Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hybridizations among Brassica napus, B. rapa and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis under open pollination conditions in the field

The rate of natural crosses occurring among the cultivated Brassica species B. napus, B. rapa, and B. juncea and their two weedy relatives B. nigra and Sinapis arvensis was studied in co-cultivation experiments under field conditions in Saskatchewan, Canada, with special reference to evaluation of the possibility of transgene escape from the cultivated to the weedy species. Natural crosses occurred among B. napus, B. rapa, and B. juncea, indicating that hybridizations among these three species do occur under field conditions. On the other hand, no natural crosses occurred between the cultivated species and B. nigra or S. arvensis. It is concluded that the crosses between the cultivated and weedy species are practically impossible under field conditions in Saskatchewan, and that the escape of transgenes from transgenic cultivars of B. napus, B. rapa and B. juncea into B. nigra and S. arvensis is basically zero in this region.     

Resynthesis of Brassica juncea through interspecific crosses between B. rapa and B. nigra

The objective of this study was to broaden the genetic base in oleiferous Brassica juncea by resynthesis, using 10 diverse parental lines of oleiferous B. rapa and two lines of B. nigra of both Indian and exotic origin. Out of 14 crosses attempted using B. rapa as the female parent, eight were successful. Embryo rescue was necessary to obtain interspecific plants. A total of 29 fertile interspecific plants were obtained after colchicine treatment. In the S₂ generation, the expression of component characters in the majority of the resynthesized plants showed a negative trend. The resynthesized B. juncea lines are being maintained through repeated selfing and selection at each generation for desirable plant types. This process will continue till the progeny lines of the desirable plants achieve uniformity.     

Genotypic Variation for Tolerance to Transient Drought During the Reproductive Phase of Brassica rapa

Brassica rapa L. is a genetically diverse parent species of the allotetraploid species, oilseed rape (B. napus) and a potential source of drought tolerance for B. napus. We examined the effect of a 13‐day drought stress period during the early reproductive phase, relative to a well‐watered (WW) control, on subsequent growth and development in nine accessions of B. rapa and one accession of Brassica juncea selected for their wide morphological and genetic diversity. We measured leaf water potential, stomatal conductance, water use, and leaf and bud temperatures during the stress period and aboveground dry weight of total biomass at maturity. Dry weight of seeds and reproductive tissue were not useful measures of drought tolerance due to self‐incompatibility in B. rapa. The relative total biomass (used as the measure of drought tolerance in this study) of the 10 accessions exposed to drought stress ranged from 47 % to 117 % of the WW treatment and was negatively correlated with leaf‐to‐air and bud‐to‐air temperature difference when averaged across the 13‐day stress period. Two wild‐type (B. rapa ssp. sylvestris) accessions had higher relative total and non‐reproductive biomass at maturity and cooler leaves and buds than other types. We conclude that considerable genotypic variation for drought tolerance exists in B. rapa and cooler leaves and buds during a transient drought stress in the early reproductive phase may be a useful screening tool for drought tolerance.     

Genetic diversity in advanced derivatives of Brassica interspecific hybrids

Genetic diversity among the 88 entries including eighty F₄ derivatives i.e., 20 each selected from Brassica crosses viz., B. juncea × B. napus, B. juncea × B. rapa var. toria, B. juncea ×B. rapa var. yellowsarson and B. tournefortii × B. juncea, and eight parent genotypes was assessed through multivariate analysis (D² statistic). Significant differences among the family groupsas well as within the family were recorded for all the 14 characters studied. The D² analysis revealed enormous diversity among the interspecific cross derivatives. The genetic distances calculated among different Brassica species revealed that B. tournefortii had maximumdiversity with B. juncea followed by B. napus, B.rapa var. toria and B. rapa var. yellow sarson.Amongst interspecific crosses, maximum diversity was noticed indescendants of cross B. tournefortii × B. juncea followed byB. juncea × B. napus, B. juncea × B.rapa var. toria and the least in the cross B. juncea ×B. rapa var. yellow sarson. These results indicated that the derivatives selected from cross of diverse parents revealed greater diversity. The clustering pattern showed that many derivatives of the cross fell into the same cluster but in many cases in spite of common ancestry many descendants of the cross spread over different clusters. The characters, namely, plant height, secondary branches per plant, days to flowering and1000-seed weight were contributed maximum towards genetic divergence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytoplasmic effects on photosynthesis and ribulose-1,5-bisphosphate carboxylase activity in Brassica species

