Analyses of a multi-parent population derived from two diverse alfalfa germplasms: testcross evaluations and phenotype-DNA associations.

In a previous study, we showed that the genetic variation present in the Medicago sativa subsp. sativa Peruvian and M. sativa subsp. falcata WISFAL germplasms could be used to improve forage yields when favorable alleles were recombined and used in hybrid combination with cultivated alfalfa. In this paper, we present testcross forage yield and fall growth data for two seasons of a C0 population generated after intermating the Peruvian x WISFAL population for several generations. In addition, we conducted marker-trait association analysis as an attempt to identify Peruvian and WISFAL genomics regions affecting the targeted traits. Five and seven genomic regions were found significantly associated with forage yield and fall growth, respectively. In the case of fall growth, alleles from both accessions were positively associated with plant height. However, more alleles from WISFAL were positively associated with forage yield than from Peruvian. WISFAL is known for its winter hardiness and genomic regions with large effects on winter survival may have masked the effect of forage yield from Peruvian. The fact that most of the genomic regions discovered in this study have been previously associated with traits involved in winter hardiness validates our findings and suggests that associations between DNA fragments and agronomic traits can be detected without the necessity of developing bi-parental mapping populations.

Population structure and combining ability of diverse Medicago sativa germplasms.

Although unadapted germplasms have been used to improve disease and insect resistance in alfalfa, there has been little effort to use these for improving forage yield. We evaluated genetic diversity and combining ability among two unadapted germplasms ( Medicago sativa ssp. sativa Peruvian and M. sativa ssp. falcata WISFAL) and three Northern U.S. adapted alfalfa cultivars. Population structure analyses indicated that the WISFAL and Peruvian germplasms were genetically distinct from the cultivars, although Peruvian was relatively closer to the cultivars. Peruvian and WISFAL germplasms were intermated to generate a novel hybrid population. This population was crossed to the three cultivars as testers, and the testcross progenies were evaluated for forage yield along with the hybrid population, the original germplasms (Peruvian, WISFAL and cultivars), testcrosses of Peruvian and WISFAL to the three cultivars and a three-way hybrid of the cultivars. The experiment was carried out in the field in Temuco, Chile and Arlington, Wisconsin, USA, and forage was harvested during two seasons. Results from these evaluations showed that hybrids between the Peruvian x WISFAL population and the cultivar testers yielded as much as the cultivar testers. Heterosis was observed between Peruvian and WISFAL, and between these germplasms and the cultivar testers, suggesting that each germplasm may contain different favorable alleles. If Peruvian and WISFAL populations contain alleles at different loci that complement cultivar testers, then combining and enriching these alleles in a single population could result in improved combining ability with alfalfa cultivars.

Characterization of a dwarf gene in Brassica rapa, including the identification of a candidate gene.

Dwarf genes have been valuable for improving harvestable yield of several crop plants and may be useful in oilseed Brassica. We evaluated a dwarf gene, dwf2, from Brassica rapa in order to determine its phenotypic effects and genetic characteristics. The dwf2 mutant was insensitive to exogenous GA(3) for both plant height and flowering time, suggesting that it is not a mutation in the gibberellin biosynthesis pathway. The dwarf phenotype was controlled by a semidominant allele at a single locus. Near-isogenic lines that were homozygous or heterozygous for dwf2 had 47.4% or 30.0% reduction in plant height, respectively, compared to the tall wild-type line, and the reduction was due to reduced internode length and number of nodes. The dwf2 homozygous and heterozygous lines had the same or significantly higher numbers of primary branches than the wild-type line, but did not differ in flowering time. The DWF2 gene was mapped to the bottom of linkage group R6, in a region having homology to the top of Arabidopsis thaliana chromosome 2. The map position of DWF2 in comparison to markers in A. thaliana suggests it is a homolog of RGA ( repressor of ga1-3), which is a homolog of the wheat "Green Revolution" gene. This dwarf gene could be used to gain more insight on the gibberellin pathway and to reduce lodging problems in hybrid oilseed Brassica cultivars.

Evaluation of Sclerotinia Stem Rot Resistance in Oilseed Brassica napus Using a Petiole Inoculation Technique Under Greenhouse Conditions.

