Next‐generation sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan)

To map resistance genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) in pigeonpea, sequencing‐based bulked segregant analysis (Seq‐BSA) was used. Resistant (R) and susceptible (S) bulks from the extreme recombinant inbred lines of ICPL 20096 × ICPL 332 were sequenced. Subsequently, SNP index was calculated between R‐ and S‐bulks with the help of draft genome sequence and reference‐guided assembly of ICPL 20096 (resistant parent). Seq‐BSA has provided seven candidate SNPs for FW and SMD resistance in pigeonpea. In parallel, four additional genotypes were re‐sequenced and their combined analysis with R‐ and S‐bulks has provided a total of 8362 nonsynonymous (ns) SNPs. Of 8362 nsSNPs, 60 were found within the 2‐Mb flanking regions of seven candidate SNPs identified through Seq‐BSA. Haplotype analysis narrowed down to eight nsSNPs in seven genes. These eight nsSNPs were further validated by re‐sequencing 11 genotypes that are resistant and susceptible to FW and SMD. This analysis revealed association of four candidate nsSNPs in four genes with FW resistance and four candidate nsSNPs in three genes with SMD resistance. Further, In silico protein analysis and expression profiling identified two most promising candidate genes namely C.cajan_01839 for SMD resistance and C.cajan_03203 for FW resistance. Identified candidate genomic regions/SNPs will be useful for genomics‐assisted breeding in pigeonpea.     

Homology of AFLP products in three mapping populations of barley

Segregation of 850 polymorphic AFLP (amplified fragment length polymorphism) fragments was followed in three different doubled haploid (DH) barley populations, Dicktoo × Morex (DM), Igri × Franka (IF) and Blenheim × E224/3 (BE), which had previously been used to construct linkage maps using other molecular markers. The final maps consisted of 310, 655 and 474 markers, of which 234, 194 and 376, respectively, were AFLPs. A comparison of profiles from the parental lines identified 51 similar-sized AFLPs segregating in both DM and IF populations, 20 in the DM and BE populations and 18 in the IF and BE populations. Eight segregated in all three. Analysis of the complete datasets for each of the populations using Joinmap V.2. indicated that in general terms each of the AFLPs which were polymorphic in more than one population mapped to the same genetic locus. The number of co-dominant markers segregating in a single population ranged from 6% for DM to 12.6% for IF. These results are discussed in the context of using AFLP in genetic linkage and diversity studies. Received: 5 November 1996 / Accepted: 10 March 1997     

Association mapping in multiple segregating populations of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

Association mapping in multiple segregating populations (AMMSP) combines high power to detect QTL in genome-wide approaches of linkage mapping with high mapping resolution of association mapping. The main objectives of this study were to (1) examine the applicability of AMMSP in a plant breeding context based on segregating populations of various size of sugar beet (Beta vulgaris L.), (2) compare different biometric approaches for AMMSP, and (3) detect markers with significant main effect across locations for nine traits in sugar beet. We used 768 F _n (n = 2, 3, 4) sugar beet genotypes which were randomly derived from 19 crosses among diploid elite sugar beet clones. For all nine traits, the genotypic and genotype × location interaction variances were highly significant (P < 0.01). Using a one-step AMMSP approach, the total number of significant (P < 0.05) marker-phenotype associations was 44. The identification of genome regions associated with the traits under consideration indicated that not only segregating populations derived from crosses of parental genotypes in a systematic manner could be used for AMMSP but also populations routinely derived in plant breeding programs from multiple, related crosses. Furthermore, our results suggest that data sets, whose size does not permit analysis by the one-step AMMSP approach, might be analyzed using the two-step approach based on adjusted entry means for each location without losing too much power for detection of marker-phenotype associations.     

