Jatropha curcas L. (Euphorbiaceae) exhibits a mixed mating system, high correlated mating and apomixis

The hierarchical mating system among and within fruits of Jatropha curcas was investigated in a base population using five microsatellite loci, employing mixed mating and correlated mating models. Open-pollinated fruits were collected from 15 randomly selected seed trees, sampling seven fruits per tree for a total of 21 seeds from each tree. We detected multilocus genotypes identical to the mother tree in 13 % of offspring, implying the occurrence of apomixis in J. curcas. The presumed apomictic individuals were excluded from the analysis of the remaining results. Evidence of substantial selfing was provided by the average multilocus outcrossing rate (t _m?=?0.683), showing that the species exhibits a mixed mating system. The outcrossing rate showed a large variation among seed trees, ranging from 0.21 to 1.0, indicating that the species is not self-incompatible. Significant differences were detected between the multilocus and the single locus outcrossing rates (t _m???t _s?=?0.347) that suggested mating among related individuals, possibly because of the presence of individuals from the same progeny (sibs) in the base population. The multilocus paternity correlation was extremely high for the population (r _p(m)?=?0.999), indicating that the progenies were manly composed of full-sibs. As a consequence of selfing and a high paternity correlation, the co-ancestry coefficient within the progeny was higher (Θ?=?0.369) than expected for panmictic populations. Our results indicated that J. curcas produces seeds asexually by apomixis and sexually by a mixed mating system, combining selfing and outcrossing.     

Short-distance pollen dispersal and high self-pollination in a bat-pollinated neotropical tree

We investigated pollen dispersal and breeding structure in the tropical tree species Caryocar brasiliense Camb. (Caryocaraceae), using genetic data from ten microsatellite loci. All adult trees (101) within a patch of 8.3 ha were sampled, and from these adults 18 open-pollinated maternal progeny arrays were analyzed (280 seeds from 265 fruits). Most fruits presented only one seed (median equal to 1.000) and mean number of ripened seeds per fruit was 1.053 (SD?=?0.828). Our results showed that C. brasiliense presents a mixed-mating system, with 11.4% of self-pollination, multilocus outcrossing rate of t _m?=?0.891?±?0.025, and high probability of full-sibship within progeny arrays (r _p?=?0.135?±?0.032). Outcrossing rate and self-pollination varied significantly among mother trees. We could detect a maximum pollen dispersal distance of ～500 m and a mean pollen dispersal distance of ～132 m. However, most pollination events (80%) occurred at distances less than 200 m. Our results also indicated that pollen dispersal is restricted to a neighborhood of 5.4 ha with rare events of immigration (～1% N _e m?=?0.35). C. brasiliense also presents a significant but weak spatial genetic structure (Sp?=?0.0116), and extension of pollen dispersal distance was greater than seed dispersal (N _b?=?86.20 m). These results are most likely due to the foraging behavior of pollinators that may have limited flight range. The highly within-population synchronous flowering, high population density, and clumped distribution reinforce pollinator behavior and affect residence time leading to a short-distance pollen dispersal.     

Age dynamics of genetic variation in an isolated population of chalk pine Pinus sylvestris var. cretacea Kalenicz. ex Kom. from Donbass

Based on analysis of variation at ten allozyme loci in three age groups (25–35, 40–80, and more than 100 years of age) of plants and in seed embryos, demographic dynamics of the gene pools was studied in a small (60.5 ha) isolated relict population of chalk pine Pinus sylvestris var. cretacea Kalenicz. ex Kom. from the steppe zone of Ukraine. The observed grenotype proportions in these tree groups were shown to fit Hardy-Weinberg expectations, while in the embryos of their seeds, an excess of homozygotes was observed at five to nine loci. The mean observed heterozygosity in the sample of old (>100 years of age) trees (H _O = 0.225) was substantially lower than in trees of the two other age groups (H _O = 0.307; 0.311), but significantly higher than in the corresponding embryo samples (H _O = 0.183–0.207). No allele and genotype heterogeneity of the maternal trees and embryos of their seeds was found. However, heterogeneity was high when the progeny of trees of different ages, particularly in pairs with old trees, were compared.     

