of markers linked to Xca1bc locus in Brassica carinata on B-7 linkage group. Intron length polymorphic markers provided a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness for marker assisted alien gene introgression into cauliflower.


Plant materials development
The accession NPC-17 (female) highly susceptible to black rot pathogen (Fig 1a) was crossed with resistant accession NPC-9 (male) (Fig 1b) to develop F 1 hybrid. The F 1 plants were selfed to generate F 2 seed and pollinated simultaneously with NPC-17 (P 1 ) and NPC-9 (P 2 ) to generate backcross generations B 1 and B 2 , respectively. All the segregating generations (F 2 , B 1 and B 2 ) were grown during October to March (2013-14) at the Research Farm of Division of Vegetable Science, ICAR-Indian Agricultural Research Institute, Pusa campus, New Delhi, India. All the plants in segregating generations (F 2 , B 1 and B 2 ) were precisely tagged and numbered after transplanting for their identity during phenotyping for disease and for drawing leaf samples for DNA extraction for genotyping. The F 2 population was used for genotyping with putatively linked markers and three segregating generations, namely F 2 , B 1 , B 2 , were employed to study genetics of black rot resistance in B. carinata.

Phenotyping of F 2 mapping population
All the segregating generations (F 2 , B 1 and B 2 ) were screened against Xcc race 1 during November to December, 2013. The mean monthly weather data (S1 Table) for experimental period (October, 2013 -March, 2014) were collected from Division of Agriculture Physics, IARI, New Delhi. The bacterial strain (Accession number, ITCC-BH-0001; Delhi isolates, C1) of Xcc race 1 [27] was obtained from the Bacteriology Unit, Division of Plant Pathology, IARI, New Delhi, India and multiplied in yeast glucose chalk agar (YGCA) media at 25°C for 3 days. The culture was carefully scrapped from the media with sterilized slide. The scraped bacterial culture was mixed in 100 ml sterilized distilled water and mixed thoroughly by vortex and final concentration of 10 8 −10 9 cfu/ml was made. The plants were first inoculated on 30 th day after sowing (DAS) by using leaf cut and dip technique [28]. The plants were inoculated by clipping the secondary veins at the margins with small scissor dipped in the bacterial suspension. The inoculation was carried out at 10 points per leaf on youngest leaves per plant in three replications. To maintain high humidity, sufficient moisture was maintained by frequent irrigation. Agrometeorological data (S1 Table) favouring disease establishment and development during phenotyping period November to December, 2013. The inoculated plants were assessed for disease reaction based on disease rating scale 0-9 and percentage of inoculated points in leaves showing symptoms were recorded as per scale given by Vicente et al. [11]. The disease reaction was recorded twice at 15 and 30 DAI and the final score of disease reaction at 30 DAI was used for grouping plants into resistant (Fig 1c) and susceptible (Fig 1d-1f) categories in segregating populations.
The total number of inoculated points and the number of points showing symptoms were recorded and the percentage of infected points were calculated as disease incidence. The severity of symptoms was assessed according to 0-9 scale based on the relative lesion size (0 = no symptoms; 1 = small necrosis or chlorosis surrounding the infection point; 3 = typical small Vshaped lesion with black veins; 5 = typical lesion half way to the middle vein; 7 = typical lesion progressing to the middle vein; and 9 = lesion reaching the middle vein). For segregation analysis plants were grouped on the basis of average disease severity and percent incidence into resistant and susceptible categories. Generally, plants with 0-5 disease severity and less than 50% disease incidence were categorized as resistant whereas those with 5-9 disease severity and more than 50% disease incidence as susceptible.

Genomic DNA isolation and primer selection
Genomic DNA was isolated from the fresh young leaf tissues of both the parents and F 2 plants using cetyl trimethyl ammonium bromide (CTAB) method [29]. The DNA was purified and quantified on 0.8% agarose gel by comparison with 50 ng/μl of standard uncut lambda (λ) DNA marker. DNA was diluted in sterile distilled water to a concentration of 25-30 ng μl -1 . A total of 364 primers including 204 simple sequence repeats (SSR) (S2 Table) and 160 intron length polymorphic (ILP) markers (S3 Table) were used for polymorphic survey between the parents NPC-17 and NPC-9. Selected 125 SSR primer-pairs equally distributed to all linkage groups (LG C1-C9 and LG B1-B8) covering both the genomes of Brassica carinata [18]. Seventy nine SSR primer sequences were retrieved from B. nigra (BB) available publicly in the Brassica microsatellite information exchange (www.brassica.info/resource/markers/ssr-exchange.php). All the SSR primers were got synthesized from SBS Genetech Co. Ltd., China. One hundred sixty ILP primer sequences information used in this study were retrieved from B. juncea covering whole B genome (B1-B8) [30] and synthesized from Eurofins MWG Operon, Ebensburg (Germany).