Identification and mapping of a locus conferring plum pox virus resistance in two apricot-improved linkage maps

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit crops in Europe and America. In particular, apricot is severely affected suffering significant fruit losses. Thus, PPV resistance is a trait of great interest for the apricot breeding programs currently in progress. In this work, two apricot maps, earlier constructed with the F₁ ‘Goldrich × Currot’ (G×C) and the F₂ ‘Lito × Lito’-98 (L×L-98) populations, have been improved including 43 and 37 new simple sequence repeat (SSR) loci, respectively, to facilitate PPV resistance trait mapping. Screening of PPV resistance on the segregating populations classified seedling phenotypes into resistant or susceptible. A non-parametric mapping method, based on the Kruskal–Wallis (KW) rank sum test, was initially used to score marker–trait association, and results were confirmed by interval mapping. Contrary to the putative digenic model inferred from the phenotypic segregations, all significant markers for the KW statistic (P < 0.005) mapped in a unique region of ~21.0 and ~20.3 cM located on the upper part of the G1 linkage group in ‘G×C’ and ‘L×L-98’ maps, respectively. According to the data, PPV resistance is suggested to be controlled by at least one major dominant locus. The association between three SSRs distributed within this region and the PPV resistance was tested in two additional populations (‘Goldrich × Canino’ and ‘Lito × Lito’-00) and breeding program parents. The marker ssrPaCITA5 showed the highest KW value (P < 0.005) in all cases, pointing out its usefulness in marker-assisted selection. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of allele specificity of comigrating AFLP markers to align genetic maps from different potato genotypes

The allele specificity of AFLP markers was assessed in five relatively unrelated potato genotypes. To this end, two diploid mapping populations of potato, F₁SH × RH and F₁AM × RH, were analysed using four and six AFLP primer combinations, respectively, recently applied to the analysis of the genetically well characterized backcross population BC_C × E. The AFLP profiles of the five parents revealed 733 AFLP markers and, when identical primer combinations were used, 131 comigrating AFLP markers were identified. After construction of five parental maps, the genomic positions of these comigrating AFLP markers were compared and 117 markers (89%) which targeted the same genomic region were assumed to be homologous. Of these putative homologues, 20 markers, each cloned from at least two genotypes, were sequenced and 19 sets of amplification products were shown to be nearly identical. The number of AFLP markers previously mapped in population BC_C × E ranged from three to eleven per chromosome, which allowed a reliable assessment of chromosome numbers from individual linkage groups obtained in populations F₁SH × RH and F₁AM × RH. The high incidence of corresponding AFLP alleles was confirmed by using an additional set of five primer combinations. The 733 AFLP markers localized provide a valuable reference collection for future mapping studies in potato. As a consequence AFLP analysis may replace more laborious locus-specific marker techniques. Received: 26 July 1996 / Accepted: 30 January 1997     

Search for nucleotide diversity patterns of local adaptation in dehydrins and other cold-related candidate genes in Scots pine (Pinus sylvestris L.)

Nucleotide variation at several cold candidate genes including seven members of the dehydrin gene family was surveyed in haplotypes of Scots pine (Pinus sylvestris) sampled in populations showing divergence for cold tolerance in Europe. Patterns of nucleotide diversity, linkage disequilibrium, and frequency spectrum of alleles were compared between north and south populations to search for signs of directional selection potentially underlying adaptation to cold. Significant differentiation between populations in allelic frequency or haplotype structure was detected at dhn1, dhn3, and abaH loci. Allelic dimorphism with no evidence of haplotype clustering by geographical distribution was found at dhn9. An excess of fixed non-synonymous mutations as compared to the outgroup P. pinaster pine species was found at dhn1. Differences in nucleotide polymorphisms were found between the members of the Kn class of dehydrin upregulated during cold acclimation (average π_sil = 0.004) as compared to the SKn class (average π_sil = 0.024). The multilocus nucleotide diversity at silent sites (θ _W = 0.009) was moderate compared to other conifer species, but higher than previous estimates for Scots pine. There was an excess of rare and high frequency derived variants as revealed by significantly negative multilocus value of Tajima’s D (D = −0.72, P < 0.01) and negative mean value of Fay and Wu H statistics (H = −0.50). The level of linkage disequilibrium decayed rapidly with an average expected r ² of 0.2 at about 200 bp. Overall, there was a positive correlation between polymorphism and divergence at ten loci when outgroup sequence was available. The discovered polymorphism will be used for further evaluation of the adaptive role of genes through association mapping studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mapping of post-flowering drought resistance traits in grain sorghum: association between QTLs influencing premature senescence and maturity

