Relationships among genetic distance,forage yield and heterozygosity in isogenic diploid and tetraploid alfalfa populations

Isogenic diploid and tetraploid alfalfa (Medicago sativa L.) was studied with molecular markers to help understand why diploid performance and breeding behavior does not always predict that of tetraploids. In a previous study of partially heterozygous alfalfa genotypes, we detected a low correlation between yields of isogenic diploid (2x) and tetraploid (4x) single-cross progenies, and genetic distances were more highly correlated with yields of tetraploids than diploids. These differences may be related to the level of RFLP heterozygosity expected among progenies derived from heterozygous parents at the two ploidy levels. The objectives of this study were to determine the relationships among genetic distance, forage yield and heterozygosity in isogenic 2 x and 4 x alfalfa populations. Four diploid genotypes were chromosome doubled to produce corresponding isogenic autotetraploids, and these genotypes were mated in 4 × 4 diallels to produce 6 single-cross families at each ploidy level for field evaluation. Allele compositions of parents were determined at 33 RFLP loci by monitoring segregation of homologous restriction fragments among individuals within progenies, and these were used to estimate RFLP heterozygosity levels for all single-cross progenies at both ploidy levels. RFLP heterozygosity rankings were identical between progenies of isogenic diploid and tetraploid parents; but significant associations (P < 0.05) between estimated heterozygosity levels and forage yield were detected only at the tetraploid level. Since tetraploid families were nearly 25% more heterozygous than the corresponding diploid families, inconsistencies in the association between molecular marker diversity and forage yields of isogenic 2 x and 4 x single crosses may be due to recessive alleles that are expressed in diploids but masked in tetraploids. The gene action involved in heterosis may be the same at both ploidy levels; however, tetraploids benefit from greater complementary gene interactions than are possible for equivalent diploids. Present address: AgResearch Grasslands, New Zealand Pastoral Agriculture Research Institute, Palmerston North, New Zealand     

Isozyme gene duplication in diploid and tetraploid potatoes

Summary Isozyme techniques allow the study of gene redundancy in different ploidy levels of potato (Solanum tuberosum). In tetraploid potatoes all isozyme loci are duplicated. No sign of structural or regulatory divergence was found, as is expected due to their tetrasomic inheritance patterns. In addition to this genetic redundancy, produced by a relatively recent polyploidization event, some additional redundancy was found for at least three enzymes even in diploid groups and species. These older duplicate genes show structural and regulatory divergence, indicating they appeared by a separate polyploidization event far in the past. Their common origin is still recognizable by both their expression in the same subcellular compartment and by the dimerizing ability of the isozymes they encode. To account for the present chromosome number x = 12 of the Solanaceae family, the most frequently found among the species, a hypothetical polyploidization event is proposed.     

RFLP analysis in chrysanthemum. I. Probe and primer development

In order to study genetic variability at the DNA level in chrysanthemum (Dendranthema grandiflora Tzvelev) PstI and HindIII genomic libraries were constructed. Probes from both libraries were tested for the presence of restriction fragment length polymorphisms (RFLPs). Of the probes from the PstI library 91% appeared to hybridize to low-copy genes, while only 35% of those from the HindIII library appeared to do so. The PstI probes were used in further analyses as 79% of them showed RFLPs, whereas the HindIII low-copy number probes gave only 14% polymorphic patterns. Because of the hexaploid character of chrysanthemum, complex patterns generally consisting of 6–12 fragments were visible on a Southern blot after hybridization. To simplify the genetic analysis, locus-specific polymerase chain reaction (PCR) primers were developed that gave simple polymorphic patterns in a number of cases. The RFLP probes and primers developed will be used in future marker-assisted selection in this polyploid crop.     

Genetic diversity in European wheat and spelt breeding material based on RFLP data