Brassica genotypes representing four different species viz. B. nigra and B. campestris (both primary diploids), B. juncea and B. carinata (both allotetraploids) were examined for photosynthesis rate (Pn), ribulose-1,5-bisphosphate carboxylase (RuBPC) activity, leaf soluble protein and chlorophyll content. B. nigra is the male and female parent of B. juncea and B. carinata, respectively. B. campestris is the maternal parent of B. juncea. Pn was significantly higher in B. nigra and B. carinata compared to the other two species. These two species also sustained Pn of the leaves for a longer period during ontogeny. When B. nigra was the male parent, the higher Pn of B. nigra was not inherited in B. juncea. On the other hand, the Pn and pattern of ontogenetic changes in B. campestris and B. juncea were more or less similar. In specific RuBPC activity on soluble protein basis, chlorophyll and soluble protein content B. carinata also followed B. nigra whereas, B. juncea followed B. campestris. These results indicated the possible maternal(cytoplasmic) influence on the inheritance of these traits. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP analysis of genetic variability among Stylosanthes guianensis accessions resistant and susceptible to the stylo anthracnose

Stylosanthes guianensis, belonging to the genus Stylosanthes, is one of the most important tropical forage legumes and is native to South and Central America and Africa. Anthracnose, caused by the fungus Colletotrichum gloeosporioides (Penz.) Sacc., is a major constraint to the extensive use of Stylosanthes as tropical forage. Forty‐two accessions of S. guianensis were assessed with amplified fragment length polymorphism (AFLP) for genetic diversity and for resistance to anthracnose. In AFLP analysis, four selective primer combinations screened from 96 primer combinations were used to analyse these accessions, and a total of 225 clear bands were used for genetic similarity (GS) analysis, showing a 95.5% level of polymorphism on average. GS from 31.0% to 95.0% among the accessions was calculated with ntsys ‐pc software. The dendrogram was constructed with unweighted pair group method of averages (UPGMA) based on the AFLP data, and five clusters were defined at 48% GS. Two typical strains of C. gloeosporioides from Stylosanthes in China were used for anthracnose resistance screening. Most of the plant accessions showed variation in the reaction to two strains, and the correlation of resistance had a value of 0.904 (P < 0.01), suggesting common resistance to the two strains. The resistance accessions were randomly distributed in different groups of UPGMA clustering. These results demonstrate that AFLP analysis is an efficient method for evaluating the genetic diversity among S. guianensis accessions.     

Assessment of transgene escape from Brassica rapa (B. campestris) into B. nigra or Sinapis arvensis

The possibility of gene transfer between Brassica rapa and the two weedy species B. nigra and Sinapis arvensis was evaluated with the special concern on transgene escape from B. rapa to these two weedy species. B. rapa cultivar Tobin was reciprocally crossed to five and four strains of B. nigra and S. arvensis, respectively, using controlled cross. A single interspecific hybrid was obtained from the cross B. rapa×B. nigra, but no other cross was successful. The fertility of this hybrid on open pollination, selfing and backcrosses was investigated. The data of the present study and the information available to date indicate that gene transfer between B. rapa and B. nigra is possible. The chance of transgene escape from B. rapa to B. nigra depends essentially on whether natural cross can occur between these two species. Gene transfer between B. rapa and S. arvensis is at the most difficult, whereas trans-gene escape directly from B. rapa to S. arvensis appears very unlikely.     

Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea

To select superior seed parents for vegetable hybrid seed production, we conducted interspecific crosses between male sterile Brassica juncea (2n = 36, AABB) and eight inbred lines of Brassica rapa (2n = 20, AA). Alloplasmic lines of B. rapa with the cytoplasm of B. juncea were developed from B. juncea × B. rapa hybrids by repeated backcrossing using B. rapa as the recurrent male parent until the BC₃ generation. Seed fertility, male sterility and chlorophyll content were investigated in these plants cultivated under four different temperature conditions (5, 10, 12 and 20°C). At 10°C, the alloplasmic lines of B. rapa with the cytoplasm of B. juncea were male sterile with partly chlorotic leaves. The alloplasmic B. rapa had lower chlorophyll a, chlorophyll b and carotenoid contents than those of the original B. rapa. The leaves recovered from chlorosis when the plants were cultivated at 20°C. An alloplasmic line of B. rapa (A6) is available as a seed parent for vegetable hybrid seed production and contributes seed fertility, slight chlorosis and stable male sterility.     

