Analysis Of Allelic Diversity Research Articles

Analysis of locus estimates of allelic diversity of STR markers in a sample of breeding bulls

Genotypes of the 11 DNA microsatellite loci of 84 bulls of seven breeds were used to evaluate 14 indicators of allelic diversity/differentiation. Traditional and multidimensional statistical methods were applied to the data matrices from the original and transformed estimates (11×14). Estimates of heterozygosity had coefficients of variability of 8-14 %, the number of alleles per locus and indicators of differentiation of breeds at the level of 20-26 %, fixation indices – 38-44 %. Statistically significant Kendall correlations (0.8-1.0) between indicators of allelic richness and heterozygosity, fixation indices, and differentiation indicators were established. The variability of the transformed estimates of diversity/differentiation indicators by loci was in the range of 6-32 %. Including by loci Eth3, Tgla122, Eth225, Bm2113 – 6-12 %, loci Inra23, Tgla126, Eth10 – 15-20 %, loci Tgla227, Sps115, Tgla53, Bm1824 – 28-32 %. The nonparametric Mann-Whitney-Wilcoxon test showed statistically significant differences in the medians of the Eth3 locus with the Bm2113 locus, the Tgla126 locus with the Eth3, Inra23, Tgla122, Eth225, Bm2113, Bm1824, Eth10 loci. The principal component analysis (PCA) identified two components with a total information content of 95,2 %. The first one took into account 59.4 % of the total variance, had the highest loads in intra-breed diversity data and was defined as an «alpha component». The second accounted for 35.8 % of the total variance, had the highest loads in inter-breed differentiation data and was defined as a «beta component». 2D-PCA-ordination showed that a characteristic grouping of loci took place for the analyzed breeds (samples), loci and measures of diversity. Loci Tgla227 and Tgla53 formed group A, group B – loci Tgla122, Eth225, Eth10, group C – loci Inra23, Bm2113 and Bm1824. The loci of the conditional group D (Eth3, Tgla126, Sps115) were defined as «untypical». Validation of ordination was confirmed by calculations on reduced data (dimension 11×7) and the method of non-metric multidimensional scaling (nMDS). The consistency of ordinations according to the Procrust test was 96 % (pperm <0.001). A similar classification of loci was obtained by cluster analysis (UPGMA) with butstrap probabilities of cluster: A – 73, B – 100, C – 73, D – 47 %. The distances and similarity indicators (S) between the profiles of loci and the «true» summary estimates for 11 loci were calculated. Loci Tgla126 and Sps115 had S ≈ 40 %, loci Tgla53 and Bm1824 – at the level of 60 %, loci Inra23, Tgla227 and Bm2113 – 70-75 %, loci Eth3, Tgla122, Eth225 and Eth10 – 84-88 %. The average absolute deviation of the estimates of diversity indicators for the four loci with S≥84 % from the «true» estimates was 3.4 %, for the four loci with S≤60 % – 12.4 %. According to component scores, a general diversity index, γLV, was calculated for each locus. Its correlation with the estimates of the Shannon/Sherwin′s γ-diversity with a 95 % probability value was in the range of 0.73-0.98, Kendall's rank correlation was 0.67 (pvalue = 0.005). The conducted research makes a certain contribution to the expansion of tools for processing molecular genetic data in the analysis of allelic diversity in subdivided populations.

Multi-locus genome-wide association studies reveal the genetic architecture of Fusarium head blight resistance in durum wheat.

