An abiotic stress response gene TaSnRK2.7-B in wheat accessions: genetic diversity analysis and gene mapping based on SNPs

TaSnRK2.7, a SnRK2 (sucrose non-fermenting1-related protein kinase 2) member of wheat, confers enhanced multi-stress tolerance in carbohydrate metabolism. Here, the nucleotide diversity of TaSnRK2.7 was investigated in 60 wheat accessions with different stress-tolerant features. One copy, named TaSnRK2.7-B, was detected in 32 accessions, which mainly exhibited stress tolerance. The TaSnRK2.7-B evolutionary dynamics were examined in these cultivars, which have been subjected to population bottlenecks and intensive selection in breeding. Nucleotide diversity (π) in the entire TaSnRK2.7-B region was 0.00148. Sliding-window analysis demonstrated that 3932-4535bp was a variation-enriched region, covering the eighth intron, the ninth exon and the 3'-flanking region. As predicted, neutrality tests revealed that the TaSnRK2.7-B protein was relatively conservative, and population bottlenecks or purifying selection may have acted on TaSnRK2.7-B. Furthermore, linkage disequilibrium between SNP loci extends across the entire TaSnRK2.7-B region. A SNP marker of TaSnRK2.7-B was developed and mapped on chromosome 2AL flanked by WMC179.4 and WMC401, which were co-located in the same or adjacent chromosome intervals with QTLs for phosphorus utilization efficiency and accumulation efficiency of stem water-soluble carbohydrates.     

Molecular differentiation of null alleles at <Emphasis Type="Italic">ZCCT</Emphasis>-<Emphasis Type="Italic">1</Emphasis> genes on the A,B, and D genomes of hexaploid wheat

A dominant allele of the vernalization gene Vrn-2 is the wild type conferring winter growth habit, whereas a recessive vrn-2 allele confers spring growth habit. The recessive vrn-2 allele is mutated due to the deletion of the complete gene (a null form) or alternation of a key amino acid in the VRN-2 protein (a nonfunctional form) in diploid wheat or tetraploid wheat. VRN-2 is also denoted ZCCT due to the presence of a zinc finger and a CCT domain in its protein. There are two paralogous ZCCT genes at the VRN-2 locus in diploid Triticum monococcum and three paralogous ZCCT genes on each of the A and B genomes in tetraploid wheat, but little is known about the allelic variation in VRN-2 in hexaploid wheat. In the study reported here, we performed a one-shot PCR to simultaneously amplify the promoter regions of the three ZCCT-1 genes from hexaploid wheat, including the 302-bp fragment from ZCCT-A1, the 294-bp fragment from ZCCT-B1, and the 320-bp fragment from ZCCT-D1. Each amplicon could be differentiated by electrophoresis in an acrylamide/bisacrylamide gel. This PCR marker for different lengths of the three ZCCT-1 genes was used to search for null alleles in hexaploid wheat. A null allele was found in each of ZCCT-A1, ZCCT-B1, and ZCCT-D1 among 74 cultivars and genetic stocks of U.S. hexaploid wheat. Among 54 Chinese wheat cultivars, breeding lines, and landraces, we identified three accessions carrying a single null allele at ZCCT-A1, three accessions carrying a null allele at ZCCT-B1, and one accession carrying a double null allele at both ZCCT-A1 and ZCCT-D1. The potential application of these natural ZCCT-1 mutant materials in wheat breeding programs and studies on the genetics of wheat is discussed.     

Genome-wide association mapping for seedling and field resistance to <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> in elite durum wheat

Key message

Genome-wide association analysis in tetraploid wheat revealed novel and diverse loci for seedling and field resistance to stripe rust in elite spring durum wheat accessions from worldwide.