Assessment of genetic diversity in selected breeding lines and cultivars of canola quality Brassica juncea and their implications for canola breeding

AFLP markers were used to assess the genetic diversity of 77 breeding lines from three of the world's major canola qualityBrassica juncea breeding programs from Canada (Agriculture and Agri-Food Canada and Saskatchewan Wheat Pool) and Australia (Agriculture Victoria). The objectives of the paper were to assess the genetic diversity within and between these three breeding programs and to assess genetic diversity of the canola quality germplasm as compared to mustard quality B. juncea. Fifteen lines of mustard quality B. juncea from India, China, Russia and Australia were also included in the investigation. Ten EcoR1/Mse1 based primer pairs generated 751 scorable fragments with an average of 26 polymorphic bands per primer pair (35%). Similarity coefficients were calculated using the Simple Matching coefficient and adendrogram was developed using the UPGMA procedure, resulting in germplasm being partitioned into five main groups. Line specific markers were discovered that have potential in enhancing the efficiency of individual breeding programs using breeding techniques like accelerated backcrossing. Further understanding the genetic diversity within and between programs has implications for future breeding and collaboration within and between the three programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

中国西部芥菜型油菜遗传多样性研究

利用23对SRAP引物、11对AFLP引物和10对SSR引物对我国西部地区的芥菜型油菜及其近缘种108份材料进行了遗传多样性分析, 共检测到313个等位变异, 3种标记的每对引物平均分别可检测到6.8、12.5和1.9个等位变异。包括白菜型和芸芥在内的108份品种间遗传相似系数在0.378~0.936之间, 103份芥菜型油菜品种间遗传相似系数在0.545~0.936之间。聚类分析结果表明, 在相似系数0.558处, 5个参照品种白菜型油菜、小白菜以及芸芥首先被聚出芥菜型油菜之外; 在相似系数0.70处, 103份芥菜型油菜可分为云贵陕南冬播(A)、关中冬播(B)、新疆I (C)、新疆II (D)和西部春播(E)五个类群, 其中A、B基本为冬播品种, C、D、E为春播品种。A类群品种间遗传差异最大, B类群其次。陕西和新疆的品种均分别被聚到3个类群, 表现出更广泛的遗传多样性。春播类型绝大部分被聚到E类群, E类群可分为3个亚类, 其中陕北及其邻近一带春播黄芥为一类, 形成一个独立的遗传群体, 群内遗传多样性较高; 西藏的10个品种为一类, 相似系数高达0.83以上, 表现出西藏品种遗传系统的独立和遗传基础的单一; 澳大利亚2个品种单独为一类, 与我国的春播品种关系较近。由此说明, 地理和生态条件是影响芥菜型油菜类群的主要因素, 我国的冬播品种间的遗传多样性高于春播品种, 陕西和新疆的芥菜型油菜遗传多样性较高。     

Analysis of bulked and redundant accessions of Brassica germplasm using assignment tests of microsatellite markers

This study was conducted to determine if Brassica germplasm bulks created and maintained by the USDA-ARS North Central Plant Introduction Station (NCRPIS) were made with genetically indistinguishable component accessions and to examine newly identified putative duplicate accessions to determine if they can be bulked. Using ten microsatellite primer pairs, we genotyped two bulks of B. rapa L. ssp. dichotoma (Roxb.) Hanelt comprising four accessions and three bulks of B. rapa L. ssp. trilocularis (Roxb.) Hanelt comprising fourteen accessions, as well as four pairs of putatively duplicate accessions of B.␣napus L. Assignment tests on ten individual plants per accession were conducted using a model-based clustering method to arrive at probabilities of likelihood of accession assignment. The assignment tests indicated that one of the two bulks of B. rapa ssp. dichotoma involves genetically heterogeneous accessions. It was observed in the B. rapa ssp. trilocularis bulks that the component accessions could be differentiated into groups, with misassignments observed most frequent within groups. In B. napus, only one of the four pairs of putative duplicates showed significant genetic differentiation. The other three pairs of putative duplicates lack differences and support the creation of bulks. The results of the assignment tests were in agreement with cluster analyses and tests of population differentiation. Implications of these results in terms of germplasm management include the maintenance and/or re-creation of some Brassica germplasm bulks by excluding those accessions identified as being unique in this study.     