Durum wheat is more susceptible to Fusarium head blight (FHB) than other types or classes of wheat. The disease is one of the most devastating in wheat; it reduces yield and end-use quality and contaminates the grain with fungal mycotoxins such as deoxynivalenol (DON). A panel of 265 Canadian and European durum wheat cultivars, as well as breeding and experimental lines, were tested in artificially inoculated field environments (2019-2022, inclusive) and two greenhouse trials (2019 and 2020). The trials were assessed for FHB severity and incidence, visual rating index, Fusarium-damaged kernels, DON accumulation, anthesis or heading date, maturity date, and plant height. In addition, yellow pigment and protein content were analyzed for the 2020 field season. To capture loci underlying FHB resistance and related traits, GWAS was performed using single-locus and several multi-locus models, employing 13,504 SNPs. Thirty-one QTL significantly associated with one or more FHB-related traits were identified, of which nine were consistent across environments and associated with multiple FHB-related traits. Although many of the QTL were identified in regions previously reported to affect FHB, the QTL QFhb-3B.2, associated with FHB severity, incidence, and DON accumulation, appears to be novel. We developed KASP markers for six FHB-associated QTL that were consistently detected across multiple environments and validated them on the Global Durum Panel (GDP). Analysis of allelic diversity and the frequencies of these revealed that the lines in the GDP harbor between zero and six resistance alleles. This study provides a comprehensive assessment of the genetic basis of FHB resistance and DON accumulation in durum wheat. Accessions with multiple favorable alleles were identified and will be useful genetic resources to improve FHB resistance in durum breeding programs through marker-assisted recurrent selection and gene stacking.

Haplotype analysis of SCM2 loci for identification of donor for lodging resistance in rice

Lodging resistance is one of the vital traits in improving the yield and sustainability of production in rice (Oryza sativa L.). A quantitative trait loci (QTL), SCM2 for enhancing the culm strength and increased spikelet number has been reported. In the present study, a diverse collection of 150 accessions from the rice 3K-RG panel were assembled and evaluated for the STRONG CULM2 locus. All the accessions under study showed a normal phenotypic distribution for culm diameter and the allelic diversity analysis for SCM2 resulted in four INDEL’s and 17 SNP’s in both coding and non-coding regions of the genome. Haplotype analysis grouped the entire population into three significant haplotype groups with 114 accessions in H1, 19 accessions in H2 and 17 accessions in H3. Haplotypes were formed using two significant SNP sites viz., 27480778 and 27481339. Dunnett’s test disclosed a significant variation between H1-H2 and H1-H3 haplotypes. Phenotypic evaluation for culm diameter in all the three haplotype groups revealed that the genotypes BR IRGA and PERUNEL in H1, CICA 9 and MOSHI in H2 and KITRANA in H3 possess a maximum culm diameter. H2 and H3 were found to be superior to H1 hence can be utilized for lodging resistance in further breeding programs. Key words: Lodging resistance, SCM2, culm strength, allelic diversity and haplotype analysis.

Deriving tropical gynoecious inbred backcross lines from exotic pickling cucumber using marker assisted backcross breeding

Indian cucumber cultivars are basically monoecious and vigorous in nature with many lateral branches but they are poor in productivity. The present study was aimed to introgress gynoecious trait (F locus) from pickling cucumber line (Gy14) into elite Indian cultivar Pusa Uday using marker assisted backcross breeding. Based on allelic diversity analysis, the allele numbers per locus ranged from 2 to 3 in BC1F3 and the observed heterozygosity (Ho) was ranged from 0.01 to 0.55. The heterozygosity expected (He) mean was 0.28 (range 0.03 to 0.50) which was higher than mean Ho (0.18) and mean polymorphic information content (PIC) for 87 markers was 0.23. Whereas the allele numbers per locus was 2 in BC2F2 and the observed heterozygosity (Ho) was ranged from 0.01 to 0.56. The mean expected heterozygosity (He) was 0.24 which was higher than mean Ho (0.17) and the mean PIC value was 0.19. The gynoecism (F locus) was introgressed in the Indian slicing cucumber cv. Pusa Uday though backcross breeding accompanied by foreground and background selections. The foreground selection was performed with one dominant SCAR marker, and 3 SSR markers (SSR 00233, SSR 15516 and SSR13251). A total of 87 polymorphic markers spanning genetic distance of 1031 cM with an average distance of 12.9 cM between adjacent markers were used for background selection. The genome recovery of the individuals in BC1F3 population was ranged from 62.64% to 93.10% while among BC2F2 plants it was ranged from 89.66% to 95.40%. Four inbred backcross lines (IBL) were selected from backcross populations (BC1F4 and BC2F3) those were purely gynoecious even at 45°C with more than 5 lateral branches. Hence, marker assisted backcross breeding served as a potential approach for precise introgression of the gynoecy (F locus) from picking cucumber to Indian slicing cucumber.

Identification and mapping of CpPM10.1, a major gene involved in powdery mildew (race 2 France of Podosphaera xanthii) resistance in zucchini (Cucurbita pepo L.).