Abstract

Improving resistance to stripe rust, caused by Puccinia striiformis f. sp. tritici, is a major objective for wheat breeding. To identify effective stripe rust resistance loci, a genome-wide association study (GWAS) was conducted using 232 elite durum wheat (Triticum turgidum ssp. durum) lines from worldwide breeding programs. Genotyping with the 90 K iSelect wheat single nucleotide polymorphism (SNP) array resulted in 11,635 markers distributed across the genome. Response to stripe rust infection at the seedling stage revealed resistant and susceptible accessions present in rather balanced frequencies for the six tested races, with a higher frequency of susceptible responses to United States races as compared to Italian races (61.1 vs. 43.1% of susceptible accessions). Resistance at the seedling stage only partially explained adult plant resistance, which was found to be more frequent with 67.7% of accessions resistant across six nurseries in the United States. GWAS identified 82 loci associated with seedling stripe rust resistance, five of which were significant at the false discovery rate adjusted P value <0.1 and 11 loci were detected for the field response at the adult plant stages in at least two environments. Notably, Yrdurum-1BS.1 showed the largest effect for both seedling and field resistance, and is therefore considered as a major locus for resistance in tetraploid wheat. Our GWAS study is the first of its kind for stripe rust resistance in tetraploid wheat and provides an overview of resistance in elite germplasm and reports new loci that can be used in breeding resistant cultivars.

     

Genetic diversity of <Emphasis Type="Italic">avenin-like b</Emphasis> genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss

Avenin-like storage proteins influence the rheological properties and processing quality in common wheat, and the discovery of new alleles will benefit wheat quality improvement. In this study, 13 avenin-like b alleles (TaALPb7D-A–M) were discovered in 108 Aegilops tauschii Coss. accessions. Ten alleles were reported for the first time, while the remaining three alleles were the same as alleles in other species. A total of 15 nucleotide changes were detected in the 13 alleles, resulting in only 11 amino acid changes because of synonymous mutations. Alleles TaALPb7D-E, TaALPb7D-G, and TaALPb7D-J encoded the same protein. These polymorphic sites existed in the N-terminus, Repetitive region (Left), Repetitive region (Right) and C-terminus domains, with no polymorphisms in the signal peptide sequence nor in those encoding the 18 conserved cysteine residues. Phylogenetic analysis divided the TaALPb7Ds into four clades. The Ae. tauschii alleles were distributed in all four clades, while the alleles derived from common wheat, TaALPb7D-G and TaALPb7D-C, belonged to clade III and IV, respectively. Alleles TaALPb7D-G and TaALPb7D-C were the most widely distributed, being present in nine and six countries, respectively. Iran and Turkey exhibited the highest genetic diversity with respect to TaALPb7D alleles, accessions from these countries carrying seven and six alleles, respectively, which implied that these countries were the centers of origin of the avenin-like b gene. The new alleles discovered and the phylogenetic analysis of avenin-like b genes will provide breeding materials and a theoretical basis for wheat quality improvement.     

Biological synthesis of quercetin 3-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine conjugate using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing <Emphasis Type="Italic">UGT78D2</Emphasis>

Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide sugar metabolic pathway in the host strain.     

Physical mapping of <Emphasis Type="Italic">puroindoline b</Emphasis>-<Emphasis Type="Italic">2</Emphasis> genes and molecular characterization of a novel variant in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L.)

The puroindoline genes (Pina and Pinb) are the functional components of the common or bread wheat (Triticum aestivum L.) grain hardness locus that are responsible for kernel texture. In this study, four puroindoline b-2 variants were physically mapped using nulli-tetrosomic lines of bread wheat cultivar Chinese Spring and substitution lines of durum wheat (Triticum turgidum L.) cultivar Langdon. Results indicated that Pinb-2v1 was on 7D of Chinese Spring, Pinb-2v2 on 7B of Chinese Spring, Pinb-2v3 on 7B of Chinese Spring and Langdon, and Pinb-2v4 on 7A of Chinese Spring and Langdon. A new puroindoline b-2 variant, designated Pinb-2v5, was identified at the puroindoline b-2 locus of durum wheat cultivar Langdon, with a difference of only five single nucelotide polymorphisms compared with Pinb-2v4. Sequencing results indicated that, in comparison with the Pinb-2v3 sequence (AM99733 and GQ496618 with one base-pair modification of G to T at 6th position, designated Pinb-2v3a) in bread wheat cultivar Witchta, the coding region of Pinb-2v3 in 12 durum wheat cultivars had a single nucleotide change from T to C at the 311th position, resulting in a corresponding amino acid change from valine to alanine at the 104th position. This new allele was designated Pinb-2v3b. The study of puroindoline b-2 gene polymorphism in CIMMYT and Italian durum wheat germplasm and discovery of a novel puroindoline b-2 variant could provide useful information for further understanding the molecular and genetic basis of kernel hardness and illustrating gene duplication events in wheat.     

