Showing papers in "Theoretical and Applied Genetics in 2000"

Linkage disequilibrium and fingerprinting in sugar beet

Abstract: It has been suggested that map information for molecular markers can be used to strengthen finterprinting analyses. The success of this strategy depends on the distribution of linkage disequilibrium over the genome. Using 451 mapped AFLP markers, we investigated the occurrence of linkage disequilibrium in nine sugar beet breeding lines. A low but significant level of linkage disequilibrium was found for unlinked markers. Only for very tigthly linked (<3 cM) markers was this level substantially higher. This implies that little is gained in utilising the map position of the markers in fingerprinting applications.

Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.)

Abstract: In order to enhance the resolution of an existing genetic map of rice, and to obtain a comprehensive picture of marker utility and genomic distribution of microsatellites in this important grain species, rice DNA sequences containing simple sequence repeats (SSRs) were extracted from several small-insert genomic libraries and from the database. One hundred and eighty eight new microsatellite markers were developed and evaluated for allelic diversity. The new simple sequence length polymorphisms (SSLPs) were incorporated into the existing map previously containing 124 SSR loci. The 312 microsatellite markers reported here provide whole-genome coverage with an average density of one SSLP per 6 cM. In this study, 26 SSLP markers were identified in published sequences of known genes, 65 were developed based on partial cDNA sequences available in GenBank, and 97 were isolated from genomic libraries. Microsatellite markers with different SSR motifs are relatively uniformly distributed along rice chromosomes regardless of whether they were derived from genomic clones or cDNA sequences. However, the distribution of polymorphism detected by these markers varies between different regions of the genome.

Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.)

Abstract: The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers developed using different methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome. We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening and 129 were derived from the analysis of rice-expressed sequence tags (ESTs) available in public DNA databases. We have evaluated the frequency of polymorphism between parental pairs of six inter- subspecific crosses and one inter-specific cross widely used for mapping in rice. Microsatellites derived from genomic libraries detected a higher level of polymorphism than those derived from ESTs contained in the GenBank database (83.8% versus 54.0%). Similarly, the other measures of genetic variability [the number of alleles per locus, polymorphism information content (PIC), and allele size ranges] were all higher in genomic library-derived microsatellites than in their EST-database counterparts. The highest overall degree of genetic diversity was seen in GA-containing microsatellites of genomic library origin, while the most conserved markers contained CCG- or CAG-trinucleotide motifs and were developed from GenBank sequences. Preferential location of specific motifs in coding versus non-coding regions of known genes was related to observed levels of microsatellite diversity. A strong positive correlation was observed between the maximum length of a microsatellite motif and the standard deviation of the molecular-weight of amplified fragments. The reliability of molecular weight standard deviation (SDmw) as an indicator of genetic variability of microsatellite loci is discussed.

Analysis of SSRs derived from grape ESTs.

Abstract: One hundred and twenty four microsatellites were isolated from analysis of 5000 Vitis expressed se- quence tags (ESTs). A diversity of dinucleotide and tri- nucleotide simple sequence repeat (SSR) motifs were present. Primers were designed for 16 of these SSRs and they were tested on seven accessions. Ten of the sixteen primer pairs resulted in PCR products of the expected size. All ten functional primers were polymorphic across the accessions studied. Polymorphisms were evident at the level of cultivars, Vitis species, and between related genera. SSRs that were from the 3' untranslated region (3'UTR) were most polymorphic at the cultivar level, the 5' untranslated region (5' UTR) SSRs were most poly- morphic between cultivars and species, and those SSRs within coding sequence were most polymorphic between species and genera. These results show that EST-derived SSRs in Vitis are useful as they are polymorphic and highly transferable. With EST SSRs being applicable to studies at several taxonomic levels, the large number of SSRs (approximately 1000) that will be available from an expanded EST database of 45 000 will have many po- tential applications in mapping and identity research.

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Abstract: Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one.

Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice

Abstract: Three major genes (Pi1, Piz-5 and Pita) for blast resistance on chromosomes 11, 6 and 12, respectively, were fine-mapped and closely linked RFLP markers identified. New markers for Pi1 and Pita were found that were flanking the genes. The three genes were pyramided using RFLP markers. A PCR-based SAP (sequence amplified polymorphism) marker was used to identify Piz-5 in the segregating population. The plants carrying the two- and three-gene combinations that were tested for resistance to leaf blast in the Philippines and India indicated that combinations including Piz-5 have enhanced resistance than when it is present alone. The genes from the pyramided lines are at present being deployed into agronomically superior ricevarieties by marker-aided selection (MAS).

Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza

Abstract: Inter simple sequence repeat (ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in Oryza. Forty two genotypes including 17 wild species, representing AA,BB,CC,EE,FF,GG,BBCC,CCDD, and HHJJgenomes, two cultivated species, Oryza sativa (AA) and Oryza glaberrima (AA), and three related genera, Porteresia coarctata, Leersia and Rhynchoryza subulata, were used in ISSR analysis. A total of 30 ISSR primers were screened representing di-, tri-, tetra- and penta-nucleotide repeats, of which 11 polymorphic and informative patterns were selected to determine the genetic diversity. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered 42 genotypes according to their respective genomes. ISSR analysis suggests that the genus Oryza may have evolved following a polyphyletic pathway; Oryza brachyantha (FF genome) is the most divergent species in Oryza and Oryza australiensis (EE genome) does not fall under the Officinalis complex. DNA profiles based on ISSR markers have revealed potential diagnostic fingerprints for various species and genomes, and also for individual accessions/cultivars. Additionally ISSR revealed 87 putative genome/species-specific molecular markers for eight of the nine genomes of Oryza. The ISSR markers are thus useful in the fingerprinting of cultivated and wild species germplasm, and in understanding the evolutionary relationships of Oryza.

Characterization of microsatellite markers in peach (Prunus persica (L.) Batsch)

Abstract: Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)n- and (CA)n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L.

A linkage map of the chickpea (Cicer arietinum L.) genome based on recombinant inbred lines from a C. arietinum×C. reticulatum cross: localization of resistance genes for fusarium wilt races 4 and 5

Abstract: An integrated molecular marker map of the chickpea genome was established using 130 recombinant inbred lines from a wide cross between a cultivar resistant to fusarium wilt caused by Fusarium oxysporum Schlecht. emend. Snyd. &. Hans f. sp. ciceri (Padwick) Snyd & Hans, and an accession of Cicer reticulatum (PI 489777), the wild progenitor of chickpea. A total of 354 markers were mapped on the RILs including 118 STMSs, 96 DAFs, 70 AFLPs, 37 ISSRs, 17 RAPDs, eight isozymes, three cDNAs, two SCARs and three loci that confer resistance against different races of fusarium wilt. At a LOD-score of 4.0, 303 markers cover 2077.9 cM in eight large and eight small linkage groups at an average distance of 6.8 cM between markers. Fifty one markers (14.4%) were unlinked. A clustering of markers in central regions of linkage groups was observed. Markers of the same class, except for ISSR and RAPD markers, tended to generate subclusters. Also, genes for resistance to races 4 and 5 of fusarium wilt map to the same linkage group that includes an STMS and a SCAR marker previously shown to be linked to fusarium wilt race 1, indicating a clustering of several fusarium-wilt resistance genes around this locus. Significant deviation from the expected 1 : 1 segregation ratio was observed for 136 markers (38.4%, P<0.05). Segregation was biased towards the wild progenitor in 68% of the cases. Segregation distortion was similar for all marker types except for ISSRs that showed only 28.5% aberrant segregation. The map is the most extended genetic map of chickpea currently available. It may serve as a basis for marker-assisted selection and map-based cloning of fusarium wilt resistance genes and other agronomically important genes in future.

QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress

Abstract: Cell-membrane stability (CMS) is considered to be one of the major selection indices of drought tolerance in cereals. In order to determine which genomic region is responsible for CMS, 104 rice (Oryza sativa L.) doubled haploid (DH) lines derived from a cross between CT9993–5-10–1-M and IR62266-42–6-2 were studied in the greenhouse in a slowly developed drought stress environment. Drought stress was induced on 50-day-old plants by withholding water. The intensity of stress was assessed daily by visual scoring of leaf wilting and by measuring leaf relative water content (RWC). The leaf samples were collected from both control (well-watered) and stressed plants (at 60–65% of RWC), and the standard test for CMS was carried out in the laboratory. There was no significant difference (P>0.05) in RWC between the two parental lines as well as among the 104 lines, indicating that all the plants were sampled at a uniform stress level. However, a significant difference (P<0.05) in CMS was observed between the two parental lines and among the population. No significant correlation was found between CMS and RWC, indicating that the variation in CMS was genotypic in nature. The continuous distribution of CMS and its broad-sense heritability (34%) indicates that CMS should be polygenic in nature. A linkage map of this population comprising of 145 RFLPs, 153 AFLPs and 17 microsatellite markers was used for QTL analysis. Composite interval mapping identified nine putative QTLs for CMS located on chromosomes 1, 3, 7, 8, 9, 11 and 12. The amount of phenotypic variation that was explained by individual QTLs ranged from 13.4% to 42.1%. Four significant (P<0.05) pairs of digenic interactions between the detected QTLs for CMS were observed. The identification of QTLs for this important trait will be useful in breeding for the improvement of drought tolerance in rice. This is the first report of mapping QTLs associated with CMS under a natural water stress condition in any crop plants.