A petiole inoculation technique was adapted for evaluating resistance of oilseed Brassica napus seedlings to Sclerotinia sclerotiorum. In the first of four experiments, four isolates of S. sclerotiorum were tested, two originating from soybean and two from B. napus. In all, 10 to 47 B. napus accessions were inoculated in the seedling stage and responses to isolates were evaluated using days to wilt (DW) and a lesion phenotype index (LP). There were no significant differences in virulence among the four isolates for DW and only slight differences for LP. However, significant differences (P < 0.0001) were observed among the B. napus accessions for DW and LP in this experiment and in subsequent experiments using one isolate. The responses of accessions were consistent among experiments and among evaluation criteria. Higher levels of resistance were found among winter-type than spring-type accessions, and among rapeseed-quality compared with canola-quality accessions. The most resistant accessions identified also were the most resistant when inoculated at the flowering stage. Terminal stems were inoculated immediately below the lowest flower and stem lesion length (SLL) was used to characterize the interaction phenotype of each accession. The petiole inoculation technique can be used successfully to differentiate oilseed B. napus germ plasm for response to S. sclerotiorum. This inoculation technique and the sources of resistance identified in this study may be used to determine inheritance resistance to S. sclerotiorum and for improving oilseed B. napus cultivars for resistance to this important pathogen.

Linkage mapping of genes controlling resistance to white rust (Albugo candida) in Brassica rapa (syn. campestris) and comparative mapping to Brassica napus and Arabidopsis thaliana.

Genes for resistance to white rust (Albugo candida) in oilseed Brassica rapa were mapped using a recombinant inbred (RI) population and a genetic linkage map consisting of 144 restriction fragment length polymorphism (RFLP) markers and 3 phenotypic markers. Young seedlings were evaluated by inoculating cotyledons with A. candida race 2 (AC2) and race 7 (AC7) and scoring the interaction phenotype (IP) on a 0-9 scale. The IP of each line was nearly identical for the two races and the population showed bimodal distributions, suggesting that a single major gene (or tightly linked genes) controlled resistance to the two races. The IP scores were converted to categorical resistant and susceptible scores, and these data were used to map a single Mendelian gene controlling resistance to both races on linkage group 4 where resistance to race 2 had been mapped previously. A quantitative trait loci (QTL) mapping approach using the IP scores detected the same major resistance locus for both races, plus a second minor QTL effect for AC2 on linkage group 2. These results indicate that either a dominant allele at a single locus (Acal) or two tightly linked loci control seedling resistance to both races of white rust in the biennial turnip rape cultivar Per. The map positions of white rust resistance genes in B. rapa and Brassica napus were compared and the results indicate where additional loci that have not been mapped may be located. Alignment of these maps to the physical map of the Arabidopsis genome identified regions to target for comparative fine mapping using this model organism.

Brassica genomics: a complement to, and early beneficiary of, the Arabidopsis sequence.

Those studying the genus Brassica will be among the early beneficiaries of the now-completed Arabidopsis sequence. The remarkable morphological diversity of Brassica species and their relatives offers valuable opportunities to advance our knowledge of plant growth and development, and our understanding of rapid phenotypic evolution.

Novel flowering time variation in the resynthesized polyploid Brassica napus.

Recent molecular data using resynthesized polyploids of Brassica napus established that genome changes can occur rapidly after polyploid formation. In this study we present data that de novo phenotypic variation for flowering time also occurs rapidly after polyploidization. Two initial polyploid plants were developed by reciprocal crosses of B. rapa and B. oleracea followed by chromosome doubling to establish two lineages, each of which was expected to be homozygous and homogeneous. Several sublineages of each lineage were advanced by self-pollination. The range in days to flower of the sixth generation plants was 39-75 and 43-64 for the two lineages. Analysis of seventh generation progeny indicated that the variation was heritable. Lines were selected and self-pollinated to the eighth generation and also testcrossed to a natural B. napus cultivar; the testcross plants were then self-pollinated. Differences in flowering time were also inherited in these advanced generations. Days to flower was significantly correlated with leaf number in each generation. The rapid evolution of new phenotypic variation, like that observed in this model system, may have contributed to the success and diversification of natural polyploid organisms.

Linkage analysis of molecular markers and quantitative trait loci in populations of inbred backcross lines of Brassica napus L.