The identification of genetic factors underlying the complex responses of plants to drought stress provides a solid basis for improving drought resistance. The stay-green character in sorghum (Sorghum bicolor L. Moench) is a post-flowering drought resistance trait, which makes plants resistant to premature senescence under drought stress during the grainfilling stage. The objective of this study was to identify quantitative trait loci (QTLs) that control premature senescence and maturity traits, and to investigate their association under post-flowering drought stress in grain sorghum. A genetic linkage map was developed using a set of recombinant inbred lines (RILs) obtained from the cross B35 × Tx430, which were scored for 142 restriction fragment length polymorphism (RFLP) markers. The RILs and their parental lines were evaluated for post-flowering drought resistance and maturity in four environments. Simple interval mapping identified seven stay-green QTLs and two maturity QTLs. Three major stay-green QTLs (SGA, SGD and SGG) contributed to 42% of the phenotypic variability (LOD 9.0) and four minor QTLs (SGB, SGI.1, SGI.2, and SGJ) significantly contributed to an additional 25% of the phenotypic variability in stay-green ratings. One maturity QTL (DFB) alone contributed to 40% of the phenotypic variability (LOD 10.0), while the second QTL (DFG) significantly contributed to an additional 17% of the phenotypic variability (LOD 4.9). Composite interval mapping confirmed the above results with an additional analysis of the QTL × Environment interaction. With heritability estimates of 0.72 for stay-green and 0.90 for maturity, the identified QTLs explained about 90% and 63% of genetic variability for stay-green and maturity traits, respectively. Although stay-green ratings were significantly correlated (r=0.22, P ≤ 0.05) with maturity, six of the seven stay-green QTLs were independent of the QTLs influencing maturity. Similarly, one maturity QTL (DFB) was independent of the stay-green QTLs. One stay-green QTL (SGG), however, mapped in the vicinity of a maturity QTL (DFG), and all markers in the vicinity of the independent maturity QTL (DFB) were significantly (P ≤ 0.1) correlated with stay-green ratings, confounding the phenotyping of stay-green. The molecular genetic analysis of the QTLs influencing stay-green and maturity, together with the association between these two inversely related traits, provides a basis for further study of the underlying physiological mechanisms and demonstrates the possibility of improving drought resistance in plants by pyramiding the favorable QTLs. Received: 10 October 1998 / Accepted: 12 July 1999     

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 × 10³ DJ per milliliter for S. carpocapsae and 1 to 8 × 10³ DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 × 10³ DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3–6 × 10³ DJs per milliliter in order to obtain >10³ parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 × 10³ DJs per milliliter to reach >2 × 10³ parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 × 10³ and 105 × 10³ per mililiter for S. feltiae.     

RETRACTED ARTICLE: Archaeal <Emphasis Type="Italic">amoA</Emphasis> Genes Outnumber Bacterial <Emphasis Type="Italic">amoA</Emphasis> Genes in Municipal Wastewater Treatment Plants in Bangkok