SSR and ILP marker analysis
Genotyping of mapping population was carried out with SSR markers by following PCR reactions in 0.2 ml thin walled sterilized PCR tubes. Amplifications were carried out in a total volume of 15 μl reaction containing 1.5 μl 10X PCR assay buffer with 17.5 mM MgCl 2 , 0.6 μl dNTP mix (10 Mm of each dATP, dTTP, dCTP and dGTP for direct use in PCR), 0.2 μl Taq DNA polymerase (5U μl -1 ), primer (10 μM) 1.5 μl each forward and reverse, 3 μl template DNA (25-30 ng) and sterile water (6.70 μl). The PCR chemicals used were from Himedia Laboratories, Mumbai, India. The PCR amplification reaction was carried out in a thermocycler (Eppendorf master cycler, Pro S model) with the initial PCR cycle at 94°C for 5 min for DNA denaturation followed by 35 cycles of 45s denaturation at 94°C, primer annealing at 50-57°C varying with primer-pairs for 1 min, 2 min extension at 72°C and final extension at 72°C for 7 min. The amplified products of SSRs were resolved by electrophoresis in 3% agarose gel (Hi media product, India) containing ethidium bromide in 1X TAE buffer (pH 8.0) at constant voltage (@ 5v/cm) for 3h. The size of amplified fragments was determined by co-electrophoresis of standard molecular weight marker. DNA profile was visualized on UV transilluminator and photographed by using Gel documentation System (Alpha Innotech, C-1000™). Intron length polymorphic marker analysis was performed by using initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min annealing at 53-62°C for 45s and elongation at 72°C for 3 min followed by a final extension at 72°C for 10 min. Amplified PCR products were analyzed on 2% w/v agarose gel. The remaining conditions set up was same as for SSR marker analysis.

Bulk segregant analysis (BSA)
All the SSR and ILP primers were used for parental polymorphism to identify informative primers. To identify molecular markers putatively linked to black rot resistance locus, bulk segregant analysis [31] was performed. Equal quantity of DNA from ten resistant and ten susceptible F 2 plants was mixed separately to create resistant and susceptible bulks. Polymorphic primers between parents and the resistant and susceptible bulks were tested on the genomic DNA of individual F 2 plants employed in creating bulks. Putatively linked markers to the gene for resistance were examined by conducting SSR and ILP markers analysis of all 212 F 2 plants.

Statistical analysis
Disease reaction of each F 2 plant to Xcc race 1 of the pathogen and data for marker were analysed by chi square (χ2) test [32] to determine goodness of fit to the expected segregation ratio of 3:1 (three resistant: one susceptible). A data matrix was generated and imported into MAP-MAKER (3.0) [33]. The linkage between markers and resistance locus has been confirmed using this software. Markers within a group were ordered using the order command with LOD of 3.0. Map distances were calculated as centi Morgans (cM) using the Kosambi mapping function [34] and loci were ordered using the 'sequence' and 'compare' commands, with an LOD threshold score of 3.0.

Genetics of black rot resistance
Inoculated susceptible plants started showing 'V' shaped symptom as light yellowish spot near the cut portion of leaf fifteen days after inoculation. All the plants in F 1 generation as well as in F 2 , B 1 and B 2 generations were scored individually for black rot disease reaction. The plants were finally scored at 30 DAI for disease reaction and grouped into resistant and susceptible categories based on disease severity and incidence (%). All the F 1 plants were resistant to black rot disease. This finding unveils dominant nature of resistance to black rot. Out of 212 F 2 plants, 162 and 50 plants were found resistant and susceptible, respectively. On the comparison of observed segregation ratio with the expected in a χ2 test, 3:1 segregation ratio was observed with a χ2 value of 0.22 (P = 0.6-0.7) that fitted well with the expected ratio (Table 1). A total of 74 plants of B 1 generation segregated as 40 resistant: 34 susceptible and fitted well with expected ratio (1:1). However, all the plants of B 2 were found to be resistant. These findings advocated monogenic dominant control of resistance to black rot in B. carinata NPC-9.