Microsatellite variability in grapevine cultivars from different European regions and evaluation of assignment testing to assess the geographic origin of cultivars

Abstract: Nine microsatellite markers (VVMD5, VVMD7, VVS2, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79 and ssrVrZAG83) were chosen for the analysis of marker information content, the genetic structure of grapevine cultivar gene pools, and differentiation among grapevines sampled from seven European vine-growing regions (Greece, Croatia, North Italy, Austria and Germany, France, Spain and Portugal). The markers were found to be highly informative in all cultivar groups and therefore constitute a useful set for the genetic characterization of European grapevines. Similar and high levels of genetic variability were detected in all investigated grapevine gene pools. Genetic differentiation among cultivars from different regions was significant, even in the case of adjacent groups such as the Spanish and Portuguese cultivars. No genetic differentiation could be detected between vines with blue and white grapes, indicating that they have undergone the processes of cultivar development jointly. The observed genetic differentiation among vine-growing regions suggested that cultivars could possibly be assigned to their regions of origin according to their genotypes. This might allow one to determine the geographical origin of cultivars with an unknown background. The assignment procedure proved to work for cultivars from the higher differentiated regions, as for example from Austria and Portugal.

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

Abstract: A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity.

Genetic bases of appearance quality of rice grains in Shanyou 63, an elite rice hybrid

Abstract: Appearance quality of the rice grain represents a major problem of rice production in many rice-producing areas of the world, especially in hybrid rice production in China. In this study, we conducted a molecular marker-based genetic analysis of the traits that are determinants of the appearance quality of rice grains, including traits specifying grain shape and endosperm opacity. The materials used in the analysis included an F2:3 population and an F10 recombinant inbred line population from a cross between the parents of Shanyou 63, the most widely grown rice hybrid in China. Molecular marker-based QTL (quantitative trait locus) analyses revealed that grain length and grain width were each controlled by a major QTL accounting for a very large proportion of the genetic variation, plus one or two minor QTLs each explaining a small proportion of the genetic variation. The major QTLs can be detected in both the F2:3 and recombinant inbred line population using both paddy rice and brown rice, whereas the minor QTLs were detected only occasionally. The QTL located in the interval of RG393-C1087 on chromosome 3 is the major locus for grain length, and the one in the interval RG360-C734a on chromosome 5 plays a major role in determining grain width. Similarly, white belly, which largely determines the opacity of the endosperm, is almost entirely controlled by a major locus on chromosome 5, located in the same genomic region as the major QTL for grain width. The implications of the results with respect to hybrid rice improvement were discussed.

Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose

Abstract: A starch granule protein, SGP-1, is a starch synthase bound to starch granules in wheat endosperm. A wheat lacking SGP-1 was produced by crossing three variants each deficient in one of three SGP-1 classes, namely SGP-A1, -B1 or -D1. This deficient wheat (SGP–1 null wheat) showed some alterations in endosperm starch, meaning that SGP-1 is involved in starch synthesis. Electrophoretic experiments revealed that the levels of two starch granule proteins, SGP-2 and -3, decreased considerably in the SGP-1 null wheat though that of the waxy protein (granule-bound starch syn- thase I) did not. The A-type starch granules were deformed. Apparent high amylose level (30.8–37.4%) was indicated by colorimetric measurement, amperometric titration, and the concanavalin A method. The altered structure of amylopectin was detected by both high- performance size-exclusion chromatography and high-performance anion exchange chromatography. Levels of amylopectin chains with degrees of polymerization (DP) 6–10 increased, while DP 11–25 chains decreased. A low starch crystallinity was shown by both X-ray diffraction and differential scanning calorimetry (DSC) analyses because major peaks were absent. Abnormal crystallinity was also suggested by the lack of a polarized cross in SGP-1 null starch. The above results suggest that SGP-1 is responsible for amylopectin synthesis. Since the SGP-1 null wheat produced novel starch which has not been described before, it can be used to expand variation in wheat starch.

