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Сельскохозяйственная биология, 2012, № 1, с. 21-32.

doi: 10.15389/agrobiology.2012.1.21eng

УДК 633:581.145:575:577.21:57.08

ASSOCIATIONS SEARCH OF MOLECULAR MARKERS WITH DETERMINANT OF BLOSSOM-TIME IN NATURAL AND ARTIFICIAL POPULATION OF Brassica rapa L.

A.M. Artem’eva¹, E.N. Rudneva¹, J. Zhao², G. Bonnema², H. Budahn³, Yu.V. Chesnokov¹

The authors studied the chromosomal loci, associated with blossom-time, in two-parental splitting population of double haploid and in pivotal collection of native and breeding varieties-populations of B. rapa, using the different types of molecular markers. Utilizing the techniques of QTL-analysis and association mapping the authors identified and mapped the chromosomal loci, situated in linkage group 2, 3, 5, 6, 7 and 10, and established the AFLP-, SSR- and S-SAP-markers, linked with blossom-time.

Keywords: Brassica rapa, AFLP, SSR, S-SAP markers, DNA markers, QTL analysis, association mapping.

 

A widespread cultivated genus Brassica includes a large group of economically important species represented by vegetable, oilseed, fodder and spice crops. Brassica rapa L. is the first highly-polymorphic domesticated species including leafy vegetables (Chinese, Japanese, Rosette and Purple cabbage, leaf turnips Komatsuna, Japanese leafy vegetables and Broccollini), oilseed crops (spring and winter Barbarea, brown and yellow Sarson, Toria) and root rape turnip (table and fodder types) (1). Several European gene banks possess large collections of Brassica species. Thus, about 3600 samples of B. rapa are registered in the database of the European Cooperative Programme for Plant Genetic Resources, which includes 19 678 certified samples from 35 collections of 24 countries (http://documents.plant.wur.nl/cgn/pgr/brasedb/) (2, 3). The collection of B. rapa of N.I. Vavilov All-Russia Research and Development Institute of Plant Growing (VIR) includes 327 samples of oilseeds, 525 samples of six leafy types and 406 samples of root turnips.
Productivity and quality of cultivated plants are closely associated with such a key developmental factor as duration of a period required to enter a reproductive phase of ontogeny. This period widely varies in B. rapa types. In Arabidopsis L., a model object of molecular studies somewhat relative to Brassica, it was shown a flowering time as a complex trait with expression involving several mechanisms (response to vernalization and photoperiod, gibberellin synthesis) and many genes determining this process (4-6). For example, FLC (Flowering locus C) and FRI (Frigida) genes are important for changing the bloom-time and response to vernalization: FLC is a repressor of flowering at vernalization, while FRI contradicts it and controls expression of FLC (7, 8) .
Multiple paralogs of FLC (BrFLC1, BrFLC2, BrFLC3, BrFLC5) operating in B. rapa similar to FLC genes of Arabidopsis have been mapped in chromosomal regions syntenic to Arabidopsis (9-11). In previous studies of QTLs controlling flowering time in B. rapa, FLC-genes have been established as candidate genes (12-14). In several populations of B. rapa grown in different geographical and climatic conditions it has been identified the main QTL with BrFLC2 as a candidate gene determining flowering time and response to vernalization located in the 2nd linkage group, and QTL with BrFLC1 – in the 10th group (14-16).