Powdery mildew resistance in zucchini is controlled by one major dominant locus, CpPM10.1. CpPM10.1 was fine mapped. The expression of candidate gene Cp4.1LG10g02780 in resistant individuals was significantly upregulated after inoculation with the powdery mildew. Powdery mildew (PM) is one of the most destructive fungal diseases, reducing the productivity of Cucurbita crops globally. PM influences the photosynthesis, growth and development of infected zucchini and seriously reduces fruit yield and quality. In the present study, the zucchini inbred line 'X10' had highly stable PM resistance, and the inbred line 'Jin234' was highly susceptible to PM in the seedling stage and adult stages. Genetic analysis revealed that PM resistance in 'X10' is controlled by one major dominant locus. Based on the strategy of QTL-seq combined with linkage analysis and developed molecular markers, the major locus was found to be located in a 382.9-kb candidate region on chromosome 10; therefore, the major locus was named CpPM10.1. Using 1,400 F2 individuals derived from a cross between 'X10' and 'JIN234' and F2:3 offspring of the recombinants, the CpPM10.1 locus was defined in a region of approximately 20.9kb that contained 5 coding genes. Among them, Cp4.1LG10g02780 contained a conserved domain (RPW8), which controls resistance to a broad range of PM pathogens. Cp4.1LG10g02780 also had nonsynonymous SNPs between the resistant 'X10' and susceptible 'Jin234.' Furthermore, the expression of Cp4.1LG10g02780 was strongly positively involved in PM resistance in the key period of inoculation. Further allelic diversity analysis in zucchini germplasm resources indicated that PM resistance was associated with two SNPs in the Cp4.1LG10g02780 RPW8 domain. This study not only provides highly stable PM resistance gene resources for cucurbit crops but also lays the foundation for the functional analysis of PM resistance and resistance breeding in zucchini.

Fine mapping identified the gibberellin 2-oxidase gene CpDw leading to a dwarf phenotype in squash (Cucurbita pepo L.)

Dwarfism is an important agronomic trait in pumpkin that can increase yield. In this study, the dwarf Cucurbita pepo L. line X10 exhibited significantly longitudinally shorter cell length in the stem than did the normal-vine line JIN234. The dwarf stature of X10 was recovered with exogenous gibberellin (GA3) application, suggesting that X10 might be sensitive to GA biosynthesis. Genetic analysis revealed that this dwarf trait is controlled by a single completely dominant locus: CpDw (Cucurbita pepo L. Dwarf). Using 1,300 F2 individuals derived from a cross between X10 and JIN234, we mapped the CpDw locus to a region of approximately 24.6 kb on chromosome 10 that contain 5 annotated genes. The high expression level of Cp4.1LG10g05910.1 and high GA2ox enzyme activity in X10 revealed that the GA 2-oxidase gene Cp4.1LG10g05910.1 is a candidate gene for CpDw. Alignment of the Cp4.1LG10g05910.1 gene revealed two nonsynonymous single nucleotide polymorphism (SNP) mutations in the two exons, as well as several SNPs and InDels in the important functional elements of promoter between parental lines. Further allelic diversity analysis of the Cucurbita spp. germplasm resources indicated that Cp4.1LG10g05910.1 may be involved in vine growth during the early developmental stage in C. pepo but not in C. maxima or C. moschata. This study provides an important theoretical basis for the genetic regulation of vine length and crop breeding in pumpkin.

Allogeneic hematopoietic stem cell transplant recipients in Spain: Human leukocyte antigen characteristics and diversity by high-resolution analysis.