A high-density linkage map of Theobroma cacao L.

Abstract: The first linkage map established by Lanaud et al. (1995) was used as a starting point to produce a high-density molecular linkage map. A mapping population of 181 progenies resulting from a cross between two heterozygous genotypes, a Forastero and a Trinitario (hybrid between Forastero and Criollo), was used for the linkage analysis. A new DNA isolation protocol was established, which allows enough good quality DNA to construct a genetic map with PCR-based markers. The map comprises 424 markers with an average spacing between markers of 2.1 cM. The marker types used were five isozymes, six loci from known function genes, 65 genomic RFLPs, 104 cDNA RFLPs, three telomeric probes, 30 RAPDs, 191 AFLPs and 20 microsatellites. The use of new marker types, AFLP and microsatellites, did not disturb the original order of the RFLP loci used on the previous map. The genetic markers were distributed over ten linkage groups and cover 885.4 cM. The maximum distance observed between adjacent markers was 16.2 cM, and 9.4% of all loci showed skewed segregation.

Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.)

Abstract: We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L) Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci Five primer pairs amplified polymorphic products of the expected size range SSR polymorphism was explored in a set of 46 olive cultivars A total of 26 alleles were detected for the five loci Heterozygosity ranged from 046 to 071 Ninety one per cent of the cultivars had unique multilocus genotypes Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’

Mapping QTLs controlling grain yield and its components on chromosome 5A of wheat

Abstract: Chromosome 5A of wheat is known to carry a number of genes affecting adaptability and productivity To localize quantitative trait loci (QTLs) controlling grain yield and its components, an RFLP map was constructed from 118 single-chromosome recombinant lines derived from the F1 between Chinese Spring (Cappelle-Desprez 5A) and Chinese Spring (Triticum spelta 5A) The map was combined with the field-trial data scored over 3 years A total of five regions in chromosome 5A contributed effects on yield traits Increases in grain yield, 50-grain weight and spikelet number/ear were determined by complementary QTL alleles from both parents The effects associated with the vernalization requirement gene Vrn-A1 or a closely linked QTL were significant only in the favorable growing season where the later-flowering vrn-A1 allele from Cappelle-Desprez 5A produced a higher tiller number/plant and spikelet number/ear The effects of the ear morphology gene q or closely linked QTL(s) were detected for grain yield and ear grain weight Three other QTLs with minor effects were dispersed along chromosome 5A These QTLs had large interactions with years due to changes in the magnitude of the significant response The alleles from T spelta, however, conferred a higher yield performance

Characterization and detection of epistatic interactions of 3 QTLs, Hd1, Hd2, and Hd3, controlling heading date in rice using nearly isogenic lines.

Abstract: To characterize quantitative trait loci (QTLs), we used marker-assisted selection (MAS) to develop three nearly isogenic lines (NILs) differing only for the presence of a single, specific QTL (QTL-NILs) –Hd1, Hd2, and Hd3 – for heading date in rice. The three lines contained the chromosomal region of the target QTL from donor variety Kasalath(indica) in the genetic background of var. Nipponbare (japonica). To analyze epistatic interactions in pairs of these QTLs, we also used MAS to develop four combined QTL-NILs with 2 of the 3 QTLs or with all 3. Different daylength treatment testing of the QTL-NILs revealed that the three QTLs control photoperiod sensitivity. Genetic analysis of F2 populations derived from crosses between the three QTL-NILs with a single QTL using molecular markers revealed the existence of epistatic interactions between Hd1 and Hd2, and Hd2 and Hd3. These interactions were also confirmed by the analysis of combined QTL-NILs under different daylength conditions. The existence of an epistatic interaction between Hd1 and Hd3 was also clarified. Based on these results, we suggest that the Kasalath allele of Hd3 does not affect photoperiod sensitivity by itself but that it is involved in enhancement of the expression of the Nipponbare alleles of Hd1 and Hd2.

