LegumeSSRdb: A Comprehensive Microsatellite Marker Database of Legumes for Germplasm Characterization and Crop Improvement

Microsatellites, or simple sequence repeats (SSRs), are polymorphic loci that play a major role as molecular markers for genome analysis and plant breeding. The legume SSR database is a webserver which contains simple sequence repeats (SSRs) from genomes of 13 legume species. A total of 3,706,276 SSRs are present in the database, 698,509 of which are genic SSRs, and 3,007,772 are non-genic. This webserver is an integrated tool to perform end-to-end marker selection right from generating SSRs to designing and validating primers, visualizing the results and blasting the genomic sequences at one place without juggling between several resources. The user-friendly web interface allows users to browse SSRs based on the genomic region, chromosome, motif type, repeat motif sequence, frequency of motif, and advanced searches allow users to search based on chromosome location range and length of SSR. Users can give their desired flanking region around repeat and obtain the sequence, they can explore the genes in which the SSRs are present or the genes between which the SSRs are bound design custom primers, and perform in silico validation using PCR. An SSR prediction pipeline is implemented where the user can submit their genomic sequence to generate SSRs. This webserver will be frequently updated with more species, in time. We believe that legumeSSRdb would be a useful resource for marker-assisted selection and mapping quantitative trait loci (QTLs) to practice genomic selection and improve crop health. The database can be freely accessed at http://bioinfo.usu.edu/legumeSSRdb/.     

Rapid Development of Microsatellite Markers for the Endangered Fish Schizothorax biddulphi (Günther) Using Next Generation Sequencing and Cross-Species Amplification

Tarim schizothoracin (Schizothorax biddulphi) is an endemic fish species native to the Tarim River system of Xinjiang and has been classified as an extremely endangered freshwater fish species in China. Here, we used a next generation sequencing platform (ion torrent PGM™) to obtain a large number of microsatellites for S. biddulphi, for the first time. A total of 40577 contigs were assembled, which contained 1379 SSRs. In these SSRs, the number of dinucleotide repeats were the most frequent (77.08%) and AC repeats were the most frequently occurring microsatellite, followed by AG, AAT and AT. Fifty loci were randomly selected for primer development; of these, 38 loci were successfully amplified and 29 loci were polymorphic across panels of 30 individuals. The H_o ranged from 0.15 to 0.83, and H_e ranged from 0.15 to 0.85, with 3.5 alleles per locus on average. Cross-species utility indicated that 20 of these markers were successfully amplified in a related, also an endangered fish species, S. irregularis. This study suggests that PGM™ sequencing is a rapid and cost-effective tool for developing microsatellite markers for non-model species and the developed microsatellite markers in this study would be useful in Schizothorax genetic analysis.     

Application of Novel Polymorphic Microsatellite Loci Identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the Genetic Characterization of Wild and Released Populations

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.     

Molecular Karyotyping on Populus simonii × P. nigra and the Derived Doubled Haploid

The molecular karyotype could represent the basic genetic make-up in a cell nucleus of an organism or species. A doubled haploid (DH) is a genotype formed from the chromosome doubling of haploid cells. In the present study, molecular karyotype analysis of the poplar hybrid Populus simonii × P. nigra (P. xiaohei) and the derived doubled haploids was carried out with labeled telomeres, rDNA, and two newly repetitive sequences as probes by fluorescence in situ hybridization (FISH). The tandem repeats, pPC349_XHY and pPD284_XHY, with high-sequence homology were used, and the results showed that they presented the colocalized distribution signal in chromosomes. For P. xiaohei, pPD284_XHY produced hybridizations in chromosomes 1, 5, 8, and 9 in the hybrid. The combination of pPD284_XHY, 45S rDNA, and 5S rDNA distinctly distinguished six pairs of chromosomes, and the three pairs of chromosomes showed a significant difference in the hybridization between homologous chromosomes. The repeat probes used produced similar FISH hybridizations in the DH; nevertheless, pPD284_XHY generated an additional hybridization site in the telomere region of chromosome 14. Moreover, two pairs of chromosomes showed differential hybridization distributions between homologous chromosomes. Comparisons of the distinguished chromosomes between hybrid and DH poplar showed that three pairs of chromosomes in the DH presented hybridization patterns that varied from those of the hybrid. The No. 8 chromosome in DH and one of the homologous chromosomes in P. xiaohei shared highly similar FISH patterns, which suggested the possibility of intact or mostly partial transfer of the chromosome between the hybrid and DH. Our study will contribute to understanding the genetic mechanism of chromosomal variation in P. xiaohei and derived DH plants.     