The contribution of ammonia-oxidizing archaea (AOA) to nitrogen removal in wastewater treatment plants (WWTPs) remains unknown. This study investigated the abundance of archaeal (AOA) and bacterial (ammonia-oxidizing bacteria (AOB)) amoA genes in eight of Bangkok’s municipal WWTPs. AOA amoA genes (3.28 × 10⁷ ± 1.74 × 10⁷–2.23 × 10¹¹ ± 1.92 × 10¹¹ copies l⁻¹ sludge) outnumbered AOB amoA genes in most of the WWTPs even though the plants’ treatment processes, influent and effluent characteristics, removal efficiencies, and operation varied. An estimation of the ammonia-oxidizing activity of AOA and AOB suggests that AOA involved in autotrophic ammonia oxidation in the WWTPs. Statistical analysis shows that the numbers of AOA amoA genes correlated negatively to the ammonium levels in effluent wastewater, while no correlation was found between the AOA amoA gene numbers and the oxygen concentrations in aeration tanks. An analysis of the AOB sequences shows that AOB found in the WWTPs limited to only two AOB clusters which exhibit high or moderate affinity to ammonia. In contrast to AOB, AOA sequences of various clusters were retrieved, and they were previously recovered from a variety of environments, such as thermal and marine environments.     

Genomic mapping and testing for quantitative trait loci in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze)

The tea industry is significant in the economies of tea-growing countries. Prospects of improving yield of made tea genomic information were explored using clones from a cross between clones TRFCA SFS150 and AHP S15/10. The 42 clones were tested in two distinct tea-growing regions in Kenya. Bulk segregant analysis was performed followed by complete genotyping. Out of 260 informative markers, 100 markers that showed 1:1 segregation were used to construct a linkage map. The map contained 30 (19 maternal and 11 paternal) linkage groups that spanned 1,411.5 cM with mean interval of 14.1 cM between loci. Based on the map, quantitative trait loci (QTL) analysis was done on yield data over 2003–2007 across the two sites, Timbilil and Kangaita. Twenty-three putative QTLs were detected, 16 in five different linkage groups for Timbilil, two in two groups for Kangaita, and the rest were associated with unassigned markers. No QTL was detected at both sites, which showed strong genotype × site interaction (G × E) but highly effective within-site heritability ([^(h)]² {\hat{h}^2} generally > 0.7). Problems of overestimated and spurious QTL effects arising from the smallness of the population should be mitigated by generally high within-site heritability. At least two unassigned markers associated with yield at Kangaita over the whole study period, suggesting potential as candidate markers for site-specific marker-assisted selections. Implications of the results with respect to mapping population, G × E, and marker-assisted selection are discussed.     

Microsatellites in Cassava (Manihot esculenta Crantz): discovery, inheritance and variability

 Fourteen microsatellites containing GA-repeats were isolated and characterized in cassava (Manihot esculenta Crantz, Euphorbiaceae). Microsatellite heterozygosity (h) was estimated in 48 accessions using (³²P)-end-labeled primers and in more than 500 accessions using fluorescence-based genotyping. Heterozygosity values ranged from 0.00 to 0.88 and the number of alleles detected varied from 1 to 15. The reproducibility of allele sizing was also assessed using fluorescence-based genotyping. The average inter-gel size difference was 1.03 nucleotides. Chi-square tests (χ²) were performed to analyse segregation distortion and the linkage between alleles segregating from either or both parents in an F₁ mapping population. Most microsatellite loci segregated in the expected 1 : 1, 1 : 2 : 1 or 1 : 1 : 1 : 1 ratio. Linkage was detected between loci segregating from either parent, and segregation distortion from the male parent was detected for locus GA-131. Approximately 80% of the microsatellites detected one or two alleles per accession, suggesting a low degree of microsatellite locus duplication, an unexpected finding for a putative allopolyploid, highly heterozygous species. The high h values of most microsatellites, their amplification in other Manihot taxa and their suitability for high-throughput, fluorescence-based genotyping, make microsatellites the marker of choice for germplasm characterization and saturation of the cassava map. Received: 4 September 1997 / Accepted 16 March 1998     

EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome

In a panel of seven genotypes, 437 expressed sequence tag (EST)-derived DNA fragments were sequenced. Single nucleotide polymorphisms (SNPs) that were polymorphic between the parents of three mapping populations were mapped by heteroduplex analysis and a genome-wide consensus map comprising 216 EST-derived SNPs and 4 InDel (insertion/deletion) markers was constructed. The average frequency of SNPs amounted to 1/130 bp and 1/107.8 bp for a set of randomly selected and a set of mapped ESTs, respectively. The calculated nucleotide diversities (π) ranged from 0 to 40.0 × 10⁻³ (average 3.1 × 10⁻³) and 0.52 × 10⁻³ to 39.51 × 10^–3 (average 4.37 × 10⁻³) for random and mapped ESTs, respectively. The polymorphism information content value for mapped SNPs ranged from 0.24 to 0.50 with an average of 0.34. As expected, combination of SNPs present in an amplicon (haplotype) exhibited a higher information content ranging from 0.24 to 0.85 with an average of 0.50. Cleaved amplified polymorphic sequence assays (including InDels) were designed for a total of 87 (39.5%) SNP markers. The high abundance of SNPs in the barley genome provides avenues for the systematic development of saturated genetic maps and their integration with physical maps. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Both R. Kota and R.K. Varshney contributed equally to this work.     

Molecular mapping and inheritance of restoration of fertility (<Emphasis Type="Italic">Rf</Emphasis>) in A4 hybrid system in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.)

Key message

We report molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea. We have also developed PCR-based markers amenable to low-cost genotyping to identify fertility restorer lines.

Abstract

Commercial hybrids in pigeonpea are based on A4 cytoplasmic male sterility (CMS) system, and their fertility restoration is one of the key prerequisites for breeding. In this context, an effort has been made to understand the genetics and identify quantitative trait loci (QTL) associated with restoration of fertility (Rf). One F₂ population was developed by crossing CMS line (ICPA 2039) with fertility restorer line (ICPL 87119). Genetic analysis has shown involvement of two dominant genes in regulation of restoration of fertility. In parallel, the genotyping-by-sequencing (GBS) approach has generated ~?33 Gb data on the F₂ population. GBS data have provided 2457 single nucleotide polymorphism (SNPs) segregating across the mapping population. Based on these genotyping data, a genetic map has been developed with 306 SNPs covering a total length 981.9 cM. Further QTL analysis has provided the region flanked by S8_7664779 and S8_6474381 on CcLG08 harboured major QTL explained up to 28.5% phenotypic variation. Subsequently, sequence information within the major QTLs was compared between the maintainer and the restorer lines. From this sequence information, we have developed two PCR-based markers for identification of restorer lines from non-restorer lines and validated them on parental lines of hybrids as well as on another F₂ mapping population. The results obtained in this study are expected to enhance the efficiency of selection for the identification of restorer lines in hybrid breeding and may reduce traditional time-consuming phenotyping activities.

     

Development and application of microsatellite markers for genomic analysis of papaya

Papaya has a relatively small genome, displays high levels of phenotypic diversity, and is amenable to transformation, making it attractive as a fruit tree model system. The high level of phenotypic diversity seen among papaya cultivars in the field does not correlate with the low levels of genotypic polymorphism thus far elucidated. The highly mutable nature of microsatellites or simple sequence repeats (SSRs) make them potentially powerful markers for distinguishing deoxyribonucleic acid (DNA) polymorphisms between closely related genotypes. Genomic research for papaya has resulted in a significant quantity of sequence data. We mined 28.1 Mb of bacterial artificial chromosomes end sequences, 5.8 Mb of complementary DNA, and 1.6 Mb of random genomic sequences for SSRs. We generated 938 SSR markers and tested for polymorphism among seven varieties that had been used to produce five mapping populations. The level of polymorphism was highest for Kaek Dum × 2H94 with 210 markers, followed by UH928 × SunUp with 194, AU9 × SunUp with 189, UH918 × SunUp with 177, and Kapoho × SunUp displaying the lowest level with 97. Variation in levels of polymorphism, motif predominance, and motif length between the genomic and genic fractions indicated differential selection pressures acting on the microsatellites in these two fractions. The microsatellites developed in this study will greatly assist in the genetic and physical mapping of the papaya genome as well as enhance breeders’ ability to improve the crop. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

A molecular marker linkage map of tetraploid alfalfa (Medicago sativa L.)