Characteristics, linkage-map positions, and allelic differentiation of Sorghum bicolor (L.) Moench DNA simple-sequence repeats (SSRs)

Abstract: Fifty one clones isolated from a size-fractionated genomic DNA library of Sorghum bicolor (L.) Moench, that had been probed with four radiolabeled di- and tri-nucleotide oligomers, were sequenced. Fifty of the clones contained one or more simple-sequence repeats (SSRs) [72% of which were (AG/TC) n SSRs] and, following analysis of the clones, polymerase-chain-reaction primer sets that amplify 38 unique SSR loci were developed. Genotyping of the 38 loci in 18 sorghum accessions, including the parents of a recombinant inbred (RI) mapping population, revealed polymorphism at 36 of the loci among the 18 accessions and at 31 of the loci (not including null alleles at two loci) between the parents of the RI population. All of the latter 31 loci were mapped. The genotypes at 17-mapped SSR loci were assayed in 190 S. bicolor accessions in order to determine δ* T , the estimated level of allelic differentiation (the estimated probability that two members of a population, chosen at random and without replacement, differ in allelic composition), at each of the loci. The mean δ* T value determined for S. bicolor overall was 0.89, the range of mean δ* T values for ten S. bicolor races was from 0.88 to 0.83, and the range of mean δ* T values for ten working groups (= sub-races) of the race caudatum, with only two exceptions, was from 0.87 to 0.79. The lowest δ* T values for six of the loci among the ten race-caudatum working groups ranged from 0.86 to 0.70; thus, the probability that different alleles will be present at one or more of these loci in two accessions chosen at random from a working group is > 0.996 when three of the loci are genotyped, and > 0.9999 when all six of the loci are genotyped. The results of this study confirm that most S. bicolor SSR loci are sufficiently polymorphic to be useful in marker- assisted selection programs and they indicate that the levels of polymorphism at some loci are high enough to allow the vast majority of S. bicolor accessions, even accessions within working groups, to be distinguished from one another by determining the genotypes at a small number, perhaps as few as a half-dozen, SSR loci.

A combined RFLP and AFLP linkage map of upland rice (Oryza sativa L.) used to identify QTLs for root-penetration ability

Abstract: Acombined RFLP and AFLP linkage map of an F6 recombinant inbred population, which was derived from a previously mapped F2 of a cross between the two drought resistant upland rice varieties Bala and Azucena, is presented. The map contains 101 RFLP and 34 AFLP markers on 17 linkage groups covering 1680 cM. Also presented is the approximate mapping position of a further four RFLP and 75 AFLP markers, which either could not be given a unique place on the map or for which the available data is not sufficient to allow confident positioning, and the result of quantitative trait locus (QTL) mapping of traits related to root-penetration ability. Root penetration was assessed by counting the number of root axes that penetrated a 3 mm-thick layer consisting of 80% wax and 20% white soft paraffin. Good root penetration would be expected to increase drought resistance where soil strength is high. Single-marker analysis revealed seven QTLs for the number of roots which penetrate the wax layer. In identical locations were seven QTLs for the ratio of penetrated to the total number of roots. Transgressive inheritance of positive alleles from Bala explained four of these QTLs. Comparison of the QTLs identified here with previous reports of QTLs for root morphology suggest that alleles which improve root penetration ability may also either make the roots longer or thicker.

Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon

Abstract: Three different types of molecular markers, RAPD, AFLP and RFLP were used to measure genetic diversity among six genotypes of Cucumis melo L. Each line represented a different melon genotype: Piel de Sapo, Ogen, PI161375, PI414723, Agrestis and C105. A number of polymorphic RAPD, AFLP and RFLP bands were scored on all materials and the genetic similarity measured. Clustering analysis performed with the three types of markers separated the genotypes into two main groups: (1) the sweet type, cultivated melons and (2) the exotic type, not cultivated melons. While the data obtained suggest that all three types of markers are equally informative, AFLPs showed the highest efficiency in detecting polymorphism.

A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes

Abstract: Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm2 filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe. A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomic applications in barley.