Backcross populations are often used to study quantitative trait loci (QTL) after they are initially discovered in balanced populations, such as F(2), BC(1), or recombinant inbreds. While the latter are more powerful for mapping marker loci, the former have the reduced background genetic variation necessary for more precise estimation of QTL effects. Many populations of inbred backcross lines (IBLs) have been developed in plant and animal systems to permit simultaneous study and dissection of quantitative genetic variation introgressed from one source to another. Such populations have a genetic structure that can be used for linkage estimation and discovery of QTL. In this study, four populations of IBLs of oilseed Brassica napus were developed and analyzed to map genomic regions from the donor parent (a winter-type cultivar) that affect agronomic traits in spring-type inbreds and hybrids. Restriction fragment length polymorphisms (RFLPs) identified among the IBLs were used to calculate two-point recombination fractions and LOD scores through grid searches. This information allowed the enrichment of a composite genetic map of B. napus with 72 new RFLP loci. The selfed and hybrid progenies of the IBLs were evaluated during two growing seasons for several agronomic traits. Both pedigree structure and map information were incorporated into the QTL analysis by using a regression approach. The number of QTL detected for each trait and the number of effective factors calculated by using biometrical methods were of similar magnitude. Populations of IBLs were shown to be valuable for both marker mapping and QTL analysis.

Genetic Analysis and Identification of Amplified Fragment Length Polymorphism Markers Linked to the alm1 Avirulence Gene of Leptosphaeria maculans.

ABSTRACT A gene-for-gene interaction was previously suggested by mapping of a single major locus (LEM 1) controlling cotyledon resistance to Leptosphaeria maculans isolate PHW1245 in Brassica napus cv. Major. In this study, we obtained further evidence of a gene-for-gene interaction by studying the inheritance of the corresponding avirulence gene in L. maculans isolate PHW1245. The analysis of segregating F(1) progenies and 14 test crosses suggested that a single major gene is involved in the interaction. This putative avirulence gene was designated alm1 after the resistance locus identified in B. napus. Amplified fragment length polymorphism (AFLP) markers were used to generate a rudimentary genetic linkage map of the L. maculans genome and to locate markers linked to the putative avirulence locus. Two flanking AFLP markers, AC/TCC-1 and AC/CAG-5, were linked to alm1 at 3.1 and 8.1 cM, respectively. Identification of markers linked to the avirulence gene indicated that the differential interaction is controlled by a single gene difference between parental isolates and provides further support for the gene-for-gene relationship in the Leptosphaeria-Brassica system.

Comparison of flowering time genes in Brassica rapa, B. napus and Arabidopsis thaliana.

The major difference between annual and biennial cultivars of oilseed Brassica napus and B. rapa is conferred by genes controlling vernalization-responsive flowering time. These genes were compared between the species by aligning the map positions of flowering time quantitative trait loci (QTLs) detected in a segregating population of each species. The results suggest that two major QTLs identified in B. rapa correspond to two major QTLs identified in B. napus. Since B. rapa is one of the hypothesized diploid parents of the amphidiploid B. napus, the vernalization requirement of B. napus probably originated from B. rapa. Brassica genes also were compared to flowering time genes in Arabidopsis thaliana by mapping RFLP loci with the same probes in both B. napus and Arabidopsis. The region containing one pair of Brassica QTLs was collinear with the top of chromosome 5 in A. thaliana where flowering time genes FLC, FY and CO are located. The region containing the second pair of QTLs showed fractured collinearity with several regions of the Arabidopsis genome, including the top of chromosome 4 where FRI is located. Thus, these Brassica genes may correspond to two genes (FLC and FRI) that regulate flowering time in the latest flowering ecotypes of Arabidopsis.

Location of the self-incompatibility locus in an RFLP and RAPD map of Brassica oleracea.

A single locus controlling self-incompatibility (SI) was located on an RFLP and RAPD linkage map of Brassica oleracea by assaying for SI phenotype and segregation of an RFLP locus detected by the cloned St gene SLG6. The map was developed using an F2 population derived from a cabbage x broccoli cross and included 112 RFLP and 47 RAPD loci arranged into nine main linkage groups covering 921 cM. A portion of the population was assayed for St reaction by pollinating with the broccoli parent. The SI reactions cosegregated precisely with the RFLP locus detected by SLG6, and this locus mapped to linkage group 2.

A bayesian approach to detect quantitative trait loci using Markov chain Monte Carlo.