Screening and identification of resistance to bacterial soft rot in Brassica rapa

Mist-chamber, field, and detached leaf inoculation procedures identified plants resistant to bacterial soft rot [Erwinia carotovora subsp. carotovora, (Ecc)] in Brassica rapa and related species. The mist-chamber seedling inoculation provided the best correlation of mean disease severity ratings with the field plant inoculation (r = 0.67^**) and was used to identify resistant materials. The optimum mist-chamber incubation conditions to distinguish the resistance of accessions were 23 ^°C and 100% RH and were used for primary screening. A total of 752 accessions of B. rapa and related Cruciferae were screened. In general, accessions of B. oleracea were more resistant than accessions of B. rapa. Within B. rapa, subspecies pekinensis and chinensis were more susceptible than other subspecies. No completely resistant material was found. In species of B. rapa, only 7% of accessions showed some degree of resistance with plant-to-plant variation within the accessions. G30444, G30449, and AVRDC2837 were identified as the most resistant materials in B. rapa by both mist-chamber and field inoculations. The resistance was correlated between an USA isolate (Geneva-1) and two Chinese isolates (RL4-1 and RL-19) of Ecc. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity in cornsalad (Valerianella locusta) and related species as determined by AFLP markers

Fifteen amplified fragment length polymorphism (AFLP) EcoRI/MseI‐based primer combinations with five selective bases (Eco RI‐ANN, MseI‐CN) were used to estimate genetic diversity among 45 line varieties of cultivated cornsalad and 19 genebank accessions classified into nine different species related to cornsalad. Polymorphic fragments were scored for calculation of Jaccard's coefficient of genetic similarity (GS). The average GS estimate in elite germplasm (GS = 0.90) was substantially higher than in exotic germplasm (GS = 0.47). UPGMA‐cluster analysis revealed genetic relationships among recently bred varieties, old varieties and genebank accessions. Analysis of molecular variance indicated almost threefold variability within sets compared with between sets due to a high level of polymorphism among wild species. Sources for increasing genetic diversity in elite germplasm of cornsalad were suggested and a duplicate among the genebank accessions was detected. AFLPs could be considered a powerful tool for genetic diversity estimation in cornsalad germplasm and are recommended for systematic fingerprinting of remaining cornsalad species.     

Pedigree, RAPD and simplified AFLP-based assessment of genetic relationships among Avena sativa L. cultivars

Two molecular marker techniques: RAPD and simplified ^PstIAFLP have been compared in order to decide on, which technique is better suited to genetic characterization of oat (Avena sativa L.) cultivars. It was investigated, if the same pattern of variability is revealed by two approaches and whether the observed molecular variability reflects pedigree-based relationships. Polymorphic RAPD and ^PstIAFLP markers were sufficient to distinguish all analysed cultivars, demonstrating the usefulness of both methods for cultivar identification. Genetic similarity estimates derived from RAPD, simplified ^PstIAFLP and combined RAPD and ^PstIAFLP data were compared with coefficients of parentage (COP). Molecular markers-based mean genetic similarities were considerably greater than mean COP value. Correlation coefficients between COP and genetic similarities calculated from RAPD, ^PstIAFLP and combined molecular data were very low and not significant. A better correlation (0.50) was found between similarity estimates derived from RAPD and ^PstIAFLP markers. Four separate dendrograms were constructed based on pedigree and molecular analyses using a neighbor-joining algorithm (NJ). The dendrograms were compared and found to be topologically different. The results of this study showed, that both molecular techniques can be conveniently used for genetic characterization of oat cultivars, however ^PstIAFLP would be the method of choice due to the higher efficiency and reproducibility. This revised version was published online in August 2006 with corrections to the Cover Date.     