Genetic mapping is carried out through as finding associations between molecular markers and traits, and resulting gene maps reflect a parallel genotypic and phenotypic diversity in natural and artificial populations, such as collections of plant species. Main techniques of these studies are QTL-analysis of target designed biparental splitting populations and associative mapping. Associative mapping of plant genes is based on detection of linkage disequilibrium (LD), which occurs in natural and breeding populations including those in collections of unrelated genotypes, as well as on the analysis of population structure at the level of individual genes and a whole genome (17). Associated pairs “marker – trait” can be detected when a linkage disequilibrium between a marker and gene controlling the trait is not completely erased by recombination (18). In such approach, genetic diversity of allelic polymorphism is compared with observed phenotypic variation. Associative mapping takes a number of advantages for QTL-analysis: as a rule, it implies a wider range of observed diversity of studied traits with no need in creation of splitting populations obtained through controlled crosses (19, 20).
 The first research on associative mapping of B. rapa from the Netherlands’ collection has resulted in establishing AFLP-markers associated with three morphological characters of a leaf, content of phytates and flowering time after a vernalization and without it (21). It was also reported about the creation of a core collection of B. rapa for associative mapping of a phenological trait “flowering time” (22).
This study was the first in Russia attempt to identify genetic loci determining the time of transition to flowering in genotypes of biparental splitting population and in samples from the core collection of Brassica rapa local varieties and breeding populations using different types of molecular markers; the obtained data of associative mapping were compared with results of QTL-analysis.
Technique. During a 3-year research, QTL-analysis was performed on plants from a biparental splitting population of dihaploid tomato lines created in Wageningen University (WUR, the Netherlands) through crossing the leafy Chinese cabbage and oilseed yellow Sarson (DH38, 68 lines). Samples for associative mapping were obtained from VIR collection of 96 populations-varieties; a complex European core collection of B. rapa was created upon 102 varieties from VIR collection and 137 samples from WUR core collection, which two include all botanical subspecies, varieties and morphological types of various eco-geographical origin.
Field phenotypic description of DH38 lines was performed under the conditions of experimental stations of VIR in Pushkin (Leningrad province) and Derbent (Dagestan), the core collection of VIR – in VIR laboratory in Pushkin, WUR collection - in Wageningen (Netherlands). The dates of following phenophases were recorded: appearance of a floral shoot in 10% plants (a start of visible transition to reproductive period) and in 75% plants of a sample, the beginning of flowering (flowering in 10% plants) and complete flowering of line / sample (flowering in 75 % plants) (23).
DH38 lines were genotyped using 326 molecular AFLP- and SSR-markers (24). To investigate genotypic variation in the two core collections, a standard PCR procedure was carried out with 13 pairs of microsatellite primers (22) and one BrFLC1-specific CAPS-marker, which represented the genetic loci for all 10 linkage groups of the species thereby providing an adequate coverage of the genome. Associative mapping of “flowering time” trait with detection of candidate genes in samples from the two collections  was performed using biallelic CAPS-marker BrFLC1 (15) located in the 10th linkage group and multiallelic SSR-marker BRH04D11-BrFLC2 (16 alleles) mapped in the 2nd linkage group. Individual genotyping of VIR core collection (96 samples) and detecting in it associations with the studying trait was done by standard methods of SSR and AFLP, and using a set of primers (21 pairs of SSR-primers, 1 CAPS-marker and 12 pairs of S-SAP-primers based on sequences of mobile genetic elements II class CACTA). CACTA-specific primers were designed upon TIR sequences of 17 and 19 bp length described by K. Alix et al. (25): BoB029L16 (CACTACAAGAAAACAGC) and a fragment BoB048N13 (CACTACAAGAAAACAGCGA). For selective amplification there were used Mse-CAC, Mse-CAT + CAG, Mse-CCG, Mse-CCT, Mse-CGT and Eco-ACA chosen in preliminary test trials.