Markov chain Monte Carlo (MCMC) techniques are applied to simultaneously identify multiple quantitative trait loci (QTL) and the magnitude of their effects. Using a Bayesian approach a multi-locus model is fit to quantitative trait and molecular marker data, instead of fitting one locus at a time. The phenotypic trait is modeled as a linear function of the additive and dominance effects of the unknown QTL genotypes. Inference summaries for the locations of the QTL and their effects are derived from the corresponding marginal posterior densities obtained by integrating the likelihood, rather than by optimizing the joint likelihood surface. This is done using MCMC by treating the unknown QTL, genotypes, and any missing marker genotypes, as augmented data and then by including these unknowns in the Markov chain cycle alone with the unknown parameters. Parameter estimates are obtained as means of the corresponding marginal posterior densities. High posterior density regions of the marginal densities are obtained as confidence regions. We examine flowering time data from double haploid progeny of Brassica napus to illustrate the proposed method.

Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.

Mapping loci controlling the concentrations of erucic and linolenic acids in seed oil of Brassica napus L.

The quality of plant oil is determined by its component fatty acids. Relatively high levels of linolenic acid reduce the oxidative stability of the oil, and high levels of erucic acid in the diet have been associated with health problems. Thus, oilseed Brassica napus cultivars with low linolenic and low erucic acid contents are highly desirable for edible oil production. In order to identify genes controlling the levels of erucic and linolenic acids, we analyzed the oil composition of 99 F1-derived doubled haploid lines from a cross between cv 'Major' (high levels of erucic and linolenic acids) and cv 'Stellar' (low levels of both fatty acids). A molecular marker linkage map of 199 loci for this population was used to identify quantitative trait loci (QTL) controlling oil composition. We identified two regions that accounted for nearly all of the phenotypic variation in erucic acid concentration and one region that accounted for 47% of the variation in linolenic acid concentration. The QTL associated with linolenic acid concentration mapped near a RFLP locus detected by a cDNA clone encoding an omega-3 desaturase, suggesting that the low linolenic acid content of 'Stellar' may be due to a mutation in this gene.

Mapping loci controlling flowering time in Brassica oleracea.

The timing of the transition from vegetative to reproductive phase is a major determinant of the morphology and value of Brassica oleracea crops. Quantitative trait loci (QTLs) controlling flowering time in B. oleracea were mapped using restriction fragment length polymorphism (RFLP) loci and flowering data of F3 families derived from a cabbage by broccoli cross. Plants were grown in the field, and a total of 15 surveys were made throughout the experiment at 5-15 day intervals, in which plants were inspected for the presence of flower buds or open flowers. The flowering traits used for data analysis were the proportion of annual plants (PF) within each F3 family at the end of the experiment, and a flowering-time index (FT) that combined both qualitative (annual/biennial) and quantitative (days to flowering) information. Two QTLs on different linkage groups were found associated with both PF and FT and one additional QTL was found associated only with FT. When combined in a multi-locus model, all three QTLs explained 54.1% of the phenotypic variation in FT. Epistasis was found between two genomic regions associated with FT. Comparisons of map positions of QTLs in B. oleracea with those in B. napus and B. rapa provided no evidence for conservation of genomic regions associated with flowering time between these species.

Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution.

Although the evolutionary success of polyploidy in higher plants has been widely recognized, there is virtually no information on how polyploid genomes have evolved after their formation. In this report, we used synthetic polyploids of Brassica as a model system to study genome evolution in the early generations after polyploidization. The initial polyploids we developed were completely homozygous, and thus, no nuclear genome changes were expected in self-fertilized progenies. However, extensive genome change was detected by 89 nuclear DNA clones used as probes. Most genome changes involved loss and/or gain of parental restriction fragments and appearance of novel fragments. Genome changes occurred in each generation from F2 to F5, and the frequency of change was associated with divergence of the diploid parental genomes. Genetic divergence among the derivatives of synthetic polyploids was evident from variation in genome composition and phenotypes. Directional genome changes, possibly influenced by cytoplasmic-nuclear interactions, were observed in one pair of reciprocal synthetics. Our results demonstrate that polyploid species can generate extensive genetic diversity in a short period of time. The occurrence and impact of this process in the evolution of natural polyploids is unknown, but it may have contributed to the success and diversification of many polyploid lineages in both plants and animals.

RFLP mapping of quantitative trait loci controlling seed aliphatic-glucosinolate content in oilseed rape (Brassica napus L).