Mapping of black rot resistance gene
Cross transferability and rapid identification of linked markers. Out of 364 (204 microsatellite and 160 ILP) primers used in parental polymorphic survey analysis, 41 distinguished the parental lines and, therefore, these were identified as polymorphic markers (S4 Table). Single plant analysis with putatively linked markers (ILPAt1g70610, SSRNa14-G02, ILPAt1g7186) confirmed resistant and susceptible individuals. These linked markers to the gene resistant to Xcc were examined by conducting SSR and ILP markers analysis of all 212 F 2 plants. Genotyping pattern of F 2 individuals with the marker ILP At1g70610 closely linked to the gene of interest marker is given in Fig 3. Observed segregation ratio of three molecular markers that fitted well with the expected ratio (Table 2) and the details of molecular markers linked to R gene conferring resistance to Xcc race 1 in B. carinata is given in Table 3.
Map construction and linkage group assignment. The linkage between markers and resistance gene (R) was confirmed using MAPMAKER (3.0) with LOD score 3.0. A linkage map of three markers along with the resistance locus covering 36.30 cM distance was developed (Fig 4).
Intron length polymorphic markers At1g70610 and At1g71865 flanked the resistant gene (R) at a genetic distance of 6.2 and 12.8 cM, respectively. However, SSR (Na14-G02) marker was far away from the resistant locus with a distance of 30.1 cM. This is the first report of identification of markers linked to black rot resistance locus (Xca1bc) in Brassica carinata. The chromosomal locations of the ILP (At1g70610 and At1g71865) and SSR (Na14-G02) markers linked to resistance gene were inferred from their position on the high-density genetic linkage map of B. carinata [18] and B. juncea [30], whereas SSR Na14-G02 marker was located at B-7 linkage group in conserved block E of B. carinata and ILP At1g70610 and At1g71865 markers were located in conserved block E of B. juncea.

Genetics of black rot resistance
Exploring the new resistance sources from A and B genomes of alien Brassica species is one of the current priority areas for black rot resistance breeding in cauliflower. The accessions 'NPC-9' and 'NPC-17' of B. carinata were found as resistant and susceptible, respectively. The accession NPC-9 could be used for transferring resistance against Xcc race 1 into commercial susceptible varieties of Brassica oleracea group. Segregation analysis for Xcc resistance gene in three populations (F 2 , B 1 , B 2 ) fitted well in expecting ratios indicated involvement of single dominant locus controlling resistance to Xcc race 1 in B. carinata accession NPC9. A pure Delhi isolate of Xcc race 1 was used for artificial inoculation, which facilitated the analysis of inheritance of resistance locus in more simplified and precise manner. On the basis of postulated gene-for-gene model, single dominant gene (R1) present in B. carinata revealed that resistance (incompatible interaction) with most of the pathogenic races differed from single dominant gene of B. oleracea (R3) for black rot resistance [2]. The co-segregation of disease reaction in individual F 2 plants with putatively linked markers viz., ILP (At1g70610, At1g71865) and SSR (Na14-G02) showed good fit to 3: 1 ratio, confirming their linkage with black rot resistance locus (Xca1bc). On the basis of phenotypic and molecular marker segregation, we reached at a conclusion that R gene controlling the Xcc race 1 resistance was inherited monogenically with dominant nature in an accession 'NPC-9' of B. carinata. Therefore, it is important and feasible to transfer this black rot resistance gene in to susceptible vegetable Brassicas through sexual/somatic hybridization attempting embryo recue and protoplast fusion techniques. Previously, a single dominant locus (Xca1) for resistance to Xcc race 1 and 4 was  reported in line PI 199947 of B. carinata [11]. The involvement of single dominant locus for resistance to Xcc has also been reported in other alien Brassica species viz. B. rapa [12], Brassica napus [35] and B. nigra [36]. However, some polygenes also played a role in controlling Xcc resistance loci in B. rapa [13][14].