Fifty-two winter wheat (Triticum aestivum L.), nine spring wheat, and 20 spelt (Triticum spelta L.) lines representing part of the European breeding germplasm, were assayed for RFLPs (restriction fragment length polymorphisms) with 56 wheat DNA clones and two barley cDNA clones. Objectives of this study were to (1) determine the level of variation for RFLPs in the wheat and spelt breeding lines, (2) characterize the genetic diversity within the European winter wheat germplasm, and (3) evaluate the usefulness of RFLP markers for pedigree analysis and the grouping of wheat and spelt lines of various origins. Seventy-three of the 166 RFLP loci detected with 58 probes and one restriction enzyme were polymorphic for the 81 lines. The percentage of polymorphic loci was greatest for the B genome (58%) and smallest for the D genome (21%). Among the 81 lines, 271 different RFLP bands were detected. RFLP band frequencies of the winter wheat lines differed considerably (0.5) from those of the spring wheat lines at five loci, and from those of the spelt lines at 17 loci. Eight cultivars that had a major impact as progenitors on the development of improved winter wheat cultivars accounted for 93% of the observed RFLP bands in winter wheat. Genetic distance (GD) estimates between two lines ranged between 0.01 and 0.21. Mean GD estimates within winter wheat (0.083), within spring wheat (0.108) and within spelt (0.096) were smaller than between spring and winter wheat (0.114), and greatest between winter wheat and spelt (0.132) and spring wheat and spelt (0.148). Principal coordinate analysis performed on GD estimates revealed a clear separation of wheat and spelt germplasm. Novel spelt lines with various proportions of wheat germplasm were positioned between wheat and traditional spelt lines. The spring wheat lines formed a distinct group at the periphery of the distribution of the winter wheat lines. Subgroupings of the winter wheat lines according to the cluster analysis were in good agreement with their origin, and lines with common ancestors were grouped together.     

RFLP variability in peanut (Arachis hypogaea L.) cultivars and wild species

Summary RFLP variability was studied in eight U.S. peanut cultivars, representing the four market types, and in 14 wild Arachis species accessions, using random genomic clones from a PstI library. Very low levels of RFLP variability were found among the allotetraploids, which included the U.S. cultivars and Arachis monticola, a wild species. The diploid wild species were very diverse, however. RFLP patterns of the allotetraploids were more complex than the diploids, and the two constituent genomes could usually be distinguished. On the basis of RFLP band sharing, A. ipaensis, A. duranensis, and A. spegazzinii appeared most closely related to the diploid progenitor species of the allotetraploids. A dendrogram of relationships among the diploid wild species was constructed based on band sharing.     

Mapping the jp (jumbo pollen) gene and QTLs involved in multinucleate microspore formation in diploid alfalfa

The objective of this research was to map the jumbo-pollen trait in diploid alfalfa. Homozygous recessive (jpjp) plants are characterized by the complete failure of post-meiotic cytokinesis during microsporogenesis resulting in 100% 4n-pollen formation. Three F₁ segregating populations were produced and analyzed for pollen-grain production and the segregation of RFLP markers. The first cross combination did not segregate for the jumbo-pollen trait, but showed a clear segregation for multinucleate (bi-, tri- and tetra-nucleate)-microspore formation. Cytological analysis showed that few plants produced 100% normal (uninucleate) microspores, whereas most of them produced multinucleate microspores at a variable frequency (0–75%). Plants with multinucleate microspores always showed a prevalence of binucleated microspores, even though some plants showed a background presence of tri- and tetra-nucleate microspores. QTL analysis based on ANOVA I and Stepwise Multiple Regression identified three QTLs with a highly significant effect on multinucleate-microspore formation. Two cross combinations, subsequently executed, showed Mendelian segregation for the jumbo-pollen trait and were effective in locating the jp gene on linkage group 6 close to the Vg1G1b RFLP locus. Interestingly, this RFLP locus was also linked to one QTL for multinucleate-microspore formation. Genetic models are discussed concerning the presence in linkage group 6 of a cluster of genes involved in multinucleate-microspore formation together with possible relationships between the jp gene and the Vg1G1b QTL. Received: 28 January 1999 / Accepted: 16 December 1999     

山羊品种间线粒体DNA限制性片段长度多态性研究

本试验利用ＡｐａⅠ、ＢａｍＨⅠ、ＢｃｌⅠ、ＢｇｌⅠ、ＢｇｌⅡ、ＣｌａⅠ、ＥｃｏＲⅠ、ＥｃｏＲⅤ、ＨａｅⅡ、ＨｉｎｄⅢ、ＫｐｎⅠＰｓｔⅠ、ＰｖｕⅡ、ＳａｃⅠ、ＳａｌⅠ、ＳｍａⅠ和ＸｈｏⅠ计１８种限制性内切酶,研究了来自欧洲,非洲及国内的５个山羊品种共计３３只个体的ｍｔＤＮＡ,共检测了２７处限制性态型,可归结为８种单倍型。结果表明,国内２个百品种的基本单倍型为ＢａｍＨⅠ－Ａ,ＢｃｌⅠ－Ｂ、ＣｌａⅠ－Ａ     