Data processing of QTL-analysis was done in a computer program MAPQTL 6.0 (26) identifying the presence and location of QTLs (candidates) in a linkage group (mapping interval 5 cM), LOD values (logarithm of odds) (P = 0,05) and degrees of variation of traits determined by corresponding QTLs for each trait and population. Significance of each LOD was evaluated in test permutations (1000 repetitions). The analysis of population structure was done in Structure 2.2 (http://pritch.bsd.uchicago.edu/software). Visualization of morphological variability in studied collections and grouping samples by phenotype were performed using principal components analysis. Analysis of variance (ANOVA) was applied to calculate associations between SSR-alleles BRH04D11-BrFLC2 and “flowering time” trait.
Analyzing associations in B. rapa samples from VIR core collection, the data were standardized according to a molecular matrix (1 – marker detected, 0 - no marker) and phenological characteristics associated with flowering time (early shooting, complete shooting and flowering) were measured by a scale from 0 to 1 and then ranked. Calculation of genetic distances and similarity analysis were performed using the program NTSYSpc, a cluster analysis – by the method of neighbor joining (NJ). As a result, the integrated dendrogram of studied traits was designed, which shows a clustered indication of molecular fragments corresponding to trait-specific markers.
Results. Variation of a trait “transition to flowering” between the lines of a splitting population DH38 was significant and depended on year of research. Thus, in the year 2007 this diversity amounted to 32-64 days,  in 2008 - 76-89 and in 2009 - 33-55 days. It have been isolated DH38 lines (numbers 46, 69, 77, 87, 142) resistant to early start of flowering, which symptom was correlated with outstanding productivity more than a half exceeding average population value.
The analysis of observed by-year changes in position of chromosomal loci (QTL) determining flowering time in DH38 population allowed to establish AFLP- and SSR-markers linked to these loci. Several QTLs have been detected in the 2nd linkage group; in 2007-2008, a large QTL was located at the top of the group right where the major QTL with BrFLC2 as a candidate gene involved in controlling flowering time and response to vernalization, the effect of which is strongly suppressed by vernalization temperature (16) (Table 1). This fact explains the absence of this QTL in a cool season of 2009. Other detected QTLs were: QTL in the center of the 2nd linkage group (the effect was observed in all three years of research) and QTLs with low LOD values (0,52-2,02) in the 7th linkage group, one of which (located in the middle of the group) was present during all 3 years under the conditions of both VIR stations in Pushkin and Dagestan (Table 1).
In the Netherlands, QTL in R07 (7th linkage group) was detected in F2 population obtained using the same parental pair. In addition, in 2008-2009, QTLs were revealed in the 3rd, 5th and 10th linkage groups, in 2007 and 2009 – in the 4th one (in close positions or at a distance from each other), which data are consistent with results obtained for the same mapping population in the Netherlands (14, 21). In 2009 in Derbent, QTL determining the start of bolting were detected in the 2nd, 3rd, 6th, 7th, 8th and 10th linkage groups. An  interesting fact – identical or very close positions of markers in the bottom of R02, in top and middle parts of R07 and in the bottom part of R10 – was revealed by analysis of data from VIR Pushkin branch and Dagestan Experimental Station. Thus, most likely these sites correspond to the most stable genomic regions associated with time of plant transition to a generative phase.