Identification of a Candidate restorer-of-fertility Gene Rf3 Encoding a Pentatricopeptide Repeat Protein for the Cytoplasmic Male Sterility in Soybean

The cytoplasmic male sterility/restorer-of-fertility (CMS/Rf) system plays a vital role in high-efficiency hybrid seed production in crops, including soybean (Glycine max (L.) Merr.). The markers linked to fertility restoration and the restorer-of-fertility (Rf) genes are essential because they can facilitate the breeding of new CMS lines and production of commercial hybrid soybean seeds. To date, several soybean Rf genes have been mapped to various genetic loci in diverse genetic populations. However, the mapping range of restorer genes remains narrow, with relatively limited practical applicability. Therefore, in the present study, F₂ and F₃ segregating populations derived from the CMS line JLCMS5A crossed with the restorer line JLR2 were developed and used for Rf3 gene fine mapping. Genetic investigation indicated that the restorer line JLR2 was controlled by a single dominant gene, Rf3. By integrating bulk-segregant analysis and next-generation sequencing, a 4 Mb region on chromosome 9 was identified, which was most likely the target region harboring the candidate gene responsible for fertility restoration. This region was further narrowed down to 86.44 Kb via fine mapping in F₂ and F₃ populations using SSR, InDel, and dCAPS markers. This region contained 10 putative genes (Glyma.09G171100–Glyma.09G172000). Finally, Glyma.09G171200, which encodes a mitochondria-targeted pentatricopeptide repeat protein, was proposed as the potential candidate for Rf3 using sequence alignment and expression analysis in restorer and CMS lines. Based on single-nucleotide polymorphisms in Glyma.09G171200, a CAPS marker co-segregated with Rf3 named CAPS1712 was developed. Our results will be fundamental in the assisted selection and creation of potent lines for the production and rapid selection of novel restorer lines.     

Identification of QTLs and a Candidate Gene for Reducing Pre-Harvest Sprouting in Aegilops tauschii–Triticum aestivum Chromosome Segment Substitution Lines

Wheat pre-harvest sprouting (PHS) causes serious losses in wheat yield. In this study, precise mapping was carried out in the chromosome segment substitution lines (CSSL) F₂ population generated by a direct cross of Zhoumai 18 (PHS-sensitive) and Aegilops tauschii accession T093 (highly PHS-resistant). Three Ae. tauschii-derived quantitative trait loci (QTLs), QDor.3D.1, QDor.3D.2, and QDor.3D.3, were detected on chromosome 3DL using four simple sequence repeats (SSR) markers and 10 developed Kompetitive allele-specific PCR (KASP) markers. Alongside these QTL results, the RNA-Seq and qRT-PCR analysis revealed expression levels of TraesCS3D01G466100 in the QDor.3D.2 region that were significantly higher in CSSLs 495 than in Zhoumai 18 during the seed imbibition treatment. The cDNA sequencing results of TraesCS3D01G466100 showed two single nucleotide polymorphisms (SNPs), resulting in two changed amino acid substitutions between Zhoumai 18 and line 495, and the 148 nt amino acid substitution of TraesCS3D01G466100, derived from Ae. tauschii T093, which may play an important role in the functioning of ubiquitin ligase enzymes 3 (E3) according to the homology protein analysis, which could lead to differential PHS-resistance phenotypes. Taken together, our results may foster a better understanding of the mechanism of PHS resistance and are potentially valuable for marker-assisted selection in practical wheat breeding efforts.     

