Allele mining and haplotype discovery in barley candidate genes for drought tolerance

In the present study, allele mining was conducted on a panel of drought related candidate genes in a set of 96 barley genotypes using EcoTILLING, which is a variant of the targeting induced local lesions in genomes (TILLING) technology. Analyzing approximately 1.5 million basepairs in barley a total number of 94 verified unique haplotypes were identified in 18 amplicons designed for 9 genes. Overall, 185 single nucleotide polymorphisms (SNPs) and 46 insertions/deletions (INDELs) were detected with a mean of 1SNP/92 bp and 1INDEL/372 bp genomic sequence. Based on overlapping haplotype sequences, markers were developed for four candidate genes (HvARH1, HvSRG6, HvDRF1, HVA1), which allows distinguishing between the main haplotypes showing either differences in amino acid sequence or which have larger INDELs in the promoter region. As “proof of concept”, the HvARH1 and HvSRG6 haplotypes were tested for the level of abscisic acid-induced gene expression in subsets of genotypes belonging to different haplotype categories. An integrated database was developed to contain information about the genes, genotypes, and haplotypes analyzed in this study. The database supplies profound information about the natural variation in the tested drought related candidate genes providing a significant asset for further mapping studies dealing with this highly polygenic trait.     

高效液相色谱法测定地黄蔗糖合成酶、蔗糖磷酸合成酶活性

为了建立地黄蔗糖合成酶、蔗糖磷酸合成酶的高效液相色谱测定方法,利用高效液相法测定蔗糖合成酶、蔗糖磷酸合成酶反应前后蔗糖含量的变化,计算出酶活性。采用Waters XBridgeTM Amide 3.5 ?m （4.6×150 mm Column）色谱柱,流动相乙腈-水（70:30）,流速0.5 mL/min,ELSD检测器：漂移管温度40℃,雾化气体30 psi,喷雾模式：冷却;地黄中蔗糖在0.128~3.2 μg的范围内,峰面积与含量呈良好线性关系（r=0.9999）,且HPLC测定两种酶活性的RSD均小于5%。HPLC法稳定、可靠、精密度高、重复性好,可作为地黄蔗糖合成酶、蔗糖磷酸合成酶活性的测定方法。     

Evaluation of a barley (Hordeum vulgare) chromosome 2 drought tolerance quantitative trait locus in genomic backgrounds of two cultivars

A barley drought tolerance Quantitatif Trait Locus (QTL) on chromosome 2 was transferred from tolerant cultivar ‘Tadmor’ to susceptible ‘Baronesse’ and ‘Aydanhanım’. Effects of this QTL on drought tolerance and other traits were studied using near-isogenic lines under controlled environments and field trials for two years. This QTL resulted in 5.0% and 9.1% improvement in leaf relative water content of ‘Baronesse’ and ‘Aydanhanım’ cultivars, respectively, under controlled environments. The QTL accelerated heading and maturity by 2.5 days in ‘Baronesse’ and by 5–6 days in ‘Aydanhanım’. It was associated with shorter stature and more ears. This QTL region increased grain yields by 1.1 and 0.6 t/ha in ‘Baronesse’ and ‘Aydanhanım’, respectively, mainly by increasing the number of tillers. There were previous reports related to yield promoting effects of this region harbouring flowering locus eps2 (barley HvCEN gene). However, sequencing of 1025 bp fragment encompassing HvCEN coding region revealed that our parents and near-isogenic lines had no Single Nucleotide Polymorphism (SNP) variation, ruling out direct involvement of eps2. These findings pointed to the possible effect of another flowering locus in the QTL region.     

Mapping QTL controlling soybean seed sucrose and oligosaccharides in a single family of soybean nested association mapping (SoyNAM) population

The meal value of Soybean for monogastric animals is determined partly by sucrose, raffinose and stachyose. Of these, sucrose is desirable, while raffinose and stachyose are indigestible, causing flatulence and abdominal discomfort. The objective of this study was to identify quantitative trait loci (QTL) controlling seed sucrose, raffinose, and stachyose in a set of 140 SoyNAM (Nested Association Mapping) recombinant inbred lines (RILs), developed from the cross between lines IA3023 and LD02‐4485. A total of 3,038 SNP markers from the Illumina SoyNAM BeadChip SNP were used to map the QTLs for sucrose and the RFOs, raffinose, and stachyose. Significant genotypic differences (p < .001) among RILs were observed for sucrose, raffinose and stachyose contents across years. A 3038 Illumina SoyNAM BeadChip SNPs identified three QTLs for sucrose, one on chromosome 1, explaining 10% variance and two on chromosome 3 each explaining 22%. Raffinose QTL was detected on chromosome 6, explaining 6% variance. The mapped QTLs were novel and spanned regions harbouring candidate genes with roles in plant growth including seed development.     