There are many studies on the polymorphism of the HLA system in healthy donor populations, such as registries of unrelated bone marrow donors. Investigations on the characterization of the HLA complex in hematopoietic stem cell transplant (HSCT) patients, however, are scarce, at least in the Spanish population. This study presents a large-scale analysis of allelic diversity and HLA distribution at a high-resolution level in 2886 patients undergoing HSCT in Spanish centres of the "Grupo Español de Trasplante Hematopoyético y Terapia Celular" during a period of 11 years. Allelic diversity analysis identified 67 HLA-A, 133 HLA-B, 60 HLA-C, 63 HLA-DRB1, 24 HLA-DQB1 and 27 HLA-DPB1 different alleles. Rare alleles were detected among which 33 alleles had not been reported in the European catalog of common and well-documented HLA alleles. Regarding the distribution of five genes-haplotypes, it was observed that the five most frequent extended haplotypes found in our population were between the most common in other Spanish populations, both in patients and in healthy subjects. However, some particular haplotypes were also detected. Bilocus associations HLA-C ~ B and -DRB1 ~ DQB1 were analyzed in order to predict the probability of finding 10/10 matched donors in registries. We found HLA-B alleles showing a great diversity of combinations with HLA-C alleles and unusual associations involving a negative predicting factor. In the field of adoptive therapies, our work supports the necessity to expand further research of TCR-engineered cells, adoptive transfer of virus-specific T-cells and vaccines to target HLA alleles other than A*02:01. HLA alleles such as A*01:01, A*03:01, A*24:02, B*44:03, B*07:02 or B*51:01, might be considered new targets due to its high frequency in our population.

Comparative characteristics of the genetic structure of the Syrian cattle breed compared to Holstein and Aberdeen-Angus breeds.

Objectives:The objective of this study was to perform a comparative analysis of allelic diversity to reveal population-genetic characteristics of animal breeds, namely Shami (SH), Holstein (HLS), and Aberdeen-Angus (A-A).Materials and Methods:The genetic materials of SH breed animals represented by wool with hair follicles were collected from 39 SH heads in Syria. Also, genetic materials of HLS breed of American selection (n = 55, HLS) and bulls and cows of A-A breed bred at breeding enterprises in Russia (n = 30, A-A) were collected. Genetic differences between the cattle groups were studied using 11 microsatellite markers. Results:The cattle breed in Syria was characterized by high genetic diversity, 107 alleles, while the average number of alleles per microsatellite locus was 9.23, which is significantly higher than that in the animals of HLS (6.18) and A-A (5.00). When analyzing the genetic equilibrium for individual locus in SH breed, a deviation from equilibrium at four loci was revealed: TGLA227, SPS115, TGLA122, and ETH225; at one locus in HLS breed: SPS115, for A-A breed: at two loci, i.e., TGLA122 and ETH225. When assessing the level of genetic consolidation, a deficiency of heterozygotes was observed in two of the three studied breeds: 4.8% for SH and 8.0% for A-A. A slight excess of heterozygotes was found in the HLS breed at the level of 0.2%. The average comparative measurement of genetic variation in different populations value for 11 loci for all breeds was 0.069, which indicates that 93.1% of the total variability is due to the intra-breed diversity, and only 6.9% is due to the differences between breeds. Conclusion:The analysis of the animals belonging to their breed has shown a 100% genetic consolidation and the compliance of individual animals with the respective breeds. The study of genetic distances, adjusted for small samples, revealed the smallest genetic distance between the SH breed and HLS breed, equaling 0.107. The A-A breed, which has its separate origin and has never been imported into the Syrian Arab Republic, adjoins this cluster as an independent branch. Microsatellites can be used as an essential criterion for assessing the population-genetic characteristics of groups of cattle of various breeds (degree of polymorphism, level of heterozygosity, fixation indices, genetic group membership).

Field Performance of Five Soybean Mutants Under Drought Stress Conditions and Molecular Analysis Using SSR Markers