Fig. 1. QTL controlling transition to flowering (2nd linkage group) in Вrassica rapa L.; LOD — common logarithm of  odds (St. Petersburg – Pushkin, 2008). Denotations: abscissa – Linkage group length, cM

The analysis of weather conditions during the years of research in both geographic areas suggests that the action of QTL located on the top of the 2nd linkage group is commonly manifested at a relatively warm weather without vernalization temperature. This assumption is consistent with the data of J. Zhao et al. (16), who described the abovementioned reduce in effect of the major QTL with BrFLC2 on the top of R02 caused by vernalization; in this case, the level of BrFLC2 transcription at all developmental stages in any plant tissues of late-flowering DH lines was higher than in early-flowering ones. A strongest suppression of expression of the QTL with BrFLC2 in both early- and late-flowering lines was achieved by vernalization of seedlings, therefore, transition to flowering is a trait determined very early in ontogeny. The authors consider BrFLC2 as a candidate gene to control flowering time and vernalization response in B. rapa (16).
  

In DH38 population there were identified QTLs exhibiting high LOD values in particular years and simultaneously affecting several important traits. For example, a QTL was located in the middle of the 2nd linkage group in 2008 which provided up to 82% diversity in flowering time (LOD - 9,95) (Fig. 1), 35% - diameter of leaf rosettes (LOD – 4,67), 18% - weight of plants, 31-40% - length of a petiole along with length and width of a leaf blade; all these traits were labeled by the only AFLP-marker R11. A QTL situated in the 7th group controls shoot diameter, plant height and weight (resp., 24, 25 and 11% diversity of these traits). Another QTL located in the 10th linkage determines shoot diameter, plant habit, leaf type, petiole length and width of a leaf blade (LOD - 1,61-3,67). The authors’ data confirm the known feature of leafy B. rapa forms – a pronounced correlation between plant size and time of transition to flowering.
Thus, manifestation of a complex quantitative trait is usually controlled by a number of QTLs located in different linkage groups. In the population DH38, QTLs determining a number of traits (transition to flowering, size of a plant and its productive organs - stem and leaf) are located in the 2nd, 3rd, 7th and 10th linkage groups where they form complexes of coadapted genes and coadapted gene complexes (18), which highlights the importance of these loci in ontogeny.
The chromosomal loci whose expression was dependent or independent on environmental conditions have been revealed as well. In the second case, QTL location in a linkage group was constant through all years of research while LOD values varied. There were also detected QTLs whose positions varied from year to year for no more than 1-6 cM, so they can be regarded as fixed loci even despite of different markers. Commonly, fixed QTLs showed low LOD values and 10-20% contribution to diversity of traits. Most likely, these are the loci associated with stability of typical morphological traits of plants, and, possibly, with their increased adaptive capacity. On the contrary, QTLs manifested only in particular years often showed high LOD and significantly provided diversity of plants under specific conditions. In the case of environmentally-independent QTLs there are usually observed high positive additive effects of genes.
To find associations between molecular markers and traits in natural heterozygous and heterogeneous populations of plants from the two core collections of Brassica rapa, the diversity of allelic polymorphism for a set of markers was compared with observed phenotypic variation in leaf length, pod length, plant habitus and flowering time. In both collections, phenotypic and genotypic variation in main groups of crops were comparable. During a genotyping, 88 polymorphic markers were detected including 86 SSR-markers and BrFLC1-specific CAPS-marker used in analysis of population structure; the number of alleles per marker varied from 2 to 16. A population structure was analyzed using the model allowing mixed (hybrid) origin of genotypes and a presence of independent frequency of alleles between subgroups. Both collections were found to carry a similar allelic diversity (in terms of the total number of alleles in each crop / group of crops – Chinese cabbage, Japanese leafy vegetables, turnips rape, Broccolini, winter and spring forms of Barbarea, Chinese oilseed crops) along with a comprehensive range of morphological types suggested by low phenotypic variation within a subspecies between the two collections.

The analysis of population structure has revealed in the integrated collection the presence of five subgroups (Fig. 2). Oilseeds from VIR collection were assumed as a separate single group, very diverse and absent in WUR collection. On the contrary, VIR collection had no Broccolini and Japanese turnips rape samples represented by WUR, so these two efficiently complemented each other. Along with it, integration of these collections allowed to form subgroups with a greater number of samples providing more accurate associations of markers with analyzed traits.

 
Associative mapping with CAPS-marker for the candidate gene BrFLC1 has shown that its alleles of 500 bp and 800 bp as well as their joint presence were significantly associated with flowering time in Chinese cabbage, Japanese cabbage, Japanese leafy vegetables and spring oilseeds from VIR collection (Table 2). An interesting fact: an additive effect of these alleles was observed in Japanese leafy vegetables and oilseeds from VIR collection, while in Chinese cabbage and turnip rape heterozygotes manifested overdominance. In Chinese cabbage and spring oilseed crops from the Netherlands there was found only a weak effect of the association of different alleles with studied traits of heterozygotes in one year of the research. Using CAPS-marker revealed only a single polymorphism, however, the studied samples most likely possess a greater number of alleles with different functions. Therefore, there is a possibility of interactions with other alleles or other genes involved in flowering, which explains the unequal level of manifestation of this trait in heterozygotes of different crops. As expected, vernalization reduced the effect of BrFLC1 since exposure to low temperatures reduces gene expression (10, 16). In turnip rape, a prolonged exposure to low temperatures promotes faster transition to flowering; indeed, even after vernalization BrFLC1-specific CAPS-marker was yet associated with flowering time in this crop. Y.X. Yuan et al. (15) also reported that BrFLC1 delayed flowering time in a collection of 121 lines by an average of 15 days. For clear understanding of effects provided by BrFLC1 gene on flowering time, it is necessary to use several SNP-markers allowing identification of different haplotypes.