Quantitative trait loci for the stay green trait in sorghum (Sorghum bicolor L. Moench): consistency across genetic backgrounds and environments

Abstract: Stay green in sorghum (Sorghum bicolor L. Moench) is characterized by the plant’s ability to tolerate post-flowering drought stress, thereby delaying the premature leaf and plant death. It contributes to normal grain filling and reduces the incidence of stalk lodging and charcoal rot disease during the late stages of grain development. Breeding for improving post-flowering drought tolerance in sorghum hybrids remains an important objective of sorghum breeders. Since evaluation of the stay green response is difficult and unreliable under field conditions, due to the timing and intensity of moisture stress and large environmental interaction, progress in improving drought tolerance by conventional breeding methods has been slow. The objective of the present study was to determine the consistency of quantitative trait loci (QTLs) controlling stay green in sorghum. We re-evaluated the Recombinant Inbred Line (RIL)-mapping population from the cross B35 x Tx7000 in two locations over 2 years and compared it with earlier reports. Analysis using the combined stay green-rating means of seven environments and the expanded molecular map reconfirmed all four stay green QTLs (Stg1, Stg2, Stg3 and Stg4) that were identified earlier by Xu et al. (2000). Similarly, comparison of the stay green QTL locations with earlier reported results indicated that all four stay green QTLs showed consistency across different genetic backgrounds. Examination of the stay green QTL profiles of the best and poorest stay-green lines indicated that three stay green QTLs, Stg1, Stg2 and Stg3, appear to be important for the expression of this trait when the percent phenotypic variation, and the consistency in different backgrounds and different environments, are considered. A significant epistatic interaction involving Stg2 and a region on linkage group C was also identified for the stay green and chlorophyll content. We concluded that Stg2 is the most important QTL controlling stay green, explaining the maximum amount of phenotypic variation. This report further strengthens our view to target the Stg2 QTL region for gene discovery in order to improve the basic understanding of the stay green phenomenon, which might be helpful in manipulating this trait not only in sorghum but also in other cereal crop species.

Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA

Abstract: Genetic diversity and relationships of 40 accessions of Ipomoea, representing ten species of series Batatas, were examined using ISSR markers and restriction-site variation in four non-coding regions of chloroplast DNA. A total of 2071 ISSR fragments were generated with 15 primers in these accessions and, on average, 52 bands per accession were amplified. Most of the primers contained dinucleotide repeats. The ISSR fragments were highly polymorphic (62.2%) among the 40 accessions studied. Restriction analysis of chloroplast (cp) DNA revealed 47 informative restriction-site and length mutations. Phylogenetic analyses of ISSR and cpDNA datasets generally revealed similar relationships at the interspecific level, but the high polymorphism of ISSRs resulted in a better separation of intraspecific accessions. However, the combined ISSR and cpDNA dataset appeared to be appropriate in resolving both intra- and interspecific relationships. Of the species examined, I. trifida was found to be the most closely related to cultivated sweetpotato, the hexaploid I. batatas, while I. ramosissima and I. umbraticola were the most distantly related to I. batatas within the series. Ipomoea triloba, hitherto considered to be one of the ancestors of sweetpotato, was only distantly related to sweetpotato based on ISSR similarity index.

Identification of genomic regions associated with stay green in sorghum by testing RILs in multiple environments

Abstract: Stay green is an important drought resistance trait for sorghum production. QTLs for this trait with consistent effects across a set of environments would increase the efficiency of selection because of its relatively low heritability. One hundred and sixty recombinant inbreds, derived from a cross between QL39 and QL41, were used as a segregating population for genome mapping and stay green evaluation. Phenotypic data were collected in replicated field trials from five sites and in three growing seasons, and analysed by fitting appropriate models to account for spatial variability and to describe the genotype by environment interaction. Interval mapping and non-parametric mapping identified three regions, each in a separate linkage group, associated with stay green in more than one trial, and two regions in single trial. The regions on linkage groups B and I were both consistently identified from three trials. The multiple environment testing was very helpful for correctly identifying QTLs associated with the trait. The utilisation of molecular markers for stay green in sorghum breeding is also discussed.

Development and characterization of microsatellite markers in black poplar (Populus nigra L.)

Abstract: Using an enrichment procedure, we have cloned and sequenced microsatellite loci from black poplar (Populus nigra L.) and developed primers for sequence-tagged microsatellite (STMS) analysis. Twelve primer pairs for dinucleotide repeats produced fragments of sufficient quality which were polymorphic in P. nigra. Some of them also showed amplification in other Populus species (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, and/or P. lasiocarpa). The best nine and (GT) (GA) microsatellite markers were tested on a set of 23 P. nigra genotypes from all over Europe. The microsatellites were highly polymorphic, with 10–19 different alleles per microsatellite locus among these 23 genotypes. WPMS08 sometimes amplified three fragments. Using the other eight marker loci, the level of heterozygosity among the plants was on average 0.71 (range 0.25–1.00). The microsatellite markers developed will be useful for screening the genetic diversity in natural populations and in gene bank collections.