Molecular mapping of greenbug (Schizaphis graminum) resistance gene Rsg1 in barley

The greenbug, Schizaphis graminum (Rondani) is an extremely damaging aphid pest of barley (Hordeum vulgare L.) particularly in the southern Great Plains of the USA. The simply inherited, dominant resistance gene Rsg1 is in all greenbug‐resistant US barley cultivars. In this study, we conducted molecular mapping of Rsg1 using an F_2:3 population derived from a cross between the greenbug‐resistant Post 90*4/R015 and susceptible CI2260 inbred lines. Segregation of host responses to greenbug biotype E infestation confirmed that a single dominant gene is responsible for greenbug resistance in Post 90*4/R015. Simple sequence repeat (SSR) markers evenly distributed along the seven barley chromosomes were employed for the construction of a framework genetic map. Linkage analysis placed the Rsg1 locus in the long arm of chromosome 3H (3HL) flanked by SSR markers Bmag0877 and GBM1420 that were 35 cM apart. Polymorphic single‐nucleotide polymorphism (SNP) markers in 3HL were identified from an Illumina GoldenGate SNP assay and used for targeted mapping to locate Rsg1 to an 8.4‐cM interval. Comparative analysis identified syntenic genomic regions in Brachypodium distachyon chromosome 2, in which 37 putative genes were annotated including a NB‐LRR‐type resistance gene homologue that may be a potential candidate gene for the Rsg1 locus of barley. Results from this study offer a starting point for fine mapping and cloning of this aphid resistance gene in barley.     

Development of two new molecular markers specific to cytoplasmic male sterility in tuber mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tumida</Emphasis> Tsen et Lee)

A novel and stable cytoplasmic male sterility CMS line of tuber mustard has been bred by subsequent backcrosses for 10 years. Two specific markers atpA and orf220 were cloned and partially characterized in our previous study (Zhang et al. 2003). In this study, two new molecular markers, orf256 and orf305/orf324, have been isolated and identified. The orf256 gene size was found to be 825 bp in CMS line and a 1,357 bp in its maintainer line. Sequence analysis indicated that the orf256 gene was an entire coding sequence and downstream of the cox1 gene. Interestingly, the 906 bp fragment, which contains part of the sequence of orf222, nad5 and orf139 genes, was found to be inserted from the 451st bp of 5′-flank of the 1,357 bp fragment. In the same way, the orf324 gene was isolated from CMS line and orf305 gene from its maintainer line. Both of them are entire coding sequences, upstream from nad3 and rps12 gene, and co-transcribed with the nad3 and rps12 genes. In addition, two molecular markers, orf256 and orf324/orf305, have been successfully converted into the SCAR markers. Subsequently, ORF256, ORF324, ORF305 protein and ORF256-M-431 fragment are predicated to contain signal peptide sequences, and ORF220 was predicated to contain signal anchor sequence. RFLP analysis results revealed that all of the molecular markers exhibited polymorphisms. Northern blot analysis indicated that the expression level of these genes in CMS line is higher than that of the maintainer line. In the mass, all of these genes are expressed lower in the leaf than that of floral organs between the CMS line and its maintainer line. The difference in expression pattern of different mitochondrial specific marker genes suggests that the abundance of mitochondrial proteins is differentially regulated in the organ/tissue development in tuber mustard. Results of this study also provide some novel and useful clues to explore the biological function of these specific marker genes in the tuber mustard.     