Fine genetic mapping of greenbug aphid-resistance gene <Emphasis Type="Italic">Gb3</Emphasis> in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

The greenbug, Schizaphis graminum (Rondani), is an important aphid pest of small grain crops especially wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) in many parts of the world. The greenbug-resistance gene Gb3 originated from Aegilops tauschii Coss. (2n = 2x = 14, genome D^tD^t) has shown consistent and durable resistance against prevailing greenbug biotypes in wheat fields. We previously mapped Gb3 in a recombination-rich, telomeric bin of wheat chromosome arm 7DL. In this study, high-resolution genetic mapping was carried out using an F_2:3 segregating population derived from two Ae. tauschii accessions, the resistant PI 268210 (original donor of Gb3 in the hexaploid wheat germplasm line ‘Largo’) and susceptible AL8/78. Molecular markers were developed by exploring bin-mapped wheat RFLPs, SSRs, ESTs and the Ae. tauschii physical map (BAC contigs). Wheat EST and Ae. tauschii BAC end sequences located in the deletion bin 7DL3-0.82–1.00 were used to design STS (sequence tagged site) or CAPS (Cleaved Amplified Polymorphic Sequence) markers. Forty-five PCR-based markers were developed and mapped to the chromosomal region spanning the Gb3 locus. The greenbug-resistance gene Gb3 now was delimited in an interval of 1.1 cM by two molecular markers (HI067J6-R and HI009B3-R). This localized high-resolution genetic map with markers closely linked to Gb3 lays a solid foundation for map based cloning of Gb3 and marker-assisted selection of this gene in wheat breeding.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Exploiting co-linearity among grass species to map the <Emphasis Type="Italic">Aegilops ventricosa-</Emphasis>derived <Emphasis Type="Italic">Pch1</Emphasis> eyespot resistance in wheat and establish its relationship to <Emphasis Type="Italic">Pch2</Emphasis>

Introgressions into wheat from related species have been widely used as a source of agronomically beneficial traits. One such example is the introduction of the potent eyespot resistance gene Pch1 from the wild relative Aegilops ventricosa onto chromosome 7DL of wheat. In common with genes carried on many other such introgressions, the use of Pch1 in commercial wheat varieties has been hindered by linkage drag with yield-limiting traits. Attempts to break this linkage have been frustrated by a lack of co-dominant PCR markers suitable for identifying heterozygotes in F₂ populations. We developed conserved orthologous sequence (COS) markers, utilising the Brachypodium distachyon (Brachypodium) genome sequence, to provide co-dominant markers in the Pch1 region. These were supplemented with previously developed sequence-tagged site (STS) markers and simple sequence repeat (SSR) markers. Markers were applied to a panel of varieties and to a BC₆ F₂ population, segregating between wheat and Ae. ventricosa over the distal portion of 7DL, to identify recombinants in the region of Pch1. By exploiting co-linearity between wheat chromosome 7D, Brachypodium chromosome 1, rice chromosome 6 and sorghum chromosome 10, Pch1 was located to an interval between the flanking markers Xwg7S and Xcos7-9. Furthermore candidate gene regions were identified in Brachypodium (364 Kb), rice (178 Kb) and sorghum (315 Kb) as a prelude to the map-based cloning of the gene. In addition, using homoeologue transferable markers, we obtained evidence that the eyespot resistances Pch1 and Pch2 on chromosomes 7D and 7A, respectively, are potentially homoeoloci. It is anticipated that the COS marker methodology could be used for the identification of recombinants in other introgressions into wheat from wild relatives. This would assist the mapping of genes of interest and the breaking of deleterious linkages to enable greater use of these introgressions in commercial varieties.     