The objective s of this research w ere to evaluate (1) the performance of soybean mutant lines under drought stress condition s, and (2) the genetic diversity and relationship among the mutant lines using SSR markers. The field evaluation was conducted during the dry season of 2011 and 2012 at the experimental F arm of Mataram University, West Nusa Tenggara, Indonesia. The field experiment was set up in a randomized block design . Ten mutant lines and two control varieties were evaluated in four replications. Genetic distance among evaluated lines were determined based on allelic diversity analysis using 40 s imple sequence repeat (SSR) loci. Under drought stress conditions, two mutant lines , K dl 3 and K dl 8 , showed a better performance compared to the other ones. The h igh yielding mutant lines were K dl 3 and K dl 8, which yielded 1.75 t ha -1 and 1.69 t ha -1 , respectively, compared to the parent and national control , Panderman 1.43 t ha -1 and Muria 1.32 t ha -1 . These mutant lines required 30.75 to 32 days to flower and 79.75 to 83.75 day to harvest with relatively short plant height 28.25 and 23.35 cm respectively. T hose mutant characters were better than those of the other three mutants, the original parents, and the control soybean species. Since the evaluated soybean mutant lines yielded more under drought stress conditions than the standard varieties, t he y can be used and registered as drought-tolerant soybean mutant s . Moreover, the evaluated soybean accessions showed a wide genetic distance. The accessions were clustered into two groups according to their genetic background, namely group I (the Panderman with three mutant lines) and group II (the Muria with two mutant lines). Twenty-three out of 40 evaluated SSR loci, including AW31, BE806, CMAC7L, S080, S126, S57, S171, S224, S285 , S 294 , S 393 , S294 , S 383, S511 , S 511, S 5 20 , S 5 40, S547, S551, S571, S577, and S578, provided polymorphic alleles between the parents and their mutants and could be used to differentiate mutants from their parents. Therefore, these loci are informative and may be use ful for further analysis of soybean mutant lines in relation to breeding program.

The Cytochrome P450 Gene CsCYP85A1 Is a Putative Candidate for Super Compact-1 (Scp-1) Plant Architecture Mutation in Cucumber (Cucumis sativus L.).

The dwarf or compact plant architecture is an important trait in plant breeding. A number of genes controlling plant height have been cloned and functionally characterized which often involve in biosynthesis or signaling of plant hormones such as brassinosteroids (BRs). No genes for plant height or vine length have been cloned in cucurbit crops (family Cucurbitaceae). From an EMS-induced mutagenesis population, we identified a super compact (SCP) mutant C257 which was extremely dwarf due to practically no internode elongation. Under dark growing condition, C257 did not undergo skotomorphogenesis and its mutant phenotype could be rescued with exogenous application of brassinolide (BL), suggesting SCP might be a BR-deficient mutant. Segregation analysis revealed a single recessive gene scp-1 that was responsible for the SCP mutation. Map-based cloning combined with a modified MutMap identified CsCYP85A1, a member of the plant cytochrome P450 monooxygenase gene family, as the most possible candidate gene for scp-1, which encodes a BR-C6-oxidase in the BR biosynthesis pathway. We show that a SNP within the second exon of scp-1 candidate gene caused the SCP phenotype. Three copies of the CsCYP85A gene are present in the cucumber genome, but only the scp-1/CsCYP85A1 gene seemed active. The expression of CsCYP85A1 was higher in flowers than in the leaves and stem; its expression in the wild type (WT) was feedback regulated by BL application. Its expression was reduced in C257 as compared with the WT. This was the first report of map-based cloning of a plant height gene in cucurbit crops. The research highlighted the combined use of linkage mapping, an improved MutMap method and allelic diversity analysis in natural populations in quick cloning of simply inherited genes in cucumber. The roles of CsCYP85A1 in regulation of internode elongation in cucumber was discussed.

Genetic dissection of seed weight by QTL analysis and detection of allelic variation in Indian and east European gene pool lines of Brassica juncea.

Seed weight QTL identified in different populations were synthesized into consensus QTL which were shown to harbor candidate genes by in silico mapping. Allelic variation inferred would be useful in breeding B. juncea lines with high seed weight. Seed weight is an important yield influencing trait in oilseed Brassicas and is a multigenic trait. Among the oilseed Brassicas, Brassica juncea harbors the maximum phenotypic variation wherein thousand seed weight varies from around 2.0g to more than 7.0g. In this study, we have undertaken quantitative trait locus/quantitative trait loci (QTL) analysis of seed weight in B. juncea using four bi-parental doubled-haploid populations. These four populations were derived from six lines (three Indian and three east European lines) with parental phenotypic values for thousand seed weight ranging from 2.0 to 7.6g in different environments. Multi-environment QTL analysis of the four populations identified a total of 65 QTL ranging from 10 to 25 in each population. Meta-analysis of these component QTL of the four populations identified six 'consensus' QTL (C-QTL) in A3, A7, A10 and B3 by merging 33 of the 65 component Tsw QTL from different bi-parental populations. Allelic diversity analysis of these six C-QTL showed that Indian lines, Pusajaikisan and Varuna, hold the most positive allele in all the six C-QTL. In silico mapping of candidate genes with the consensus QTL localized 11 genes known to influence seed weight in Arabidopsis thaliana and also showed conserved crucifer blocks harboring seed weight QTL between the A subgenomes of B. juncea and B. rapa. These findings pave the way for a better understanding of the genetics of seed weight in the oilseed crop B. juncea and reveal the scope available for improvement of seed weight through marker-assisted breeding.