AFLP genetic diversity analysis in Russian wheat aphid resistant wheat accessions

Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), is an important insect pest which causes severe economic losses in wheat (Triticum spp.). Among the various U.S. RWA biotypes, biotype 1 (RWA1) and biotype 2 (RWA2) are the most prevalent and most virulent on cultivated genotypes. Although many sources of resistance to these biotypes are available among landraces, their relatedness should be characterized to permit their more efficient use in breeding programs. In this study, 38 hexaploid accessions resistant to biotype 1 and/or biotype 2 were evaluated for genetic diversity based on amplified fragment length polymorphisms (AFLP). Fifteen AFLP selective primer combinations were used to genotype these accessions, resulting in 893 amplicons. Of these, 274 (30.6%) informative polymorphic bands were used for genetic diversity analysis. Genetic similarity coefficients ranged from 0.47 to 0.87 among the resistant accessions, indicating high genetic diversity among them. Cluster analysis grouped the 38 accessions into two major clusters, I and II, including resistant lines for RWA1 and RWA2. The study indicated that accessions in the National Small Grains Collection conferring RWA1 or RWA2 resistance comprise a diversified population which should support introgression efforts and provide genetic diversity for future breeding for RWA resistance.     

Cytogenetics of Brassica juncea×Brassica rapa hybrids and patterns of variation in the hybrid derivatives

Interspecific hybridization is an important tool to elucidate intergenomic relationships, transfer characters across species and develop synthetic amphidiploids, and it has been widely applied for improving Brassicas. The objective of the present study was to create genetic variability in Brassica through interspecific hybridization. Crosses between Brassica juncea (AABB, 2n= 36), and Brassica rapa (AA, 2n = 20) vars toria, yellow sarson, and brown sarson were attempted, and the hybrid derivatives were advanced to the F4 generation. Hybrids were obtained from the crosses B. juncea× toria and B. juncea× yellow sarson. The F₁ plants were vigorous and intermediate to the parents in many morphological traits. The meiotic study of AAB hybrids showed 10 II + 8 I in the majority (71.8%) of cells analysed. A maximum of 12 and a minimum of seven bivalents were also observed in a few cells. The occurrence of multivalent associations (trivalents to pentavalents) at diakinesis/metaphase I and a bridge‐fragment configuration at anaphase I were attributed to homoeology between A and B genomes. A high percentage of plants resembling B. juncea was observed in the F₂ generation. Transgressive segregation in both directions was found for plant height, primary branches, main raceme length, siliquae on main raceme, siliqua intensity, seeds per siliqua and seed yield. There were significant differences for the 14 characters in the F₄ derivatives. Moderate to high estimates of phenotypic and genotypic coefficients of variation, broad‐sense heritability, and expected genetic advance were found for seed yield, 1000‐seed weight, siliquae per plant, seeds per siliqua and days to flowering. Intergenomic recombination, reflected as wide variation in the hybrid progenies, permitted the selection of some useful derivatives.     

Inheritance of the resistance to Leptosphaeria maculans of Brassica nigra and B. juncea in near-isogenic lines of B. napus

The inheritance of resistance to Leptosphaeria maculans was studied in near-isogenic lines derived from asymmetric somatic hybrids between Brassica napus+Brassica nigra and Brassica napus+Brassica juncea, respectively. The hybrids had been backcrossed to B. napus for seven generations before the genetic segregation of the blackleg resistance was determined. The results of the inheritance studies suggested that one single dominant allele controls the resistance in the Brassica napojuncea line, whereas two independent dominant loci were found in the Brassica naponigra line. Total leaf DNA from the near-isogenic lines was isolated and 89 loci were detected by hybridization to 66 restriction fragment length polymorphism (RFLP) markers previously mapped in the B. nigra genome. Out of the 89 loci, eight loci were detected in the B. naponigra line and six were found in the B. napojuncea line. RFLP markers co-segregating with blackleg resistance in adult leaves were also found. Two markers associated with linkage group 5 and 8, respectively, of the B genome were found in the B. naponigra line and one marker was associated with linkage group 2 in the B. napojuncea line.