Development of PCR markers for <Emphasis Type="Italic">Tamyb10</Emphasis> related to <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">1,</Emphasis> red grain color gene in wheat

The grain color of wheat affects not only the brightness of flour, but also tolerance to preharvest sprouting. Grain color is controlled by dominant R-1 genes located on the long arm of hexaploid wheat chromosomes 3A, 3B, and 3D (R-A1, R-B1, and R-D1, respectively). The red pigment of the grain coat is composed of catechin and proanthocyanidin (PA), which are synthesized via the flavonoid biosynthetic pathway. We isolated the Tamyb10-A1, Tamyb10-B1, and Tamyb10-D1 genes, located on chromosomes 3A, 3B, and 3D, respectively. These genes encode R2R3-type MYB domain proteins, similar to TT2 of Arabidopsis, which controls PA synthesis in testa. In recessive R-A1 lines, two types of Tamyb10-A1 genes: (1) deletion of the first half of the R2-repeat of the MYB region and (2) insertion of a 2.2-kb transposon belonging to the hAT family. The Tamyb10-B1 genes of recessive R-B1 lines had 19-bp deletion, which caused a frame shift in the middle part of the open reading frame. With a transient assay using wheat coleoptiles, we revealed that the Tamyb10 gene in the dominant R-1 allele activated the flavonoid biosynthetic genes. We developed PCR-based markers to detect the dominant/recessive alleles of R-A1, R-B1, and R-D1. These markers proved to be correlated to known R-1 genotypes of 33 varieties except for a mutant with a single nucleotide substitution. Furthermore, double-haploid (DH) lines derived from the cross between red- and white-grained lines were found to necessarily carry functional Tamyb10 gene(s). Thus, PCR-based markers for Tamyb10 genes are very useful to detect R-1 alleles.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

Genetic mapping of <Emphasis Type="Italic">Stb19</Emphasis>, a new resistance gene to <Emphasis Type="Italic">Zymoseptoria tritici</Emphasis> in wheat

Key message

A new and dominant R gene Stb19 is identified from a soft wheat cultivar ‘Lorikeet’ and was mapped on the distal region of chromosome 1DS. Two tightly linked KASP markers were also discovered and validated for molecular-assisted breeding programs.

Abstract

A new R gene, designated as Stb19, provides resistance to Zymoseptoria tritici in wheat. This new dominant gene resides on the short arm of chromosome 1D, exhibiting complete resistance to three Z. tritici isolates, WAI332, WAI251, and WAI161, at the seedling stage. A genetic linkage map, based on an F_2:3 population of ‘Lorikeet’ and ‘Summit,’ found the Stb19 gene at a 9.3 cM region on 1DS, closely linked with two Kompetitive Allele-Specific PCR markers, snp_4909967 and snp_1218021. Further, the two markers were tested and validated in another F_2:3 population and 266 different wheat accessions, which gave over 95% accuracy of resistance/susceptibility prediction. Combined with the physical location of the identified SNPs and the previous evidence of gene order on chromosome 1DS (centromere–Sr45–Sr33–Lr21–telomere), Stb19 is proposed to be located between Sr33 and Lr21. Thus, the newly discovered Stb19 along with the KASP markers represents an increase in genetic resources available for wheat breeding resistance to Z. tritici.

     

Use of the <Emphasis Type="Italic">pyrG</Emphasis> gene as a food-grade selection marker in <Emphasis Type="Italic">Monascus</Emphasis>

Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5′-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.     