Fracture energy of ultra-high-performance fiber-reinforced concrete at high strain rates

The fracture energy of ultra-high-performance fiber-reinforced concrete (UHPFRC) at high strain rates (5–92 s^− 1) was investigated, and specimens with 1–1.5% fibers exhibited very high fracture energy (28–71 kJ/m²). Evaluation of the rate effects on the UHPFRC fracture resistance, including fracture strength (f_t), specific work-of-fracture (W_S), and softening fracture energy (W_F), indicated that f_t and W_S were highly sensitive to strain rate, whereas W_F was not. The effects of fiber type, volume content, specimen shape and fiber blending on the fracture resistance at high and static strain rates differed significantly: 1) smooth fibers exhibited higher f_t and W_S at high rates than twisted fibers; 2) higher fiber volume content did not clearly generate higher W_S and W_F at high rates; 3) notched specimens generally exhibited higher fracture resistance than un-notched samples at both static and high rates; and 4) UHPFRC blending two fibers produced higher W_S and W_F than UHPFRC with mono fiber at high rates.     

Isolation and Characterization of Novel Genomic and EST-SSR Markers in Coreoperca whiteheadi Boulenger and Cross-Species Amplification

We described and characterized 11 expressed sequence tag (EST)-derived simple sequence repeats (SSR) and seven genomic (G)-derived SSRs in Coreoperca whiteheadi Boulenger. The EST-SSRs comprised 62.2% di-nucleotide repeats, 32.2% tri-nucleotide repeats and 5.5% tetra-nucleotide repeats, whereas the majority of the G-SSRs were tri-nuleotide repeats (81.4%). The number of alleles for the 18 loci ranged from 3 to 6, with a mean of 3.8 alleles per locus. The observed (Ho) and expected heterozygosities (He) values ranged from 0.375 to 1.000, and 0.477 to 0.757, respectively. The polymorphic information content (PIC) values ranged from 0.466 to 0.706. The mean values number of alleles, Ho, He, and PIC of EST-SSRs were higher than those of the G-SSRs. Four microsatellite loci deviated significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction and no significant deviations in linkage disequilibrium (LD) were observed. These loci are the first to be characterized in C. whiteheadi and should be useful in the investigation of a genetic evaluation for conservation. Compared with 11 loci in C. whiteheadi, 37 potential polymorphic EST-SSRs were found in Siniperca chuatsi (Basilewsky), which will provide a valuable tool for mapping studies and molecular breeding programs in S. chuatsi.     

Fine Mapping and Gene Analysis of restorer-of-fertility Gene CaRfHZ in Pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) is a common biological phenomenon used in hybrid production of peppers (Capsicum annuum L.). Although several restorer-of-fertility (Rf) genes of pepper CMS lines have been mapped, there is no report that the Rf gene with clear gene function has been isolated. Here, pepper CMS line HZ1A and its restorer line HZ1C were used to construct (HZ1A × HZ1C) F₂ populations and map the Rf gene. A single dominant gene CaRfHZ conferred male fertility according to inheritance analysis. Using sterile plants from (HZ1A × HZ1C) F₂ populations and bulked segregant analysis (BSA), the CaRf_HZ gene was mapped between P06gInDel-66 and P06gInDel-89 on chromosome 6. This region spans 533.81 kb, where four genes are annotated according to Zunla-1 V2.0 gene models. Based on the analysis of genomic DNA sequences, gene expressions, and protein structures, Capana06g002968 was proposed as the strongest candidate for the CaRf_HZ gene. Our results may help with hybrid pepper breeding and to elucidate the mechanism of male fertility restoration in peppers.     

Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F_2–3 generations and RNA-seq of F₄ generation detected a common fragment, i.e., 18,915,234–19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.     

Solvothermal Synthesis of Ternary Sulfides of Sb2 − x Bi x S3 (x = 0.4, 1) with 3D Flower-Like Architectures

Flower-like nanostructures of Sb_{2 − x}Bi_xS₃(x = 0.4, 1.0) were successfully prepared using both antimony diethyldithiocarbamate [Sb(DDTC)₃] and bismuth diethyldithiocarbamate [Bi(DDTC)₃] as precursors under solvothermal conditions at 180 °C. The prepared Sb_{2 − x}Bi_xS₃ with flower-like 3D architectures were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS), high-resolution transmission electron microscopy (HRTEM), and selected area electron diffraction (SAED). The flower-like architectures, with an average diameter of ~4 μm, were composed of single-crystalline nanorods with orthorhombic structures. The optical absorption properties of the Sb_{2 − x}Bi_xS₃ nanostructures were investigated by UV–Visible spectroscopy, and the results indicate that the Sb_{2 − x}Bi_xS₃ compounds are semiconducting with direct band gaps of 1.32 and 1.30 eV for x = 0.4 and 1.0, respectively. On the basis of the experimental results, a possible growth mechanism for the flower-like Sb_{2 − x}Bi_xS₃ nanostructures is suggested.     