Temporal trends in the diversity of UK wheat

Abstract: The common assertion that scientific plant breeding leads to a narrowing in crop diversity has been examined. We have characterised the dominant UK winter wheat varieties from the period 1934–1994 using two types of PCR-based DNA profiling (AFLPs, amplified fragment length polymorphisms, and SSRs, simple-sequence repeats, microsatellites), seed storage protein analysis and morphological descriptors. The varieties were grouped into a series of decadal groups on the basis of their first appearance on the ’Recommended List’, and by analysis of molecular variance it was shown that an overwhelming proportion of the overall observed variance occurred within, rather than between, decades. A further range of statistical indices provided little evidence for any significant narrowing of overall diversity over the time studied. Principal co-ordinate analysis showed that the diversity in the time periods overlapped and that the most modern group of varieties encompassed the majority of the diversity found in earlier decades. The consistent indication is that plant breeding has resulted, over time, in a qualitative, rather than a quantitative, shift in the diversity of winter wheat grown in the UK.

Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato

Abstract: Reliable and easy to use techniques for chromosome identification are critical for many aspects of cytogenetic research. Unfortunately, such techniques are not available in many plant species, especially those with a large number of small chromosomes. Here we demonstrate that fluorescence in situ hybridization (FISH) signals derived from bacterial artificial chromosomes (BACs) can be used as chromosome-specific cytogenetic DNA markers for chromosome identification in potato. We screened a potato BAC library using genetically mapped restriction fragment length polymorphism markers as probes. The identified BAC clones were then labeled as probes for FISH analysis. A set of 12 chromosome-specific BAC clones were isolated and the FISH signals derived from these BAC clones serve as convenient and reliable cytological markers for potato chromosome identification. We mapped the 5S rRNA genes, the 45S rRNA genes, and a potato late blight resistance gene to three specific potato chromosomes using the chromosome-specific BAC clones.

Methods of constructing core collections by stepwise clustering with three sampling strategies based on the genotypic values of crops.

Abstract: A genetic model with genotype×environment (GE) interactions for controlling systematical errors in the field can be used for predicting genotypic values by an adjusted unbiased prediction (AUP) method. Mahalanobis distance, calculated based on the genotypic values, is then applied to measure the genetic distance among accessions. The unweighted pair-group average, Ward’s and the complete linkage methods of hierarchical clustering combined with three sampling strategies are proposed to construct core collections in a procedure of stepwise clustering. A homogeneous test and t-tests are suggested for use in testing variances and means, respectively. The coincidence rate (CR%) for range and the variable rate (VR%) for the coefficient of variation are designed to evaluate the property of core collections. A worked example of constructing core collections in cotton with 21 traits was conducted. Random sampling can represent the genetic diversity structure of the initial collection. Preferred sampling can keep the accessions with special or valuable characteristics in the initial collection. Deviation sampling can retain the larger genetic variability of the initial collection. For better representation of the core collection, cluster methods should be combined with different sampling strategies. The core collections based on genotypic values retained larger genetic variability and had superior representatives than those based on phenotypic values.

A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe

Abstract: Endosperm hardness in wheat (Triticum aes- tivum L.) is determined by one major genetic factor, the Hardness (Ha) gene on the short arm of chromosome 5D. Grain hardness has previously been reported to re- sult from either a failure to express puroindoline a (Pina-D1b ) or a glycine to serine mutation at position 46 in puroindoline b (Pinb-D1b). In this study, which in- volves a large survey of 343 wheat genotypes of mostly Northern European origin, we report two new mutations in puroindoline b associated with hard endosperm. These were characterized as involving a leucine to proline change at position 60 (Pinb-D1c), and a tryptophan to arginine change at position 44 ( Pinb-D1d), respectively. While the former seems to be widely distributed in germplasm around the world, the latter was only found in three winter wheats from Sweden and Netherlands. As discussed in the paper, the three known mutations in pu- roindoline b can be considered "loss-of-function" muta- tions (i.e. soft to hard), and structural analysis may serve to predict that their dramatic effect on wheat grain tex- ture is a result of reduced lipid-binding ability.