Genetic variation and population structure of a threatened timber tree <Emphasis Type="Italic">Dalbergia cochinchinensis</Emphasis> in Cambodia

Dalbergia cochinchinensis Pierre ex Laness. (Fabaceae) is a commercially important tree in Southeast Asia. Although this species is under legal protections, illegal logging and disorderly developments have reduced its populations, and the conservation of this species is currently of much concern. In this study, we determined nucleotide sequences at six chloroplasts and ten nuclear loci in four populations of D. cochinchinensis in Cambodia, followed by population genetic analyses. The average silent nucleotide diversity over the nuclear loci, excluding one with an exceptionally high value, was 0.0057 in the entire population, and the mean F _ST across the nuclear loci between each population pair was between 0.135 and 0.467. Thus, the nucleotide diversity in the studied populations was not low compared with that in other tree species, and the level of population differentiation was high. Neutrality test statistics indicated a recent reduction of population size and a subdivision of the population within this species. The divergence times and migration rates were estimated with a likelihood-based method assuming the isolation with migration model. Based on the results, the three populations split 68,000–138,000 years ago, possibly corresponding to the start of the last glacial period, and the level of gene flow among the populations was very low thereafter. Moreover, after the split, population sizes were reduced considerably. Notably, the nucleotide diversity in an insertion sequence in a noncoding region of nuclear C4H was much higher than the mean nucleotide diversity in silent sites across other nuclear genes, indicating that the region was affected by selection.     

Allele-specific CAPS marker in a <Emphasis Type="Italic">Ve1</Emphasis> homolog of <Emphasis Type="Italic">Capsicum annuum</Emphasis> for improved selection of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.     

Functional markers for <Emphasis Type="Italic">xa5</Emphasis>-mediated resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>, L.)

The recent cloning of several agronomically important genes has facilitated the development of functional markers. These markers reside within the target genes themselves and can be used with great reliability and efficiency to identify favorable alleles in a breeding program. Bacterial blight (BB) is a severe rice disease throughout the world that is controlled primarily through use of resistant cultivars. xa5 is a race-specific, recessive gene mediating resistance to BB. It is widely used in rice breeding programs throughout the tropics. Due to its recessive nature, phenotypic selection for xa5-mediated resistance is both slow and costly. Previously, marker assisted selection (MAS) for this resistance gene was not efficient because it involved markers that were only indirectly linked to xa5 and ran the risk of being separated from the trait by recombination. Recently, the cloning of the gene underlying this trait made it possible to develop functional markers. Here we present a set of CAPS markers for easy, quick and direct identification of cultivars or progeny carrying xa5-mediated resistance and provide evidence that these markers are 100% predictive of the presence of the xa5 allele. These markers are expected to enhance the reliability and cost-effectiveness of MAS for xa5-mediated resistance.     

Genetic variation in populations of <Emphasis Type="Italic">Allothrombium pulvinum</Emphasis> (Acari: Trombidiidae) from Northern Iran revealed by mitochondrial <Emphasis Type="Italic">cox</Emphasis>I and nuclear rDNA <Emphasis Type="Italic">ITS</Emphasis>2 sequences

Allothrombium pulvinum Ewing is a common natural enemy of aphids and some other arthropods. So far, there are no studies that have addressed genetic variation of this predatory mite. We investigated genetic variation of A. pulvinum across its whole known range in Iran. A 410 bp portion of the mitochondrial cytochrome c oxidase subunit I gene (coxI) and 797–802 bp portion of the internal transcribed spacer 2 of rDNA (ITS2) were sequenced for 55 individuals from 11 populations, resulting in 12 and 26 haplotypes, respectively. In the coxI region, haplotype and nucleotide diversities varied among populations from 0.00 to 0.90 and from 0.0000 to 0.0110, respectively. In the ITS2 region they varied from 0.20 to 0.91 and from 0.0006 to 0.0023, respectively. For both gene regions the highest haplotype and nucleotide diversities were detected in population Mahmoud Abad from northern Iran. Statistically significant population differentiation (F _ST) was detected in most pair-wise population comparisons. The results of population differentiation for both gene regions were generally congruent indicating that A. pulvinum from Iran consists of genetically different populations. This suggests that A. pulvinum comprises at least two geographically distinct populations or even more than one species. This study is an initial step towards understanding genetic variation of A. pulvinum, a taxon for which little molecular information is available. More intensive sampling and analysis of additional DNA regions are necessary for more detailed classification of this taxon.     