Influence of single-nucleotide polymorphisms in the gene encoding granule-bound starch synthase I on amylose content in Vietnamese rice cultivars

Amylose content is one of the most important factors influencing the physical and chemical properties of starch in rice. Analysis of 352 Vietnamese rice cultivars revealed a wide range of variation in apparent amylose content and the expression level of granule-bound starch synthase. On the basis of single-nucleotide polymorphisms (SNP) at the splicing donor site of the first intron and in the coding region of the granule-bound starch synthase I gene, Waxy gene, alleles can be classified into seven groups that reflect differences in apparent amylose content. The very low and low apparent amylose content levels were tightly associated with a G to T in the first intron whereas intermediate and high amylose was associated with a T genotype at SNP in exon 10. The correlation between the combination of T genotype at SNP in the first intron, C in exon 6, or C in exon 10 was predominant among low amylose rice varieties. Our analysis confirmed the existence of Wx^op allele in Vietnamese rice germplasm. The results of this study suggest that the low amylose properties of Vietnamese local rice germplasm are attributable to spontaneous mutations at exons, and not at the splicing donor site.     

Allelic variation, sequence determination and microsatellite screening at the XGWM261 locus in Chinese hexaploid wheat (Triticum aestivum) varieties

Wheat microsatellite XGWM261, due to its closely linked to the dwarfing gene Rht8, has been adopted as the diagnostic molecular marker of Rht8. Screening 408 Chinese and 98 exotic varieties showed 13 allele variants in locus of XGWM261, with 6 alleles only to be found in Chinese varieties and 2 only in exotic varieties, respectively. Sequencing results of the 13 alleles revealed their absolute fragment sizes with 216, 212, 210, 206, 204, 202, 200, 196, 194, 192, 190, 174, and 164 bp, respectively. Allelic distribution analysis showed that the 204, 192, 174, and 164 bp alleles were prevailing in Chinese varieties, and the diagnostic 192 bp allele to Rht8 had a very high percentage in the Yellow and Huai River Valleys Facultative Wheat Zone than in the Northern Winter Wheat Zone in China. The GT → AC substitution at position 35 was found in 216, 200, and 174 bp alleles. Moreover, the AG insertion immediately at the end of CT-repeat region was also found in 216, 200, 174, and 164 bp alleles.     

Single nucleotide polymorphism genotyping of the barley waxy gene by polymerase chain reaction with confronting two-pair primers

A high‐throughput single nucleotide polymorphism (SNP) genotyping procedure was developed to select amylose‐free barley mutants whose waxy genes had a C‐ to T‐base substitution in exon 5, which converted Gln‐89 of the wild‐type gene into a termination codon. An F₂ population carrying an amylose‐free waxy gene was checked for segregation. Polymerase chain reaction with confronting two‐pair primers (PCR‐CTPP) produced allele‐specific PCR products that have different sizes and are inherited in a co‐dominant manner. Two alleles of the barley waxy gene with SNP were correctly identified in parental strains using the PCR‐CTPP procedure. Segregation of the SNP as detected by PCR‐CTPP in an F₂ population fitted the expected 1:2:1 ratio. The PCR‐CTPP procedure can provide a time saving and cost‐effective alternative to derived cleaved amplified polymorphic sequence in marker‐assisted selection.     

花生蔗糖合酶基因AhSuSy的克隆和干旱胁迫表达分析

蔗糖合酶(sucrose synthase, SuSy)是蔗糖代谢途径中的关键酶, 在植物生长、发育和渗透调节过程中起着重要的作用。本研究利用同源克隆、RACE和TAIL-PCR相结合的方法从花生叶片中分离了蔗糖合酶基因, 命名为AhSuSy (GenBank登录号JF346233)。该基因cDNA序列全长2 790 bp, 包含一个2 421 bp的开放阅读框(ORF), 5′端非编码区57bp, 3¢端非编码区为312 bp。根据编码区预测AhSuSy编码806个氨基酸, 与大豆、拟南芥、玉米等植物中相关蛋白的氨基酸序列同源性达75%以上; 成功构建了该基因的原核表达载体pET32a-AhSuSy, 在IPTG诱导下得到92 kD左右的蛋白, 与理论值一致。半定量RT-PCR分析表明AhSuSy在花生根、茎、叶中均有表达。利用10%PEG模拟干旱处理花生幼苗, 分析胁迫后AhSuSy的转录水平, 同时测定蔗糖合酶活性和蔗糖含量, 发现三者均表现为处理后4~12 h逐渐升高, 相关性分析显示花生中蔗糖含量与蔗糖合酶活性的相关系数达0.993(P=0.007<0.01), 处理后12~24 h逐渐降低。推测该基因响应干旱调控, 在花生抗旱胁迫中可能起着一定的作用。     

