Co-dominant Simple Sequence Repeat Markers Research Articles

Molecular Characterization of Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Collections from Nine Countries.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. To understand the worldwide distribution of its molecular groups, as well as the diversity, differentiation, and migration of the Pst populations, 567 isolates collected from nine countries (China, Pakistan, Italy, Egypt, Ethiopia, Canada, Mexico, Ecuador, and the U.S.) in 2010–2018 were genotyped using 14 codominant simple sequence repeat markers. A total of 433, including 333 new multi-locus genotypes (MLGs), were identified, which were clustered into ten molecular groups (MGs). The MGs and country-wise populations differed in genetic diversity, heterozygosity, and correlation coefficient between the marker and virulence data. Many isolates from different countries, especially the isolates from Mexico, Ecuador, and the U.S., were found to be identical or closely related MLGs, and some of the MGs were present in all countries, indicating Pst migrations among different countries. The analysis of molecular variance revealed 78% variation among isolates, 12% variation among countries, and 10% variation within countries. Only low levels of differentiation were found by the pairwise comparisons of country populations. Of the 10 MGs, 5 were found to be involved in sexual and/or somatic recombination. Identical and closely related MLGs identified from different countries indicated international migrations. The study provides information on the distributions of various Pst genetic groups in different countries and evidence for the global migrations, which should be useful in understanding the pathogen evolution and in stressing the need for continual monitoring of the disease and pathogen populations at the global scale.

Parasexual reproduction in Alternaria solani: Simple sequence repeat molecular evidence for haploidization

ABSTRACT Multiple alleles were constantly detected in Alternaria solani isolates by simple sequence repeat (SSR) analysis, and sectors were also observed in their subcultures. These preliminary results and observations point to a possible parasexual cycle in A. solani. In this study, codominant SSR markers were used as molecular markers on the chromosomes of A. solani and single-conidium subculture was used to simulate the mitosis process of A. solani in nature. The number of alleles at locus As-95236 changed from 2 to 1 as a molecular marker for haploidy of parasexuality of A. solani. Fifty monosporic F1 strains were tested. The results showed that two parent strains lost allele with a haploid probability of 38%. For F2 strains, the results showed that all four F1 strains lost allele with a haploid probability of 75%. Since sexual recombination of A. solani has not been found so far, the allele lost in the subcultures of A. solani isolates provides molecular evidence for the existence of parasexual reproduction in A. solani.

Molecular Characterization of International Collections of the Wheat Stripe Rust Pathogen Puccinia striiformis f. sp. tritici Reveals High Diversity and Intercontinental Migration.

Puccinia striiformis f. sp. tritici causes stripe rust (yellow rust), one of the most important wheat diseases worldwide. To understand the genetic variation of the pathogen in a global scale, 283 P. striiformis f. sp. tritici isolates collected from 16 countries in eight geographic regions were genotyped using 24 codominant simple sequence repeat markers. The overall collection had a high level of genetic diversity, and the diversity levels in the Asian populations were generally higher than those of the other regions. Heterozygosity of isolates ranged from 0 to 75%, with an average of 46%. Mean heterozygosity in individual countries ranged from 34 to 59%. A total of 265 multilocus genotypes (MLGs) were detected, which were classified into eight molecular groups. Some of the molecular groups were present in all geographic regions. Moreover, many isolates from different regions were found to be identical or very closely related MLGs. Analysis of molecular variance revealed high variation within countries and intermediate variation between countries, but it revealed low and insignificant variation among geographic regions. Pairwise comparisons of regional populations detected considerable effective migrants and only low to moderate levels of differentiation. The molecular genotypes had a moderate level of correlation with the virulence phenotypes, and some of the molecular/virulence groups contained isolates from different continents. The results indicate tremendous migrations of P. striiformis f. sp. tritici and warrant the development of management strategies considering the global pathogen population.