Mapping of black rot resistance
Information regarding genetics of race-specific resistance and location of resistant gene provides vital assistance in marker assisted gene pyramiding for development of durable resistance. The rapid identification of putatively linked markers viz. ILP (At1g70610, At1g71865) and SSR (Na14-G02) was facilitated by using BSA approach as used earlier by several researchers [31,[37][38]. Bulk segregant analysis (BSA) using microarrays and extreme array mapping (XAM) have recently been used to rapidly identify genomic regions associated with the phenotypes in multiple species by Becker et al. [39]. The level of polymorphism (15.19%) of microsatellite primers was found to be low in B. carinata, however this could have been low estimation because of the use of agarose (3%) gel, which can be further increased by the use of high resolution gel electrophoresis such as metaphor and PAGE. In spite of the fact that ILP markers are cross transferable from one species to another, their information in B. carinata is very less [18]. The ILP markers derived from B genome of B. juncea were found to have high percentage of cross transferability rates (93.75%) in B. carinata which confirmed the already available information that ILP markers have high transferability rates among related plant species. However, Yadava et al. [40] reported lower level of cross-transferability from 'B' genome derived STMS markers as compared to those derived from 'A' and 'C' genomes of Brassica species. 95% ILP markers of cowpea (Vigna unguiculata (L.) Walp.) were also reported to be transferable to other Vigna species by Gupta et al. [41]. This high transferability of ILP markers may be due to the conserved nature of exons in Brassica species. These findings are also in line with those reported in rice [26], foxtail millet [42] and Tomato [43]. The usefulness of ILP markers across species and genera is mainly  Table 2. Segregation analysis of molecular markers with resistance locus (Xca 1bc).

Marker
Observed F  because of their highly conserved exonic and variable intronic regions which decrease their developmental cost and increase utility. Across-species transferability variation in Brassica genomic SSR and ILP markers developed earlier that harmonizes with the existing evolutionary relationship among the Brassica species and genera. High transferability of these markers to B. carinata (93.75%) suggested their utility in these large genome amphidiploid species in which no sequence information is available. It reduces the cost and efforts required for marker development and enrich the resources for various genotyping applications in these species. Therefore, an elaborate and intensive polymorphism survey using a large set of such markers for construction of high-density genome map of this specie is required. The first sequenced plant species A. thaliana shares a common ancestor with the family Brassicaceae and provides a good opportunity in understanding the genome structure and evolutionary pattern. Although systematic efforts have been made with the map-based cloning of resistance genes against blackleg disease [44], but there has been no such advancement in case of black rot [2]. In the present study, the resistance locus Xca1bc was found to be flanked by ILP markers At1g70610 and ILP At1g71865 with close linking to the former. The SSR (Na14-G02) marker was found to be located on B-7 linkage group in conserved block E of B. carinata [18], whereas ILP (At1g70610 and At1g71865) markers were found in conserved block E of B. juncea [30]. The nomenclature of linkage groups corresponds with the designation of each linkage group in the B genome of B. juncea [30,45]. The organization of B. juncea linkage groups based on the genomic blocks was identified by Schranz et al. [46]. However, it was hypothesized that black rot resistance probably originates from the B genome [11]. These flanking ILP markers will be more useful with great efficiency in marker assisted alien gene introgression into Brassica oleracea. Previously, Tonguc et al. [47] identified RAPD markers associated with black rot resistance in B. oleracea breeding lines derived from B. carinata accession PI 199947 and found that all associated markers significantly deviated from the expected 3:1 ratio. Soengas et al. [13] identified one major QTL on A06 and two additional QTLs on A02 and A09 controlling the resistance to most of the races of the pathogen. Artemeya et al. [14] also reported QTLs against different four Xcc races (2013) in B. rapa. Molecular mapping of agronomically important traits has also been started recently after publication of first linkage map of B. carinata by Guo et al. [18] who mapped the loci for petal colour (Pc) and anther colour (Ac) on linkage groups B6 and C9, respectively. They also identified four QTLs for seed colour which were related to the C genome of B. carinata. Mapping of QTLs for flower development and construction of a dense genetic linkage map in Brassica carinata was carried out by Zou et al. [19] in a doubled haploid population based on DArT-Seq™ markers.

Conclusions
The conclusion from the present study, therefore, is that resistance to black rot disease in B. carinata is governed by single dominant gene. ILP markers provide a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness. Resistance gene is flanked by two linked ILP markers viz., At1g70610 and At1g71865 which will be a vital practical application in marker assisted selection with high efficiency. Therefore, to identify some more closely linked markers, this study has to be continued further by employing more DNA based markers to map the black rot resistance locus (Xca1bc) present in the resistant source'NPC-9' genotype of B. carinata and validation of these flanked ILP markers would be required in other potential resistant sources. Hence, search for new gene(s) in alien Brassica species and identification of tightly linked molecular markers would be of immense importance in the context of developing pre-breeding black rot resistant genetic stocks/lines in C genome Brassicas, especially in Cauliflower, Cabbage and Broccoli through marker assisted backcross breeding.
Supporting Information S1