Haplotype structure around the nud locus in barley and its association with resistance to leaf stripe (Pyrenophora graminea)

The naked/hulled kernel trait is controlled in barley by a single gene called nud, on chromosome 7H. The first aim of this work was use bulked segregant analysis to find, new PCR‐based markers linked to nud for marker‐assisted selection (MAS). A new SCAR marker (sJ14) was developed, which is useful for introgressing the naked trait. This, and three other SCARs, were placed on the ‘Proctor’ × ‘Nudinka’ map to detail a 0.9‐cM fragment tagging nud. In order to evaluate the haplotypes around the nud locus, a phenotypically differentiated collection of naked/hulled genotypes was characterized by means of the above markers. Eight different marker haplotypes were found in the breeding germplasm, and a new allele for the marker sKT7 was found. The same barley collection has been surveyed for resistance/susceptibility to leaf stripe (Pyrenophora graminea), in order to investigate any possible association between this and other traits. The naked/hulled seed trait was not associated with resistance/susceptibility to the fungus.     

Genome-wide association studies in a barley (Hordeum vulgare) diversity set reveal a limited number of loci for resistance to spot blotch (Bipolaris sorokiniana)

Spot blotch caused by Bipolaris sorokiniana is an important disease in barley worldwide, causing considerable yield losses and reduced grain quality. In order to identify QTL conferring resistance to spot blotch, a highly diverse worldwide barley set comprising 449 accessions was phenotyped for seedling resistance with three isolates (No 31, SH 15 and SB 61) and for adult plant resistance at two locations (Russia and Australia) in two years. Genotyping with the 50 k iSelect barley SNP genotyping chip yielded 33,818 informative markers. Genome-wide association studies (GWAS) using a compressed mixed linear model, including population structure and kinship, revealed 38 significant marker-trait associations (MTA) for spot blotch resistance. The MTA corresponded to two major QTL on chromosomes 1H and 7H and a putative new minor QTL on chromosome 7H explaining between 2.79% and 13.67% of the phenotypic variance. A total of 10 and 14 high-confidence genes were identified in the respective major QTL regions, seven of which have a predicted involvement in pathogen recognition or defence.     

Genotypic variation in polyphenol content of barley grain

The polyphenol content in pearl barley, which is highly correlated to a browning reaction after heat treatment, was investigated using 1,347 cultivated barley varieties (H. vulgare) and two wild accessions (H. vulgare subsp. spontaneum) collected from different areas of the world. The polyphenol content in the cultivated barley shows a wide variation ranging from 0.19 to 0.75 mg/g with a nearly normal frequency distribution. The polyphenol content in the hulless varieties from Japan and Korea was low. On the other hand, the polyphenol content in wild barley was about two times higher than the average value recorded in cultivated barley. Based on HPLC analysis, five lowest-polyphenol content local varieties do not represent proanthocyanidin-free mutants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Tissue-specificity of maize sucrose synthase gene promoters in maize tissues after particle bombardment

The reporter gene encoding β-glucuronidase (GUS) driven by either of the two maize sucrose synthase gene (Sh1 and Sus 1) promoters was introduced and expressed in various maize tissues via particle bombardment. Transient gene expression was examined by histochemical assays. It was found that the two SS promoters directed differential GUS expression. In the developing kernel, the Sh1 promoter was active only in the upper and central parts of the endosperm. In contrast, strong GUS activity controlled by the Sus1 promoter was detected in various types of cells, including the aleurone cells, the subaleurone endosperm cells, the scutellar cells of the embryo and the pericarp cells. Both promoters showed similar expression patterns in vegetative tissues. This revised version was published online in August 2006 with corrections to the Cover Date.     