Protein markers and seed size variation in common bean segregating populations

Selection and random genetic drift are the two main forces affecting allele frequencies in common bean breeding programs. Therefore, knowledge on allele frequency changes attributable to these forces is of fundamental importance for breeders. The changes in frequencies of alleles of biochemical markers were examined in F₂ to F₇ populations derived from crosses between cultivated Mesoamerican and Andean common bean accessions (Phaseolus vulgaris L.). Biochemical markers included the seed proteins phaseolin, lectin and other seed polypeptides, and six isozymes. The Schaffer’s test detected a high significant linear trend of the 63% of the polymorphic loci studied, meaning that directional selection was acting on those loci. Associations between seed size traits, phaseolin seed-storage protein and isozyme markers were detected based on the comparisons of the progeny genotypic means. In the interracial populations the intermediate form PhaH/T, b6, and Rbcs ⁹⁸ alleles had a positive effect on seed size. In the inter-gene pool populations, a higher transmission of Mesoamerican alleles in all loci was showed, although the Andean alleles Pha^T, Skdh ¹⁰⁰ , Rbcs ⁹⁸ , and Diap ¹⁰⁰ showed positive effects on seed weight. Our results suggest that phaseolin and other seed proteins markers are linked to loci affecting seed size. These markers have good potential for improving the results of the selection and should be considered as a strategy for germplasm enhancement and to avoid the reduced performance of the inter-gene pool populations.     

Sampling properties of the heterozygote-excess estimator of the effective number of breeders

The effective number of breeders (N _b) for a cohort of progeny can be estimated from an excess of heterozygotes that arises in progeny produced by finite numbers of parents. In principle, N _b is a simple function of the standardized deviation (D) of the proportion of heterozygous progeny from its expectation under random mating. We explored the sampling properties of this D-estimator of N _b through computer simulation. The accuracy of the D-estimator is remarkably robust to variation in numbers of alleles and loci and the presence of rare alleles, though precision can be low if, relative to a given N _b, the sample of progeny or the cumulative number of independent alleles (n _ci) sampled is too small. For N _b up to 30 parents, acceptable accuracy is achieved with sample sizes of 200 or more progeny and 80 or more independent alleles; for N _b of 50–100, a sample of 500–1,000 progeny and 450–900 independent alleles are required for similar accuracy and precision. Though the estimator is most applicable for the situation of random union of gametes (as may occur in some marine invertebrates or fish, for example), it works for other mating systems (monogamous or polygamous pairings, polygyny), when the effective number of breeders is small (N _b ≤ 20). Simulations reveal small overestimation biases with smaller sample sizes, rare alleles, or highly polymorphic loci (≥10 alleles). Despite this bias, multiallelic loci are preferable to many loci with few alleles, which have larger sampling errors.     

The self-incompatibility locus (S) and quantitative trait loci for self-pollination and seed dormancy in sunflower

Wild populations of common sunflower (Helianthus annuus L.) are self-incompatible and have deep seed dormancy, whereas modern cultivars, inbreds, and hybrids are self-compatible and partially-to-strongly self-pollinated, and have shallow seed dormancy. Self-pollination (SP) and seed dormancy are genetically complex traits, the number of self-compatibility (S) loci has been disputed, and none of the putative S loci have been genetically mapped in sunflower. We genetically mapped quantitative trait loci (QTL) for self-incompatibility (SI), SP, and seed dormancy in a backcross population produced from a cross between an elite, self-pollinated, nondormant inbred line (NMS373) and a wild, self-incompatible, dormant population (ANN1811). A population consisting of 212 BC₁ progeny was subsequently produced by backcrossing a single hybrid individual to NMS373. BC₁ progeny produced 0–838 seeds per primary capitula when naturally selfed and 0–518 seeds per secondary capitula when manually selfed and segregated for a single S locus. The S locus mapped to linkage group 17 and was tightly linked to a cluster of previously identified QTL for several domestication and postdomestication traits. Two synergistically interacting QTL were identified for SP among self-compatible (ss) BC₁ progeny (R²=34.6%). NMS373 homozygotes produced 271.5 more seeds per secondary capitulum than heterozygotes. Germination percentages of seeds after-ripened for 4 weeks ranged from 0% to 100% among self-compatible BC₁S₁ families. Three QTL for seed dormancy were identified (R²=38.3%). QTL effects were in the predicted direction (wild alleles decreased self-pollination and seed germination). The present analysis differentiated between loci governing SI and SP and identified DNA markers for bypassing SI and seed dormancy in elite × wild crosses through marker-assisted selection.Electronic Supplementary Material Electronic supplementary material is available for this article at     