A screening of BrFLC2allelic variants with the target created SSR-marker BrH04D11-BrFLC2 revealed its significant correlation with flowering time in three (211, 213 and 215 bp) of 16 BrFLC2 alleles relatively frequent in turnip rape but rare or absent in other crops. These alleles similarly segregate in DH38 population, which was proved by detection of a major QTL in BrFLC2 locus (14). Delayed transition to flowering in turnip rape can be explained by these “late” alleles, which is useful for practical breeding. A set of markers can be used to isolate the samples for crossing different crops / types in order to prolong the period before flowering. These findings also explain why paralogs BrFLC were assumed as candidate genes during a study of different material (biparental crosses involve alleles of only two original forms) (13, 14). Sequencing BrFLC2 alleles in samples with unequal SSR-alleles can help to clarify whether these alleles serve as reliable markers for BrFLC2 haplotypes or such association is disturbed by evolutionary processes when marker alleles and haplotypes subject to unequal selection pressure and segregate independent from each other.

Thus, both markers (CAPS and SSR) are largely associated with flowering time, but their effects are limited to a few crops and/or alleles. Therefore, to perform an accurate associative mapping, population (collection) must be increased to 400 samples, as is suggested by statistical calculations, and phenotyping and genotyping can be simplified by fixation of heterogeneous samples through creation of homozygous lines using microspore culture.
Variation of the trait “transition to flowering” was great in 96 samples of VIR collections under the conditions of Pushkin branch, though the range of diversity changed insignificantly and averaged 19-85 days by years. Turnip rape, winter Barbarea and Japanese cabbage developed by a two-year type with appearance of a floral stem on the 210th -245th day.
In VIR collection, three SSR-markers associated with flowering time were found: early flowering - 165 bp allele of a biallelic marker Br372 in the 7th linkage group, late flowering - 195 bp allele of a multiallelic marker VS89 (9 alleles) in the 6th linkage group and, notably, 202 bp allele of a multiallelic marker BrH04D11-FLC2 (16 alleles) in the 2nd linkage group. Two molecular markers created upon the elements class II CACTA were associated with early flowering (no data on location in the genome) (Fig. 3, 4). On average, in the group of samples carrying Br372-165 marker of early transition to flowering, bolting phenophase started on the 52nd day, in samples without the marker - on the 112th day, in samples with a marker CAT-CAG-700-88 - on the 58th day, and in ones without this marker - 27 days later, in samples carrying CGT-700-480 and without it - on the 53rd and 108th days, resp. (Table 3). The marker of late transition to flowering BC89-195 allowed to isolate a group of genotypes with late start of bolting (on the 137th day) while the samples without this marker entered this phase on the 45th day; using a marker BrH04D11-FLC2, it was revealed a group of samples with a start of bolting on, respectively, the 77th and 59th days. It can be assumed that molecular markers and CACTA SSR-markers will serve as an effective tool for applied genetics and breeding work.

 

Thus, artificial biparental splitting and natural populations were analyzed to detect associations between different types of molecular markers and a trait “transition to flowering”, which has contributed to similar results and allowed to detect and map several chromosomal loci located in the 2nd, 3rd, 5th, 6th, 7th and 10th linkage groups of Brassica rapa, as well as to establish AFLP-, SSR- and S-SAP-markers associated with flowering time. Practical importance of the these markers (especially easily detectable microsatellite markers) is suggested by their use for a large scale screening of collection samples including pre-breeding studies.

The authors express their sincere gratitude to N.V. Kocherina for assistance in calculations of data obtained through QTL-analysis, as well as to G.L. Chudova and K.A. Artemieva for their help in agricultural work on collections.


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1N.I. Vavilov All-Russia Research and Development Institute of Plant Growing, St. Petersburg 190000, Russia, e-mail: yu.chesnokov@vir.nw.ru, akme11@yandex.ru; 2Laboratory of Plant Breeding, Wageningen University, P.O. Box 386, 6700 AJ Wageningen, the Netherlands; 3Institute for Breeding Research on Horticultural Crops of JKI,   Erwin-Baur-str., 27, D-06484 Quedlinburg, Germany

Received August 22, 2011