用RFLP标记分析甘蓝型油菜的遗传多样性

以甘蓝型油菜的２８个基因组探针和两种限制性内切酶对包括４６个中国品种、９个欧洲品种在内的５９个甘蓝型油菜品种（系）的ＲＦＬＰ标记进行了分析。在放射自显影胶片上,共检测到４１０条具多态性的分子杂交带,表明甘蓝型油菜中存在着极为丰富的遗传变异。聚类分析结果表明,在相似性为４５％的水平上,可把中国甘蓝型油菜划分为６组：胜利油菜组、跃进油菜组、中油８２１组、远缘种质组、优质油菜组和变异不详组。欧洲冬油菜与以上６组存在着较显著的遗传距离。主成分分析的结果与上述分组较为一致。以上结果表明,对于扩大中国甘蓝型油菜的遗传基础,欧洲冬油菜无疑是一个重要的种质资源。另一方面,用典型的中国甘蓝型油菜与欧洲冬油菜配制的杂交种,较易产生强大的杂种优势。从对已进行了染色体定位的６１条放射自显影带的分析看,无论是上述分组内,还是分组间,ＲＦＬＰ的相对差异均主要表现在Ａ基因组中。讨论了致使Ａ基因组遗传变异较大的可能因素。     

miR156靶基因SPL在植物中的系统分布和进化模式分析

Squamosa promoterbinding proteinlike genes （SPLs）在植物发育过程中具有重要作用。很多SPLs被miR156调节,然而,对于它们在植物中的系统分布和进化模式还知之甚少。本文对9个测序物种（藻类,苔藓,石松,单子叶和双子叶植物）的183个SPLs进行了生物信息学分析。结果表明miR156应答元件（MREs）仅在陆生植物SPLs中发现,藻类中不存在。系统进化分析显示陆生植物SPLs分为两大分支：group I和group II。 MiR156靶基因仅分布于group II,表明它们有着共同的祖先。Group II进一步分为7个亚支（IIaIIg）,miR156靶基因分布在除IId外的其余6个亚支的特定SPLs。系统分类与基因结构的相关性反映了SPL靶基因结构上的变化。在进化过程中,它们可能发生外显子的丢失且伴随MRE的丢失。另外,基因重复对SPL靶基因的丰度变化影响很大,尤其是被子植物与低等植物分歧后它们数量明显增加。以拟南芥为模式植物分析发现串联重复和片段重复是SPL靶基因扩张的主要机制。     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

Quantitative genetic studies at the tetraploid and hexaploid levels in alfalfa

Summary Two sets of diallel crosses involving seven tetraploid and five hexaploid alfalfa lines, previously selected for seed and forage yield, are studied. On the basis of combining ability and heritability estimates the following conclusion is drawn. The dominant and epistatic interactions (heterozygosity) are the major components in the expression of variability for yield and characters associated with yield in tetraploid populations. In hexaploid populations, however, there is an appreciable amount of additive variance, and the additive effect of genes along with interaction of various forms may account for the expressed variability for all the characters studied. Better genes and desirable interactions (possibly through heterozygosity) are the two essential genetic components of yield. The selection of better genes is feasible, but by itself will not produce the desired results. Future improvement may therefore depend on the utilization of heterosis and other forms of interactions. There is, in general, a very strong genetic correlation between yield and its components, and therefore, when a direct selection for yield is not feasible, the selection program may rely on a number of closely related characters.     

Molecular diversity in pineapple assessed by RFLP markers

Pineapple, Ananas comosus (L.) Merr, is the third most important tropical fruit cultivated in all tropical and subtropical countries. Pineapple germplasm includes all seven species of the genus Ananas and the unique species of the related genus Pseudananas. A knowledge of its diversity structure is needed to develop new breeding programs. Restriction fragment length polymorphism (RFLP) was used to study molecular diversity in a set of 301 accessions, most of which were recently collected. This sample was analysed using 18 homologous genomic probes. Dissimilarities were calculated by a Dice index and submitted to Factorial Analysis. The same data were represented as a diversity tree constructed with the score method. Pseudananas sagenarius displayed a high polymorphism and shares 58.7% of its bands with Ananas. Within Ananas, variation appears continuous and was found mostly at the intraspecific level, particularly in the wild species Ananas ananassoides and Ananas parguazensis. As for the cultivated species, Ananas comosus appears relatively homogeneous despite its wide morphological variation and Ananas bracteatus, which is grown as a fence and for fruit, appears still much less variable. By contrast Ananas lucidus, cultivated by the Amerindians for fiber, displays a high polymorphism. This tree displayed a loose assemblage of numerous clusters separated by short distances. Most species were scattered in various clusters, a few of these being monospecific. Some accessions which had not been classified, as they shared morphological traits typical of different species, re-group with one or the other, and sometimes with both species in mixed clusters. No reproductive barrier exists in this germplasm and these data indicate the existence of gene flow, enhancing the role of effective sexual reproduction in a species with largely predominant vegetative mutiplication. Received: 8 March 2000 / Accepted: 14 April 2000     