No loss of genetic diversity in small and isolated populations of Medicago sativa subsp. falcata

Molecular allozyme markers of three polymorphic isozymes were used to estimate the genetic diversity among the seed progeny in fragmented Estonian populations of sickle medic Medicago sativa ssp. falcata L. depending on the population size and the isolation degree. Genetic diversity H_e was high in all populations, ranging between 0.795 and 0.893. No correlation between the genetic diversity measures and population size or isolation distance was found. Even the smallest population had equally high genetic diversity as about a hundred times larger population. Genetic differentiation of populations into two major groups was associated with the geographic position of populations, except one remote population. Elimination of seed progeny of reduced fitness by embryo abortion and continuous yearlong contribution of the highly heterozygous progeny through the soil seed bank are considered as important supplementary factors that have contributed to maintaining high levels of genetic diversity in populations of sickle medic in addition to its autotetraploid nature and perennial life form.     

Quantitative trait loci governing carotenoid concentration and weight in seeds of chickpea (Cicer arietinum L.)

Chickpea is a staple protein source in many Asian and Middle Eastern countries. The seeds contain carotenoids such as beta-carotene, cryptoxanthin, lutein and zeaxanthin in amounts above the engineered beta-carotene-containing golden rice level. Thus, breeding for high carotenoid concentration in seeds is of nutritional, socio-economic, and economic importance. To study the genetics governing seed carotenoids in chickpea, we studied the relationship between seed weight and concentrations of beta-carotene and lutein by means of high-performance liquid chromatography in segregating progeny from a cross between an Israeli cultivar and wild Cicer reticulatum Ladiz. Seeds of the cross progeny varied with respect to their carotenoid concentration (heritability estimates ranged from 0.5 to 0.9), and a negative genetic correlation was found between mean seed weight and carotenoid concentration in the F₃. To determine the loci responsible for the genetic variation observed, the population was genotyped using 91 sequence tagged microsatellite site markers and two CytP450 markers to generate a genetic map consisting of nine linkage groups and a total length of 344.6 cM. Using quantitative data collected for beta-carotene and lutein concentration and seed weight of the seeds of the F₂ population, we were able to identify quantitative trait loci (QTLs) by interval mapping. At a LOD score of 2, four QTLs for beta-carotene concentration, a single QTL for lutein concentration and three QTLs for seed weight were detected. The results of this investigation may assist in improving the nutritional quality of chickpea.     

Biochemical genetics of hybrid sunfish: Differential survival of heterozygotes

Allelic segregation in reciprocal backcrosses involving the largemouth bass (Micropterus salmoides) and the F₁ hybrid (largemouth bass × smallmouth bass, M. dolomieui) was investigated to determine the extent of euheterosis and luxuriance. The frequencies of allelic isozymes encoded in the lactate dehydrogenase E, malate dehydrogenase B, and isocitrate dehydrogenase loci were determined for reciprocal backcross progeny subjected to different selection pressures. The progeny of the backcross (male F₁ × female largemouth bass) underwent a rapid loss of heterozygous individuals in a natural pond environment. When the offspring of this same mating were placed in artificial pools, where cannibalism is the main source of mortality, heterozygosity was advantageous. There was a marked correlation of increased heterozygosity at these enzyme loci with an increased growth rate. None of the above responses to selection was observed when the F₁ hybrid served as the maternal parent in the reciprocal backcross. A maternal factor in the egg cytoplasm may influence the expression of heterosis.     

Parentage determination of mandarin fish (Siniperca chuatsi) based on microsatellite DNA markers

In this study, ten microsatellite loci were chosen to estimate the parentage of 260 progeny in five mandarin fish (Siniperca chuatsi) full-sib families. Simulation based on allele frequency date demonstrated the combined exclusion power would be over 97% if the number of loci was up to nine. Based on the information from these nine loci, 98% of progeny were unambiguously allocated to their putative parental pairs in the parentage analysis. The assignment success rate by the real data set was lower than that predicted by the simulations, with 94% of progeny assigned correctly. The discrepancies might be caused by a scoring error or allelic dropout caused by poor quality genomic DNA. Moreover, 69 progeny were selected randomly for the double-blind test, the result indicated that 95% of the progeny can be correctly assigned to their families. This study provided a microsatellite-based approach for parentage assignment in S. chuatsi, and that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

Genetic dissection of clonally inherited genomes of poeciliopsis. I. Linkage analysis and preliminary assessment of deleterious gene loads