A mutation in Waxy gene affects amylose content,starch granules and kernel characteristics of barley (Hordeum vulgare)

Waxy barley referred to as low‐amylose or amylose‐free has special advantages in nutrition composition and food processing. Waxy gene encoding granule–bound starch synthase I (GBSSI) is responsible for amylose synthesis in barley. The G³⁹³⁵‐to‐T in Waxy gene has been previously found in amylose–free barley. In this study, G³⁹³⁵‐to‐T was proved to co‐segregate with the waxy phenotype of barley, but has no obvious effect on GBSSI catalytic activity and starch chain length distribution. However, recombinant inbred lines with G³⁹³⁵‐to‐T in Waxy gene are of significant modification in starch granules morphology and pasting properties, increase of grain β‐glucan content, and decrease of thousand kernel weight along with lower kernel width. A polymerase chain reaction with confronting two–pair primers marker was developed for economic and efficient screening of G³⁹³⁵‐to‐T. This study provides the basis for cultivar improvement of waxy barley then fully developing its potential value and utility in food processing.     

Genetic variation and associations involving Fusarium head blight and deoxynivalenol accumulation in cultivated oat (Avena sativa L.)

Resistance in oats (Avena sativa L.) to Fusarium graminearum was phenotyped in 424 spring oat lines from North America and Scandinavia and genotyped with 2974 SNP markers. Fusarium head blight (FHB), deoxynivalenol (DON) content, days to flowering (DTF) and days to yellow maturity (DTM) were scored in field trials in 2011–12. Trials with phenotypic ranges from 1 to 30 ppm, and sufficient accuracy were obtained by an augmented design and spawn inoculation. Discriminant analysis–PCA identified the different gene pools, with overlaps corresponding to known pedigrees and germplasm exchanges. Structure was negligible and GWAS (genomewide association study) was done using mixed linear models in TASSEL or partial least‐squares regression (PLSR). PLSR allows simultaneous analyses of several phenotypes (environments and/or traits) and is a promising tool for GWAS in plants and should be tested in species with sequenced genomes. FHB was associated with phenology QTLs, due to very susceptible early lines from the Midwest. Lines with consistently low DON (and early heading) were identified. Six QTLs for DON were not associated with earliness, including three QTLs reported previously.     

Rph23: A new designated additive adult plant resistance gene to leaf rust in barley on chromosome 7H

We report on a new adult plant resistance (APR) gene Rph23 conferring resistance to leaf rust in barley. The gene was identified and characterized from a doubled haploid population derived from an intercross between the Australian barley varieties Yerong (Y) and Franklin (F). Genetic analysis of adult plant field leaf rust scores of the Y/F population collected over three successive years indicated involvement of two highly additive genes controlling APR, one of which was named Rph23. The gene was mapped to chromosome 7HS positioned at a genetic distance 36.6 cM. Rph23 is closely linked to marker Ebmac0603, which is flanked by markers bPb‐8660 and bPb‐9601 with linkage distances of 0.8 and 5.1 cM, respectively. A PCR‐based marker was optimized for marker‐assisted selection of Rph23, and on the basis of this marker, the gene was postulated as being common in Australian and global barley germplasm. Pedigree and molecular marker analyses indicated that the six‐rowed black Russian landrace ‘LV‐Taganrog’ is the likely origin of Rph23.     

Molecular mapping of the leaf rust resistance gene Rph7 in barley

Leaf rust of barley, caused by Puccinia hordei Otth, is an important foliar disease in most temperate regions of the world. Sixteen major leaf rust resistance (Rph) genes have been described from barley, but only a few have been mapped. The leaf rust resistance gene Rph7 was first described from the cultivar ‘Cebada Capa’ and has proven effective in Europe. Previously mapped restriction fragment length polymorphism (RFLP) markers have been used to determine the precise location of this gene in the barley genome. From the genetic analysis of a ‘Bow‐man’/‘Cebada Capa’ cross, Rph7 was mapped to the end of chromosome 3HS, 1.3 recombination units distal to the RFLP marker cMWG691. A codominant cleaved amplified polymorphic site (CAPS) marker was developed by exploiting allele‐specific sequence information of the cMWG691 site and adjacent fragments of genomic DNA. Based on the large amount of polymorphism present in this region, the CAPS marker may be useful for the marker‐assisted selection of Rph7 in most diverse genetic backgrounds.