RFLP analysis of phylogenetic relationships and genetic variation in the genus Lycopersicon

Summary Forty single-copy, nuclear probes of known chromosomal position were used to examine restriction fragment length polymorphism in the tomato genus Lycopersion. The probes were from three libraries: one cDNA, and two genomic libraries ne genomic made with EcoRI and the other with PstI. Total DNA from 156 plants representing eight species was cut with five different restriction enzymes and scored in 198 probe-enzyme combinations. Genetic distances between accessions (populations) and species were calculated from the resultant restriction patterns and proportion of shared bands. Accessions belonging to the same species largely clustered together, confirming their current classification. However, one mountain accession, classified as L. peruvianum var. humifusum (LA2150), was sufficiently distinct from the other accessions of L. peruvianum that it may qualify as a separate species L. esculentum and L. pimpinellifolium were the least clearly differentiated, possibly reflecting introgressive hybridization, known to have been promoted by man in recent history. Dendrograms constructed from cDNA versus genomic clones were nearly identical in their general grouping of species. The dendrograms revealed two major dichotomies in the genus: one corresponding to mating behavior [self-compatible (SC) versus self-incompatible (SI) species] and the other corresponding to fruit color (red versus green-fruited species). The ratio of withinversus between-accession diversity was much lower for SC species, indicating that most of the diversity within these species exists between populations, rather than within populations. Overall, the amount of genetic variation in the SI species far exceeded that found in SC species. This result is exemplified by the fact that more genetic variation could be found within a single accession of one of the SI species (e.g., L. peruvianum) than among all accessions tested of any one of the SC species (e.g., L. esculentum or L. pimpinellifolium). Results from this study are discussed in relationship to germ plasm collection/utilization and with regard to the use of RFLPs in tomato breeding and genetics.     

Restriction fragment variation in the nuclear and chloroplast genomes of cultivated and wild Sorghum bicolor

Summary Fifty-six accessions of cultivated and wild sorghum were surveyed for genetic diversity using 50 low-copy-number nuclear DNA sequence probes to detect restriction fragment length polymorphisms (RFLPs). These probes revealed greater genetic diversity in wild sorghum than in cultivated sorghum, including a larger number of alleles per locus and a greater portion of polymorphic loci in wild sorghum. In comparison to previously published isozyme analyses of the same accessions, RFLP analysis reveals a greater number of alleles per locus. Furthermore, many RFLP alleles have frequencies between 0.25–0.75, while the vast majority of isozyme alleles are either rare (< 0.25) or near fixation (> 0.75). Correlations between genetic and geographic distances among the accessions were stronger when calculated with RFLP than with isozyme data. Systematic relationships revealed by nuclear and chloroplast restriction site analysis indicate that cultivated sorghum is derived from the wild ssp. arundinaceum. The portion of the wild gene pool most genetically similar to the cultivars is from central-northeastern Africa. Previous published data also suggested that this is most likely the principal area of domestication of sorghum. Introgression between wild and cultivated sorghum was inferred from disconcordant relationships shown by nuclear and chloroplast DNA markers. Introgression apparently occurs infrequently enough that the crop and its wild relatives maintain distinct genetic constitutions.     

Genetic diversity in Australian wheat varieties and breeding material based on RFLP data

 Restriction fragment length polymorphisms (RFLPs) have been used to characterise the genetic diversity of wheat (Triticum aestivum) germplasm. One hundred and twenty-four accessions comprising all major Australian wheat varieties and lines important for breeding purposes were assayed for RFLPs with clones of known genetic location and selected to give uniform genome coverage. The objectives of this study were to determine RFLP-based genetic similarity between accessions and to derive associations between agronomically significant traits and RFLP phenotypes. Ninety-eight probes screened against genomic DNA digested with five restriction endonucleases detected a total of 1968 polymorphic fragments. Genetic similarity (GS) calculated from the RFLP data ranged from 0.004 to 0.409 between accessions, with a mean of 0.18. Cluster analysis based on GS estimates produced four groupings that were generally consistent with available pedigree information. Comparisons of the RFLP phenotypes of accessions containing disease resistance genes present on introgressed alien segments enabled the identification of specific alleles characteristic of these regions. Associations were derived for a range of stem-rust, leaf-rust and yellow-rust resistance genes. These results suggest that RFLP analysis can be used for the characterisation and grouping of elite breeding material of wheat and RFLP profiling can identify chromosome segments associated with agronomic traits. Received: 10 March 1997 / Accepted: 28 July 1997     