A genetic linkage map was constructed for an F₁ genotype of auto-tetraploid alfalfa (Medicago sativa L.) using two backcross populations of 101 individuals each and 82 single-dose restriction fragments segregating in each population. The percentages of marker loci deviating from Mendelian ratios were considerably less than reported for inbred diploid mapping populations (4–9% compared to 18–54%), probably due to the greater buffering capacity of autotetraploids against the effects of deleterious recessive alleles. Four homologous coupling-phase cosegregation groups were detected for seven of the eight linkage groups of diploid alfalfa and aligned using probes in common. No cosegregation groups were found for linkage group 7 due to the lack of polymorphisms in this cross. A composite map was generated by integrating the four homologous cosegregation groups and consisted of 88 loci on seven linkage groups covering 443 cM. The locus map-orders and distances were in general agreement with those found in diploid alfalfa. The mapping population segregates for winterhardiness, fall dormancy, and freezing tolerance; and the map will be used to locate genomic regions affecting these traits. Received: 9 December 1998 / Accepted: 22 June 1999     

Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.)

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) can be a major economic problem to apple growers in most parts of the world, and resistance breeding provides a sustainable means to control this pest. We report molecular markers for three genes conferring WAA resistance and placing them on two linkage groups (LG) on the genetic map of apple. The Er1 and Er2 genes derived from ‘Northern Spy’ and ‘Robusta 5,’ respectively, are the two major genes that breeders have used to date to improve the resistance of apple rootstocks to this pest. The gene Er3, from ‘Aotea 1’ (an accession classified as Malus sieboldii), is a new major gene for WAA resistance. Genetic markers linked to the Er1 and Er3 genes were identified by screening random amplification of polymorphic deoxyribonucleic acid (DNA; RAPD) markers across DNA bulks from resistant and susceptible plants from populations segregating for these genes. The closest RAPD markers were converted into sequence-characterized amplified region markers and the genome location of these two genes was assigned to LG 08 by aligning the maps around the genes with a reference map of ‘Discovery’ using microsatellite markers. The Er2 gene was located on LG 17 of ‘Robusta 5’ using a genetic map developed in a M.9 × ‘Robusta 5’ progeny. Markers for each of the genes were validated for their usefulness for marker-assisted selection in separate populations. The potential use of the genetic markers for these genes in the breeding of apple cultivars with durable resistance to WAA is discussed.     

Presence of defeated qualitative resistance genes frequently has major impact on quantitative resistance to Melampsora larici-populina leaf rust in P. × interamericana hybrid poplars

Qualitative resistance to Melampsora larici-populina leaf rust inherited from North American species Populus deltoides did not allow for durable control of this pathogen in interspecific hybrid cultivars. Despite significant levels of strain-specificity, quantitative resistance would exert lower selection pressures on the pathogen populations, and hence could be more durable. Previous studies restricted to a large P. × interamericana (i.e., P. deltoides × Populus trichocarpa) F₁ family revealed that the presence of R₁, a segregating defeated qualitative resistance gene inherited from P. deltoides, had major beneficial effects on quantitative resistance. The present study was based on 14 F₁ families from a 4 × 5 P. deltoides × P. trichocarpa factorial mating design where at least four defeated qualitative resistances segregate 1:1. Even though quantitative resistance assessments were conducted in the laboratory with a M. larici-populina strain able to overcome these qualitative resistances, their presence had a significant effect on the mean level and on the genetic variability for quantitative resistance. One unprecedented result is the identification of a defeated qualitative resistance which presence is associated with lower levels of quantitative resistance. Possible inferences on the nature of the genetic relationship between both resistance types are discussed.     

Exploiting rice–sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 × E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice–sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.