Current state of the genetic polymorphism in spring barley (Hordeum vulgare L.) from Russia assessed by the alleles of hordein-coding loci

Starch gel electrophoresis was performed to study the polymorphism of hordeins encoded by the Hrd A, Hrd B, and Hrd F loci in 211 varieties of spring barley. For 41 of these varieties, the genetic formulas were established for the first time. In the two samples of varieties, the comparative analysis of allelic diversity and allele frequencies of hordein-coding loci was carried out. The first sample consisted of 101 spring barley varieties approved for the use on the territory of the Russian Federation in 1999, while the second sample included 160 spring barley varieties that were approved in 2014; 49 of these varieties were common for both samples. It is demonstrated that the current tendency to reduction of the proportion of heterogeneous spring barley varieties is mainly due to the introduction of foreign varieties homogeneous for the hordein-coding loci. At the same time, there is an increase in polymorphism of hordein-coding loci in modern spring barley varieties. The number of alleles for the Hrd A locus increased by five alleles, and for the Hrd B locus, by nine alleles. Along with the alleles recorded earlier in barley landrace populations and varieties bred in 20th century, three novel alleles of the Hrd A locus and four alleles of the Hrd B locus were identified. The number of alleles of the Hrd F locus remained unchanged (four), and the changes in their frequencies were small. At the same time, the changes in frequency observed for some alleles of the Hrd A and Hrd B loci were statistically significant. All newly identified alleles of hordein-coding loci were found with low frequencies (from 0.003 to 0.006), so despite the increased number of alleles, no statistically significant increase in genetic diversity in terms of μ and PIC indices was observed.

Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne.

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome-wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L.perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L.perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.

Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae)

Analyses of genetic structure and phylogenetic relationships illuminate the origin and domestication of modern crops. Despite being an important worldwide vegetable, the genetic structure and domestication of carrot (Daucus carota) is poorly understood. We provide the first such study using a large data set of molecular markers and accessions that are widely dispersed around the world. • Sequencing data from the carrot transcriptome were used to develop 4000 single nucleotide polymorphisms (SNPs). Eighty-four genotypes, including a geographically well-distributed subset of wild and cultivated carrots, were genotyped using the KASPar assay. • Analysis of allelic diversity of SNP data revealed no reduction of genetic diversity in cultivated vs. wild accessions. Structure and phylogenetic analysis indicated a clear separation between wild and cultivated accessions as well as between eastern and western cultivated carrot. Among the wild carrots, those from Central Asia were genetically most similar to cultivated accessions. Furthermore, we found that wild carrots from North America were most closely related to European wild accessions. • Comparing the genetic diversity of wild and cultivated accessions suggested the absence of a genetic bottleneck during carrot domestication. In conjunction with historical documents, our results suggest an origin of domesticated carrot in Central Asia. Wild carrots from North America were likely introduced as weeds with European colonization. These results provide answers to long-debated questions of carrot evolution and domestication and inform germplasm curators and breeders on genetic substructure of carrot genetic resources.