Isolation and Characterisation of 11 Polymorphic Microsatellite Markers in Papaver rhoeas L. (Corn Poppy), a Major Annual Plant Species from Cultivated Areas

Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (F_ST = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.     

Quantitative Trait Loci Mapping for Bacterial Blight Resistance in Rice Using Bulked Segregant Analysis

Oryza meyeriana is highly resistant to rice bacterial blight (BB) and this resistance trait has been transferred to cultivated rice (O. sativa) using asymmetric somatic hybridization. However, no resistance genes have yet been cloned. In the present study, a progeny of the somatic hybridization with high BB resistance was crossed with a rice cultivar with high BB susceptibility to develop an F₂ population. Using bulked segregant analysis (BSA), 17 polymorphic markers that were linked to rice BB resistance were obtained through scanning a total of 186 simple sequence repeats (SSR) and sequence-tagged site (STS) markers, evenly distributed on 12 chromosomes. A genetic linkage map was then constructed based on the 17 linkage markers and the F₂ segregating population, which was followed by mapping for quantitative trait loci (QTLs) for BB resistance. Three QTLs were identified on chromosomes 1, 3 and 5, respectively, and the alleles of the resistant parent at any of the QTLs increased BB resistance. All of the three QTLs had a strong effect on resistance, explaining about 21.5%, 12.3% and 39.2% of the resistance variance, respectively. These QTLs were different from the loci of the BB resistance genes that have been identified in previous studies. The QTLs mapped in this work will facilitate the isolation of novel BB resistance genes and their utilization in rice resistance breeding.     

Studies with inorganic precipitate membranes—XII: Consideration of membrane field strength and energetics of permeation

Electrolytic transfort processes occuring across parchment supported membranes have been described by Nernst Planck flux equation taking into account the membrane resistance R_m, membrane potential E_metc.E_m values for various electrolytes display very interesting phenomena. In the case of 1:1 electrolyte the E_m values are all positive, while in the case of (2:1) and (3:1) electrolytes surface charge reversal takes place. The diffusion rate sequence and selectivity of the membrane for different uni- bi- and trivalent cations was found to be primarily dependent on the difference in the hydration energies of counter ions in the external solution. On the basis of Eisenman-Sherry theory the diffusion rate sequence of alkali metal cations point towards the weak field strength of the fixed charge groups. Various thermodynamic parameters, ΔH^≠, ΔF^≠ and ΔS^≠ were evaluated by applying the theory of absolute reactions rates to the diffusion process through parchment supported membranes. The values of ΔS^≠ were found to be negative, indicating that diffusion takes place with partial immobilization in the membrane phase. The relative partial immobility was found to increase with increase in the valence of the ions constituting the electrolyte. A formal relation between ΔH_hydration, ΔF_hydration and ΔS_hydration of cations with the corresponding values of ΔH^≠, ΔF^≠ and ΔS^≠ for diffusion, was also found to exist for these membranes.     

Does Indole Alkaloid Gramine Confer Resistance in Barley to Aphid Rhopalosiphum Padi?

Accessions of cultivated barley (Hordeum vulgare ssp. vulgare) and its wild progenitor Hordeum vulgare ssp. spontaneum (H. ssp. spont.) were screened for gramine content at the seedling stage. H. ssp. spont. generally had higher gramine concentration compared with cultivated spring barley. Thus gramine concentrations might be raised in modern barley through crossings with selected H. ssp. spont. accessions and repeated back-crossings (BC) of selected offspring to cultivated barley. In the present study, the barley cultivar Lina was used as the recurrent parent. Lina was exceptional among the two-rowed barleys in that it contained moderate levels of gramine, whereas most of the others were very low in gramine. Chromosome-doubled haploid lines (DHs) from the first generation (F₁) had a skewed distribution towards higher gramine concentrations and so had the first back-cross generation (BC₁F₁-DHs). A hairy plant surface, another character proposed to confer resistance to aphids, was also found among some of the plants in the breeding material. BC₁F₁-DHs with a high proportion of the Lina genome, as determined in an analysis of PCR-based molecular markers, in addition to high gramine concentrations and hairy plant bases in two cases were tested for resistance to the barley pest Rhopalosiphum padi. However, based on aphid performance and preference tests, there were no indications that either high gramine concentrations or hairiness conferred resistance to R. padi when compared with Lina and a variety very low in gramine (Golf). The pattern was the same when the F₁ generation was evaluated in aphid performance tests along with Lina, Golf, and the six H. ssp. spont. parents. Aphid weight was consistently low on only one of the six H. ssp. spont. parents. Since previous reports of a positive relationship between gramine concentrations and resistance to R. padi were based on studies in Chile and Japan, a Chilean R. padi population was compared with three Swedish populations, but the responses of all four populations were similar.     