Molecular mapping of a recessive stripe rust resistance gene yrMY37 in Chinese wheat cultivar Mianmai 37

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) in many cool and temperate regions of the world. Resistant cultivars are the most effective, economical and environmentally friendly means of controlling the disease. Chinese wheat cultivar Mianmai 37 is resistant at all growth stages to all prevalent Chinese Pst races including v26, a new Pst race virulent to resistance genes Yr24/Yr26. Genetic analysis of the F1, F2 and F2:3 populations from a cross between Mianmai 37 and susceptible line Mingxian 169 indicated that resistance to Pst race v26 was conferred by a single recessive gene, temporarily designated yrMY37. Simple sequence repeat (SSR) marker analysis placed yrMY37 on chromosome 7BL near the centromere. Eight co-dominant genomic SSR markers, Xgwm400, Xwmc758, Xwmc476, Xgwm297, Xbarc267, Xwmc364, Xwmc696 and Xwmc396, were linked with yrMY37. The closest SSR loci Xbarc267 and Xgwm297 flanked the resistance gene at 0.38 and 0.78 cM, respectively. A seedling test with 22 Pst races indicated that the reaction patterns of Mianmai 37 were different from those of lines carrying resistance genes Yr2, Yr6, Yr67 and YrZH84 on chromosome 7B. As there is no recessive stripe rust resistance gene reported on 7BL so far, we believe that yrMY37 is most likely a new stripe rust resistance gene.

Registration of BARBREN-713 Germplasm Line of Upland Cotton Resistant to Reniform and Root-Knot Nematodes

Cotton (Gossypium hirsutum L.) germplasm line BARBREN-713 (Reg. No. GP-987, PI 671965) was developed and released by the USDA–ARS, Mississippi Agricultural and Forestry Experiment Station, Texas A&M AgriLife Research, and Cotton Incorporated in 2012. The objective of the release was to provide public and private breeders with an agronomically desirable germplasm that is resistant to both the reniform nematode (Rotylenchulus reniformis Linford and Oliveira) and the root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood]. The line also has excellent seedling vigor in fields infested with nematodes and fungal root rot pathogens, such as Thielaviopsis basicola, Rhizoctonia solani and Fusarium spp. Resistance to reniform nematode was transferred from G. barbadense GB713 (PI 608139) and is associated primarily with the Ren2GB713 gene on chromosome 21. Resistance to root-knot nematode was transferred from the germplasm line M-315 RNR (PI 592514), ‘LA 887’, or ‘Acala Nem-X’ and is associated primarily with the Mi-1 gene on chromosome 11. The codominant simple sequence repeat markers, BNL 3279_105 and CIR 316_202, respectively, were closely linked to the resistance genes. A single nucleotide polymorphism marker, Gl-187401, also was developed to detect the Ren2GB713 gene. In controlled environment assays, BARBREN-713 suppressed reproduction of both nematodes by 90% or more. It also reduced reniform nematode populations in the field at eight locations in four states. The line has fiber quality similar to M-315 RNR but yielded more than M-315 RNR in reniform nematode-infested fields.

High-resolution mapping of the dull fruit skin gene D in cucumber (Cucumis sativus L.)

Dull/glossy fruit skin is a highly valuable external quality trait that affects the market value of cucumbers. In this study, genetic analysis showed that one single dominant gene, D (dull fruit skin), determines the dull fruit skin trait in cucumber. By combining bulked segregant analysis with 11 published polymorphic molecular markers on chromosome 5, the D/d gene was preliminarily mapped between markers SCZ69 and SSR16203, at genetic distances of 0.3 and 0.6 cM, respectively. Subsequently, a larger F2 (S06 × S94) population (842 individuals in total) was used for high-resolution mapping of the D/d gene. Finally, the D/d gene was fine-mapped between markers SSR37 and SSR112, at a physical distance of 244.9 kb (containing 31 candidate genes), using eight newly developed polymorphic simple sequence repeat (SSR) markers between SCZ69 and SSR16203. Based on semi-quantitative RT-PCR analysis, the possible candidate gene D was identified as Csa016880 or Csa016887. Meanwhile, validity analysis of the markers SSR37 and SSR112 was performed with 72 dull/glossy fruit lines, and showed that the two co-dominant SSR markers could be used for marker-assisted selection of the dull/glossy fruit trait in cucumber breeding. Moreover, this study will be helpful for cloning of the D gene in cucumber.

Pinus monticola pathogenesis-related gene PmPR10-2 alleles as defense candidates for stem quantitative disease resistance against white pine blister rust (Cronartium ribicola)