Molecular Discrimination ofMycobacterium bovisin São Paulo, Brazil

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, is the most common agent of cattle tuberculosis, a zoonosis that causes losses in meat and milk production in several countries. In order to support epidemiological studies aimed at controlling the disease, several methods for molecular discrimination of M. bovis isolates have recently been developed. The most frequently used are spacer oligonucleotide typing (spoligotyping), mycobacterial interspersed repetitive units (MIRU), and exact tandem repeat (ETR), but they all have different discriminatory power. In the present study, allelic diversity was calculated for each MIRU and ETR locus, and the Hunter-Gaston discriminatory index (HGI) was calculated for spoligotyping, 10 MIRUs, and 3 ETRs, in 116 isolates of M. bovis obtained from cattle. The analysis of allelic diversity indicated that MIRUs 16, 26, and 27, and ETRs A, B, and C, showed the greatest diversity between the assayed loci. The HGIs for each of the techniques were: spoligotyping=0.738381; MIRU=0.829835; and ETR=0.825337. The associations of the methods' improved discriminatory power were: spoligotyping+MIRU=0.930585; spoligotyping+ETR=0.931034; and MIRU+ETR=0.953373. The greatest discriminatory power was obtained when the three techniques were associated (HGI=0.98051). Considering the analyses of the present study, spoligotyping should be the first method to be used because it differentiates M. bovis from the other members of the Mycobacterium tuberculosis complex. As the associations of MIRU and ETR with spoligotyping resulted in nearly identical HGIs, ETR seems to be the best choice after spoligotyping, because it is faster and more economical than MIRU. Finally, MIRU should be the last method used. In spite of this finding, the choice of the method used should be based on the discriminatory power necessary for the objective at hand.

Genetic diversity and adaptability of Sarotherodon melanotheron (Cichlidae) in coastal ecosystem

We analysed the genetic diversity of nine populations of two subspecies of Sarotherodon melanotheron using allozymes in relation to the existence of environmental pressures. The genetic variability of Sarotherodon melanotheron melanotheron in the Ivorian ecosystem (A = 1.23, He = 0.04 and P(95%) = 11.11) was lower than that of Sarotherodon melanotheron heudelotii of the estuaries in Gambia and Saloum (A = 1.68, He = 0.08 and P(95%) = 22.23). The analysis of allelic diversity revealed that the individuals of S. m. melanotheron subspecies from a much polluted environment (lagoon of Grand-Lahou or Ebrié) presented a relatively higher frequency of allele GPI-2*80 than those of a less polluted environment (Aby lagoon and Ayamé lake). The allele IDH-2*90 was expressed only in the specimens of S. m. heudelotii found in the Saloum estuary, a very saline environment compared to the estuary of Gambia. A deficit of heterozygosis was observed with the tilapia Sarotherodon melanotheron. This study confirmed the possibility of using allozymes as biomarkers and bioindicators of environmental disturbances.

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.

A new method for the partition of allelic diversity within and between subpopulations

A method is proposed for the analysis of allelic diversity in the context of subdivided populations. The definition of an allelic distance between subpopulations allows for the partition of total allelic diversity into within- and between-subpopulation components, in a way analogous to the classical partition of gene diversity. A new definition of allelic differentiation, A ST , between subpopulations results from this partition, and is contrasted with the concept of allelic richness differentiation. The partition of allelic diversity makes it possible to establish the relative contribution of each subpopulation to within and between-subpopulation components of diversity with implications in priorisation for conservation. A comparison between this partition and that corresponding to allelic richness is illustrated with an example. Computer simulations are used to investigate the behaviour of the new statistic A ST in comparison with F ST for a finite island model under a range of mutation and migration rates. A ST has less dependence on migration rate than F ST for large values of migration rate, but the opposite occurs for low migration rates. In addition, the variance in the estimates of A ST is higher than that of F ST for low mutation rates, but the opposite for high mutation rates.

Sequence-based genotyping of the sheep MHC class II DRB1 locus

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.

Genotypic diversity and migration of clonal lineages of Botrytis cinerea from chickpea fields of Bangladesh inferred by microsatellite markers

An analysis of allelic diversity at nine microsatellite loci provided an insight into the population structure of Botrytis cinerea from four fields (sampled in 2003 and 2004) that represented important regional locations for chickpea production in Bangladesh. Although three populations were limited by sample size after clone‐correction, a total of 51 alleles were amplified among 146 B. cinerea isolates from Bangladesh, which revealed a high amount of within‐population and overall genetic diversity (HS = 0·48 and HT = 0·54, respectively). The percentage of maximal genotypic diversity (G) ranged between populations (G = 23–40), with a total of 69 haplotypes detected (G = 25). Bayesian cluster analysis depicted two major clusters distributed among the four Bangladesh populations, indicating population admixture from two origins that have spread throughout these regions. Genotype flow between regions was detected and indicated the spread of clonal lineages, consistent with relatively low differentiation among the four populations (mean GST = 0·1, P < 0·05). These results highlighted the potential threat of host resistance breakdown as a result of considerable genetic diversity, genotype flow and the evolutionary potential of B. cinerea.