用RFLP方法研究陕西省主要小麦品种遗传多样性及其演变

研究用ＲＦＬＰ方法,对我国主要小麦产区,陕西省４０年代以来广为种植的小麦品种的遗传多样性进行了研究,结果表明,占总带数２６．２％的ＨＩＮＤＩＩＩ酶的ＲＦＬＰ带具有遗传多态性。２４个小麦品种平均遗传相似系数为０．９４８。５３．８％的探针显示了多态性。平均多态性信息含量指数为０．２５２。陕西省小麦品种遗传多样性以７０年代为最高,之后开始降低。     

Seed-storage-protein loci in RFLP maps of diploid, tetraploid, and hexaploid wheat

 Linkages between high- and low-molecular-weight (M_r) glutenin, gliadin and triticin loci in diploid, tetraploid and hexaploid wheats were studied by hybridization of restriction fragments with DNA clones and by SDS-PAGE. In tetraploid and hexaploid wheat, DNA fragments hybridizing with a low-M_r glutenin clone were mapped at the XGlu-3 locus in the distal region of the maps of chromosome arms 1AS, 1BS, and 1DS. A second locus, designated XGlu-B2, was detected in the middle of the map of chromosome arm 1BS completely linked to the XGli-B3 gliadin locus. The restriction fragments mapped at this locus were shown to co-segregate with B subunits of low-M_r glutenins in SDS-PAGE in tetraploid wheat, indicating that XGlu-B2 is an active low-M_r glutenin locus. A new locus hybridizing with the low-M_r clone was mapped on the long arm of chromosome 7A^m in diploid wheat. No glutenin protein was found to co-segregate with this new locus. Triticin loci were mapped on chromosome arms 1AS, 1BS, and 1DS. A failure to detect triticin proteins co-segregating with DNA fragments mapped at XTri-B1 locus suggests that this locus is not active. No evidence was found for the existence of Gli-A4, and it is concluded that this locus is probably synonymous with Gli-A3. Recombination was observed within the multigene gliadin family mapped at XGli-A11 (1.2 cM).1 Although these closely linked loci may correspond to the previously named Gli-A1 and Gli-A5 loci, they were temporarily designated XGli-A1.1 and XGli-A1.2 until orthology with Gli-A1 and Gli-A5 is established. Received: 25 March 1997 / Accepted: 23 June 1997     

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn ₁ and nn ₂ )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F₁ plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F₂ population segregating the nn ₁ and nn ₂ genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F₁ plants.     

RFLP studies of genetic relationships among inbred lines of the cultivated sunflower,Helianthus annuus L.: evidence for distinct restorer and maintainer germplasm pools

One-hundred-and-eighty-one nuclear DNA probes were used to examine restriction-fragment length polymorphism in inbred lines of the cultivated sunflower (Helianthus annuus L.). The probes were from six libraries: two genomic libraries — one made with PstI and the other with HindIII, and four cDNA libraries — from etiolated plantlets, green leaves, ovaries, petals and anthers. Total DNA from 17 inbred lines representing an overview of the genetic stocks of sunflower, including restorer and maintainer lines of the classical cytoplasmic male sterility, was digested with four different restriction enzymes and probed in 331 probe-enzyme combinations. Of 181 clones analysed, 73 probes were found to be polymorphic. Genetic distances between inbreds were calculated from the resultant proportion of shared bands and submitted to principal component analysis and the UPGMA tree-making method. The RFLP analysis allowed a clear differentiation between restorer and maintainer lines of the cytoplasmic male sterility, together with a grouping of some of the genotypes from the same origin. The analysis of the accuracy of distance estimation as a function of the number of probe-enzyme combinations used, indicates that 40–50 combinations ensure a confidence level of near 95%. Considering the inbreds as representatives of the range of cultivated inbreds, estimates of gene diversity, as well as estimates of average gene diversity between and within the sets of restorer and maintainer lines, were calculated. Estimation of gene diversity showed that the available genetic variability in cultivated sunflower, based on allelic frequencies, is lower than that of other plants (H=0.20). Moreover, we show that the proportion of genetic variability due to the difference between maintainer and restorer lines (D_m) is about 2%.