Cloning and sequence diversity analysis of <Emphasis Type="Italic">GmHs1</Emphasis><Superscript><Emphasis Type="Italic">pro-1</Emphasis></Superscript> in Chinese domesticated and wild soybeans

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is the most important pathogen in soybean production worldwide and causes substantial yield losses. An apparent narrow genetic base of SCN resistance was observed in current elite soybean cultivars, and searching for novel SCN resistance genes as well as novel resistance sources rather than focusing on the two important genes rhg1 and Rhg4 has become another major objective in soybean research. In the present paper we report a 1,477 bp Hs1 ^pro-1 homolog, named GmHs1 ^pro-1 . This gene was cloned from soybean variety Wenfeng 7 based on information for Hs1 ^pro-1 , a beet cyst nematode resistance gene in sugar beet. It has two domains, Hs1pro-1_N and Hs1pro-1_C, both of which are believed to confer resistance to nematodes. Of the 1,477 bp sequence in GmHs1 ^pro-1 , an open reading frame of 1,314 bp, encoding a protein with 437 amino acids, was flanked by a 5′-untranslated region of 27 bp and a 3′-untranslated region of 135 bp. Fourteen single-nucleotide polymorphisms (SNPs) were observed in 44 soybean accessions including 23 wild soybeans, 8 landraces, and 13 soybean varieties (or lines), among which 5 in wild soybeans and 3 in landrace accessions were unique. Sequence diversity analysis on the 44 soybean accessions showed π = 0.00168 and θ = 0.00218 for GmHs1 ^pro-1 ; landraces had the highest diversity, followed by wild soybeans, with varieties (or lines) having the lowest. Although we did not detect a significant effect of selection on GmHs1 ^pro-1 in the three populations, sequence diversity, unique SNPs, and phylogenetic analysis indicated a slight domestication bottleneck and an intensive selection bottleneck. High sequence diversity, more unique SNPs, and broader representation across the phylogenetic tree in wild soybeans and landraces indicated that wild collections and landrace accessions are invaluable germplasm for broadening the genetic base of elite soybean varieties resistant to SCN. C. Yuan and G. Zhou contributed to this paper equally.     

Patterns of nucleotide sequence variation in <Emphasis Type="Italic">ICAM1</Emphasis> and <Emphasis Type="Italic">TNF</Emphasis> genes in twelve ethnic groups of India: roles of demographic history and natural selection

We have studied DNA sequence variation in and around the genes ICAM1 and TNF, which play functional and correlated roles in inflammatory processes and immune cell responses, in 12 diverse ethnic groups of India, with a view to investigating the relative roles of demographic history and natural selection in shaping the observed patterns of variation. The total numbers of single nucleotide polymorphisms (SNPs) detected at the ICAM1 and TNF loci were 29 and 12, respectively. Haplotype and allele frequencies differed significantly across populations. The site frequency spectra at these loci were significantly different from those expected under neutrality, and showed an excess of intermediate-frequency variants consistent with balancing selection. However, as expected under balancing selection, there was no significant reduction of F _ST values compared to neutral autosomal loci. Mismatch distributions were consistent with population expansion for both loci. On the other hand, the phylogenetic network among haplotypes for the TNF locus was similar to expectations under population expansion, while that for the ICAM1 was as expected under balancing selection. Nucleotide diversity at the ICAM1 locus was an order of magnitude lower in the promoter region, compared to the introns or exons, but no such difference was noted for the TNF gene. Thus, we conclude that the pattern of nucleotide variation in these genes has been modulated by both demographic history and selection. This is not surprising in view of the known allelic associations of several polymorphisms in these genes with various diseases, both infectious and noninfectious.