White pine blister rust, caused by the invasive fungus Cronartium ribicola, has been responsible for extremely high mortality of native western white pine (Pinus monticola) and other five-needle pines in natural stands throughout western North America. The presence of this non-native fungus has also led to greatly restricted use of western white pine for reforestation. A few families of defense proteins have been found as functional candidates involved in tree resistance to rust infection. Here we report genetic variation of a gene encoding a family 10 pathogenesis-related (PR) protein (PmPR10-2) in open-pollinated seed families with different levels of stem quantitative disease resistance (QDR). Six novel alleles and five common genotypes were identified inside the PmPR10-2 locus: these genetic variations included 33 single nucleotide polymorphisms (SNPs) throughout the gene regions and copy variation of a rare octanucleotide simple sequence repeat (SSR), 5′-AATTATTT-3′, in the gene intron. PmPR10-2 exhibited a moderate level (average r 2 = 0.42) of linkage disequilibrium. Two-thirds of the identified SNPs and the SSR marker were significantly associated with stem QDR levels. The PmPR10-2 genotype (H3:H3) exhibited the highest level of stem QDR (P < 0.01). Cost-effective and co-dominant SSR markers were developed and used for genotyping the PmPR10-2 locus using simple PCR, providing a potential molecular tool for accelerating screening efforts of white pine resistance against C. ribicola.

Next generation DNA sequencing technology delivers valuable genetic markers for the genomic orphan legume species, Bituminaria bituminosa.

BackgroundBituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species.Results432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme.ConclusionsB. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.

Classification and diversity of sacred and American Nelumbo species: the genetic relationships of flowering lotus cultivars in Japan using SSR markers

The water lotus, genusNelumbo, contains two species, the sacred (Nelumbo nucifera) and American lotuses (Nelumbo lutea). Hundreds of flowering lotus cultivars are currently known. However, their classification is unclear. For the classification ofNelumbocultivars, in addition to 35 simple sequence repeat (SSR) markers recently developed, we have developed 17 and 16 of newNelumboSSR markers from SSR-enriched genomic libraries and expressed sequence tag (EST) data, respectively. Out of these 68 SSRs, along with SSRs recently published by others, 52 showed clear polymorphisms in 98Nelumbosamples. A total of 300 alleles were observed, ranging from 2 to 11 alleles per locus, with an average of 5.77. Alleles specific for the American lotus-derived cultivars and a cluster of the American lotus-derived cultivars on a neighbour-joining tree confirmed genetic differences betweenN. luteaandN. nucifera. In addition, a possible differentiation between Chinese and Japanese cultivars was also suggested. Parentage analysis using the SSR markers confirmed four known parentages and predicted currently-unknown parentages of six cultivars. The present data have demonstrated that site-specific, co-dominant SSR markers enable more accurate classification, identification and comparison ofNelumbospecies.

QTL mapping of Sclerotinia midstalk-rot resistance in sunflower.

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F3 families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F2 genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F3 families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h2=0.55-0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.

Inheritance and diversity of simple sequence repeat (SSR) microsatellite markers in various families of Picea abies.

A large number of sequence-specific SSRs were screened by using electrophoresis on metaphore agarose gels with the bands visualized by ethidium bromide staining. Many SSRs appeared as codominant and many as dominant markers, with presence or absence of bands. A simple Mendelian inheritance pattern for most codominant and dominant SSR loci was found. For many codominant SSR markers, null alleles were detected. The proportion of dominant microsatellites detected in this study (close to 50 %) was much higher than that commonly reported in many other studies. A high proportion of dominant markers together with a high frequency of codominant markers with null alleles may represent two important limitations for the use of microsatellites in different studies. On the other hand, many polymorphic codominant SSR microsatellite markers were found to be highly repeatable, and can be used for population studies, seed certification, quality control of controlled crosses, paternity analysis, pollen contamination, and mapping of QTL in related families. In this paper, we report on the inheritance pattern and diversity of codominant and dominant SSR microsatellites in seven families of Picea abies sharing a common mother.

Polymorphism of simple sequence repeats reveals gene flow within and between ectomycorrhizal Suillus grevillei populations.

• Gene flow within and between two populations of the ectomycorrhizal fungus Suillus grevillei is shown here using co-dominant simple sequence repeat (SSR) markers. • Co-dominant SSR markers were developed for S. grevillei. Distribution and frequency of alleles at the three codominant SSR markers were analysed within two S. grevillei populations in two Larix Kaempteri stands located 700m apart. • Among eight SSR loci isolated from S. grevillei, five loci (designated SG1-5) were polymorphic and SG1-3 were co-dominant. Genets (73) previously identified by inter-simple sequence repeat markers at the Larix stands were divided by the combination of SG1-3 into 22 genotypes. Most of the SSR genotypes were spatially clustered, indicating that the dispersal distance of S. grevillei spores was relatively short. • There was no conspicuous genetic differentiation within or between the two S. grevillei populations, indicating extensive gene flow. The spread of alleles within or between populations might be by repeats of short-distance spore dispersal rather than long-distance spore dispersal.