We report the RFLP mapping of quantitative trait loci (QTLs) which regulate the total seed aliphaticglucosinolate content in Brassica napus L. A population of 99 F1-derived doubled-haploid (DH) recombinant lines from a cross between the cultivars Stellar (low-glucosinolate) and Major (high-glucosinolate) was used for singlemarker analysis and the interval mapping of QTLs associated with total seed glucosinolates. Two major loci, GSL-1 and GSL-2, with the largest influence on total seed aliphatic-glucosinolates, were mapped onto LG 20 and LG 1, respectively. Three loci with smaller effects, GSL-3, GSL-4 and GSL-5, were tentatively mapped to LG 18, LG 4 and LG 13, respectively. The QTLs acted in an additive manner and accounted for 71 % of the variation in total seed glucosinolates, with GSL-1 and GSL-2 accounting for 33% and 17%, respectively. The recombinant population had aliphatic-glucosinolate levels of between 6 and 160 µmoles per g(-1) dry wt of seed. Transgressive segregation for high seed glucosinolate content was apparent in 25 individuals. These phenotypes possessed Stellar alleles at GSL-3 and Major alleles at the four other GSL loci demonstrating that low-glucosinolate genotypes (i.e. Stellar) may possess alleles for high glucosinolates which are only expressed in particular genetic backgrounds. Gsl-elong and Gsl-alk, loci which regulate the ratio of individual aliphatic glucosinolates, were also mapped. Gsl-elong-1 and Gsl-elong-2, which control elongation of the α-amino-acid precursors, mapped to LG 18 and LG 20 and were coincident with GSL loci which regulate total seed aliphatic glucosinolates. A third tentative QTL, which regulates side-chain elongation, was tentatively mapped to LG 12. Gsl-alk, which regulates H3CS-removal and side-chain de-saturation, mapped to LG 20.

Mapping loci controlling vernalization requirement in Brassica rapa.

Brassica cultivars are classified as biennial or annual based on their requirement for a period of cold treatment (vernalization) to induce flowering. Genes controlling the vernalization requirement were identified in a Brassica rapa F2 population derived from a cross between an annual and a biennial oilseed cultivar by using an RFLP linkage map and quantitative trait locus (QTL) analysis of flowering time in F3 lines. Two genomic regions were strongly associated with variation for flowering time of unvernalized plants and alleles from the biennial parent in these regions delayed flowering. These QTLs had no significant effect on flowering time after plants were vernalized for 6 weeks, suggesting that they control flowering time through the requirement for vernalization. The two B. rapa linkage groups containing these QTLs had RFLP loci in common with two B. napus linkage groups that were shown previously to contain QTLs for flowering time. An RFLP locus detected by the cold-induced gene COR6.6 cloned from Arabidopsis thaliana mapped very near to one of the B. rapa QTLs for flowering time.

Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. campestris).

Construction of a detailed RFLP linkage map of B. rapa (syn. campestris) made it possible, for the first time, to study individual genes controlling quantitative traits in this species. Ninety-five F2 individuals from a cross of Chinese cabbage cv 'Michihili' by Spring broccoli were analyzed for segregation at 220 RFLP loci and for variation in leaf, stem, and flowering characteristics. The number, location, and magnitude of genes underlying 28 traits were determined by using an interval mapping method. Zero to five putative quantitative trait loci (QTL) were detected for each of the traits examined. There were unequal gene effects on the expression of many traits, and the inheritance patterns of traits ranged from those controlled by a single major gene plus minor genes to those controlled by polygenes with small and similar effects. The effect of marker locus density on detection of QTL was analyzed, and the results showed that the number of QTL detected did not change when the number of marker loci used for QTL mapping was decreased from 220 to 126; however, a further reduction from 126 to 56 caused more than 15% loss of the total QTL detected. The detection of putative minor QTL by removing the masking effects of major QTL was explored.

Mapping loci controlling vernalization requirement and flowering time in Brassica napus.

Rapeseed cultivars (Brassica napus L.) can be classified into annual and biennial groups according to their requirement for vernalization in order to induce flowering. The genetic control of these phenotypic differences is not well understood, but this information could be valuable for the design of breeding approaches to accelerate rapeseed improvement. In order to map loci controlling this variation, a doubled haploid population, derived from a cross between annual and biennial cultivars, was evaluated for vernalization requirement and days-to-flowering in a replicated field experiment using three treatments: no vernalization, 4 weeks of vernalization and 8 weeks of vernalization. A linkage map of 132 RFLP loci was used to locate loci controlling these traits. Marker segregation in one region of linkage group 9 was strongly associated with the annual/biennial growth habit in the unvernalized treatment and with days-to-flowering in all three treatments. Two other regions with smaller effects on days-to-flowering were also identified.