Theoretical considerations suggest that a high load of deleterious mutations should accumulate in asexual genomes. An ideal system for testing this hypothesis occurs in the hybrid all-female fish Poeciliopsis monacha-lucida. The hybrid genotype is retained between generations by an oogenetic process that transmits only a nonrecombinant haploid monacha genome to their ova. The hybrid genotype is re-established in nature by fertilization of these monacha eggs with sperm from a sexual species, P. lucida. The unique reproductive mechanism of these hybrids allows the genetic dissection of the clonal monacha genome by forced matings with males of P. monacha. The resultant F₁ hybrids and their backcross progeny were examined to determine the amount and kinds of genetic changes that might have occurred in two clonal monacha genomes.—Using six allozyme markers, four similar linkage groups were identified in each clonal genome. Segregation and assortment at these loci revealed no apparent differences between monacha genomes from sexually and clonally reproducing species. Mortality of F₁ and backcross progeny revealed differences between the two clonal genomes, suggesting that deleterious genes may accumulate in genomes sheltered from recombination.     

Genetic diversity and seed production in Santa Lucia fir (Abies bracteata), a relict of the Miocene Broadleaved Evergreen Forest

Santa Lucia fir (Abies bracteata), is a unique fir, the sole member of the subgenus Pseudotorreya. It is a relict of the Miocene broadleaved evergreen sclerophyll forest, and is now restricted to a highly fragmented range in the Santa Lucia Mountains of central coastal California. Expected heterozygosity for 30 isozyme loci in 18 enzyme systems, averaged over six populations that spanned the species’ north–south range, was only 0.036. Despite a fragmented range and isolated populations, differentiation (F _ST) was only 0.080 for mature trees, and the number of migrants per generation (Nm) was 2.88 or 3.83, depending on the method of estimation. F _ST for embryos was lower, 0.025, and Nm correspondingly higher, 9.75. Nei’s genetic distances were small and unrelated to geographic distances between populations. The proportion of full seeds per cone was only 0.082–0.488, depending on population, which suggests a high incidence of selfing followed by embryo abortion. However, the level of accumulated inbreeding, F _IS, in mature trees was low, only 0.049. By contrast, F _IS for embryos was 0.388, which indicates a high proportion of selfed progeny, in agreement with the low seed yields. The difference in inbreeding coefficients between seed trees and their progeny suggest that most inbreds are eliminated before maturity and, therefore, seed production, already low, overestimates the true potential for regeneration of these populations. These results have implications for conservation.     

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B₅ medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R₁ generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R₂ generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.     

Genetic diversity and mating system of <Emphasis Type="Italic">Pinus brutia</Emphasis> var. <Emphasis Type="Italic">stankewiczii</Emphasis> Sukacz. in small localities of Sudak (Crimea)

The comparative analysis of genetic variation at 12 polymorphic isozyme loci, as well as the mating system, has been carried out in mature trees and their seed progenies in three small localities of Pinus brutia var. stankewiczii Sukacz. in the region between the town of Sudak and settlement of Novyi Svet in Crimea. We found that embryos maintain the same allelic diversity as parent trees; however, their observed heterozygosity, on average, is lower by 37.4%. The significant deviation of genotype distribution from the expected ratios caused by the deficiency of heterozygotes was observed at 8 out of 12 loci. The multilocus estimate of the out-crossing rate (t _m ) in populations varied from 68.9 to 94.9% with the average of 80.7%.     

Genetic variability of maternal plants and embryos of their seeds in the populations of <Emphasis Type="Italic">Pinus kochiana</Emphasis> Klotzsch ex Koch in Crimea

Samples of trees and seed embryos of five Pinus kochiana Klotzsch ex Koch populations in Crimea were comparatively studied at 12 allozymic loci for its genetic variability and mating system. It has been shown that the embryos of seeds reproduce the allelic diversity of maternal plants, but the level of their observed heterozygosity (H_o) is significantly lower, i.e., 0.286 vs. 0.189, respectively. Unlike maternal trees, embryos are characterized by a significant divergence between the observed distribution of genotypes and the theoretically expected one according to the Hardy–Weinberg law for the majority of analyzed loci. The frequency of cross pollination in populations varied from 0.384 to 0.673 according to single-locus (t_s) estimates and from 0.639 to 0.841 according to multilocus (t_m) estimates.     