A DFT Study of Pyrrole-Isoxazole Derivatives as Chemosensors for Fluoride Anion

The interactions between chemosensors, 3-amino-5-(4,5,6,7-tetrahydro-1H-indol-2-yl)isoxazole-4-carboxamide (AIC) derivatives, and different anions (F⁻ Cl⁻, Br⁻, AcO⁻, and H₂PO₄⁻) have been theoretically investigated using DFT approaches. It turned out that the unique selectivity of AIC derivatives for F⁻ is ascribed to their ability of deprotonating the host sensors. Frontier molecular orbital (FMO) analyses have shown that the vertical electronic transitions of absorption and emission for the sensing signals are characterized as intramolecular charge transfer (ICT). The study of substituent effects suggests that all the substituted derivatives are expected to be promising candidates for fluoride chemosensors both in UV-vis and fluorescence spectra except for derivative with benzo[d]thieno[3,2-b]thiophene fragment that can serve as ratiometric fluorescent fluoride chemosensor only.     

Genetic Mapping of ms1s,a Recessive Gene for Male Sterility in Common Wheat

The utilization of heterosis is an important way to improve wheat yield, and the production of wheat hybrid seeds mainly relies on male-sterile lines. Male sterility in line 15 Fan 03 derived from a cross of 72,180 and Xiaoyan 6 is controlled by a single recessive gene. The gene was mapped to the distal region of chromosome 4BS in a genetic interval of 1.4 cM and physical distance of 6.57 Mb between SSR markers Ms4BS42 and Ms4BS199 using an F₂ population with 1205 individuals. Sterile individuals had a deletion of 4.57 Mb in the region presumed to carry the Ms1 locus. The allele for sterility was therefore named ms1s. Three CAPS markers were developed and verified from the region upstream of the deleted fragment and can be used for ms1s marker-assisted selection in wheat hybrid breeding. This work will enrich the utilization of male sterility genetic resources.     

Disease Resistance and Molecular Variations in Irradiation Induced Mutants of Two Pea Cultivars

Induced mutation is useful for improving the disease resistance of various crops. Fusarium wilt and powdery mildew are two important diseases which severely influence pea production worldwide. In this study, we first evaluated Fusarium wilt and powdery mildew resistance of mutants derived from two elite vegetable pea cultivars, Shijiadacaiwan 1 (SJ1) and Chengwan 8 (CW8), respectively. Nine SJ1 and five CW8 M₃ mutants showed resistant variations in Fusarium wilt, and the same five CW8 mutants in powdery mildew. These resistant variations were confirmed in M₄ and M₅ mutants as well. Then, we investigated the genetic variations and relationships of mutant lines using simple sequence repeat (SSR) markers. Among the nine effective SSR markers, the genetic diversity index and polymorphism information content (PIC) values were averaged at 0.55 and 0.46, which revealed considerable genetic variations in the mutants. The phylogenetic tree and population structure analyses divided the M₃ mutants into two major groups at 0.62 genetic similarity (K = 2), which clearly separated the mutants of the two cultivars and indicated that a great genetic difference existed between the two mutant populations. Further, the two genetic groups were divided into five subgroups at 0.86 genetic similarity (K = 5) and each subgroup associated with resistant phenotypes of the mutants. Finally, the homologous PsMLO1 cDNA of five CW8 mutants that gained resistance to powdery mildew was amplified and cloned. A 129 bp fragment deletion was found in the PsMLO1 gene, which was in accord with er1-2. The findings provide important information on disease resistant and molecular variations of pea mutants, which is useful for pea production, new cultivar breeding, and the identification of resistance genes.