Isozyme analysis of progeny derived from (Allium fistulosum ×Allium cepa) ×Allium cepa

Summary Relatively large quantities of seed were obtained from the interspecific backcross (A. fistulosum xA. cepa) ×A. cepa allowing, for the first time, an extensive study of the heritable traits exhibited by backcross progeny. Two backcross populations, BC1034 and BC1040, distinguished by differentA. fistulosum parents, were characterized for the isozyme markersIdh-1, Adh-1, andPgi-1. Statistical methods are described to calculate cell probabilities for a mixed population of F₂ and BC₁ progeny, using an estimate of the fraction of F₂ progeny in the population derived from the isozyme data. Cell probability distributions were calculated for a mixed population with independent pairs of loci and a mixed population with nonindependent pairs of loci. The isozyme lociIdh-1 andPgi-1 appear to be linked, with a map distance estimated at 33 centimorgans (cM) in BC1034 and 42 cM in BC1040. The probability distribution model for linked loci did not account for all of the distorted segregation ratios inIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The cytological literature does not support linkage betweenIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The distorted segregation ratios for these pairs of loci are likely the result of genetic incompatibilities between the two species.Journal Article No. 1578, Agricultural Experiment Station, New Mexico State University, Las Cruces, NM, USA     

A new method for the identification and isolation of genes essential forArabidopsis thaliana seed germination

Seed viability, dormancy and germination efficiency are very important aspects of the life cycle of plants and their potential to survive and spread in the environment. To characterize the genes controlling these processes, we have devised a technique for the selection of mutants impaired in seed germination. Selection for such a trait is complicated by physiological factors that interact with these processes and affect seed germination efficiency. The distinction between low seed germination potential due to physiological factors that interfere with seed maturation or germination and germination deficiency due to genetic factors was based on screening for tagged mutations.Arabidopsis thaliana T-DNA primary transformants obtained by an in planta transformation technique are all heterozygotes. We screened for lack of germination of 1/4 of the seeds in the progeny of independent transformants, and simultaneously for the abnormal segregation (2:1 instead of 3:1) of a kanamycin resistance marker carried by the T-DNA inserted into the genome of these primary transformants in the plants that germinate. This yielded several mutants affected in the germination processes. One of the mutants, designated ABC33, was further characterized. Once the viable embryos from non-germinating seeds were removed from their testa, they grew and displayed a dwarf phenotype which could be complemented by providing gibberellic acid. A genetic and molecular analysis, based on the characterization of the flanking genomic sequences of the T-DNA insert, showed that ABC33 is a new loss-of-function allele at theGA ₁ locus.     

Dwarf mutations in grass pea (Lathyrus sativus L.): origin, morphology, inheritance and linkage studies

Induction of mutation has been used to create additional genetic variability in grass pea (Lathyrus sativus L.). During the ongoing investigations on different induced-morphological mutants, the author detected three types of dwarf mutants in grass pea. One mutant, designated as dwf1 type was earlier identified in colchicine-induced C₂ generation of grass pea variety BioR-231 while the other two, designated as dwf2 and dwf3 were isolated in 250 Gy and 300 Gy gamma ray irradiated M₂ progeny of variety ‘BioR-231’ and ‘Hooghly Local’, respectively. As compared to their parental varieties (controls), all the three mutants manifested stunted, erect and determinate stem, early maturity and tolerance to pod shattering habit. The mutants differed from each other, as well as with controls, in number of primary branches, nature of stipules and internodes, length of peduncle, leaflet and seed coat colour, seed yield and seed neurotoxin content. The three dwarf mutants were monogenically recessive and bred true in successive generations. F₂ segregation pattern obtained from the crosses involving the three mutants indicated that dwarf mutation in grass pea was controlled by two independent non-allelic genes, assigned as df1 (for dwf1 type), df2 (for dwf2 type) and df3 (for dwf3 type), with the df1 locus being multiple allelic. Primary trisomic analyses revealed the presence of df1/df2 locus on the extra chromosome of trisomic type I, whereas df3 was located on the extra chromosome of type III. Linkage studies involving five other phenotypic markers suggested linked association of df1/df2 locus with lfc (leaflet colour) and wgn (winged internode) and df3 locus with cbl (seed coat colour). Both the loci; however, assorted independently with flower colour and stipule character. The dwarf types can be utilized as valuable tools for further cytogenetic research and breeding of grass pea.