Identifying alleles of <Emphasis Type="Italic">Viviparous-1B</Emphasis> associated with pre-harvest sprouting in micro-core collections of Chinese wheat germplasm

Wheat pre-harvest sprouting (PHS) is an undesired trait, which often reduces yield and downgrades end-use quality of grain. Viviparous-1B (Vp-1B), a regulator gene located on chromosome 3B, has previously been proved to be involved in inducing grain dormancy of wheat. In order to obtain some new or useful alleles associated with PHS tolerance of white-grained wheat, we developed a gene-specific marker (Vp1-b2) to identify allelic variations of Vp-1B using denaturing PAGE in micro-core collections of Chinese wheat and landraces. As a main component observed genetic variation for PHS, seed dormancy evaluated by germination index (GI) was determined at dough-yellow ripening stage in the present study. The results indicated that six alleles of Vp-1B, in our study, were discovered among 276 Chinese wheat varieties. Of these alleles, two variants were validated to be novel alleles and designated as Vp-1Be and Vp-1Bf, respectively. By investigating the association between allelic variations of Vp-1B and seed dormancy, we found allele of Vp-1Ba always inclined to weak seed dormancy and susceptibility to PHS. Up to 62.2% genotypes carrying the allele had high GI value with a range of 0.51–1.00, only 14.4% genotypes had low GI value under 0.30. On the contrary, other variants such as Vp-1Bb, Vp-1Bc, Vp-1Bd, Vp-1Be and Vp-1Bf mostly occurred in varieties with higher PHS tolerance, which average of GI values were 0.204, 0.227, 0.296, 0.256 and 0.186, respectively. In Chinese wheat germplasms, Vp-1Ba and Vp-1Bc showed the most widespread distribution followed by Vp-1Bb; other alleles fell into less used varieties. Our research confirmed rich allelic variation of Vp-1B occurred in micro-core collections of Chinese wheat and landraces, which may be useful for improving PHS tolerance as breeding parents.     

乌拉尔图小麦Viviparous-1基因的单倍型及其表达特性研究

该研究采用PCR和半定量RT-PCR方法,对乌拉尔图小麦（Triticum urartu）休眠基因Viviparous-1A（Vp-1A）的单倍型进行分析,并通过建立系统进化树,对Vp-1A基因在乌拉尔图小麦、普通小麦和其他近缘种间的系统发育关系进行分析。结果表明：（1）在20份乌拉尔图小麦中共发现4种新等位变异类型,分别命名为TuVp-1Abgi、TuVp-1Adfi、TuVp-1Aefi和TuVp-1Acgh。与普通小麦的TaVp-1Aa(AJ400712)基因相比,这4种单倍型主要是在第3内含子中有多个TTC不同重复,在第2和第5内含子中存在序列的缺失以及SNPs;（2）用ABA处理种子胚后,4种不同单倍型材料的mRNA表达水平发生变化,表明这4种单倍型对ABA敏感性不同;（3）乌拉尔图小麦中Vp-1A基因不同单倍型,在第2、第3和第5内含子中碱基序列的插入和缺失,影响了Vp-1A基因的表达特性及对ABA的敏感性,从而影响种子休眠特性;（4）鉴定和分析发现,乌拉尔图小麦TuVp-1Adfi单倍型可作为小麦穗发芽抗性资源。     

Validating a novel allele of <Emphasis Type="Italic">viviparous-1</Emphasis> (<Emphasis Type="Italic">Vp-1Bf</Emphasis>) associated with high seed dormancy of Chinese wheat landrace,Wanxianbaimaizi

Pre-harvest sprouting (PHS) can easily lead to yield losses of wheat and downgrading of grain quality in wheat-growing areas where long periods of wet weather occur frequently during harvest. As a main component of PHS, seed dormancy is often evaluated by germination index (GI). Previous researches have proved allelic variations of Vp-1B to have a close relationship with dormancy of white-grained wheat. In the present study, a mapping population covering 157 recombinant inbred lines was developed from a cross of two white-grained varieties, Wanxianbaimaizi and Jing411. Wanxianbaimaizi is a strongly dormant landrace carrying a novel allele, Vp-1Bf; whereas, Jing411 is a non-dormant variety with wild allele, Vp-1Ba. Our objective was to validate the association between the novel allele and seed dormancy using the population. The results of sequences alignment indicated an insertion of 193 bp and a deletion of 109 bp were both identified in the novel allele, respectively, compared with wild allele in Jing411. Here, the deletion was first detected. As for lines possessing Vp-1Ba, the average GI value was 0.584, significantly higher than that of lines holding Vp-1Bf. Among three genotypes, Vp-1Bf allele was generally corresponded to the lowest average of GI value (0.195), and the highest dormancy; in addition, lines with heterozygous genotype often showed intermediate GI value (0.356). Of 92 RILs with Vp-1Ba, over 70 lines showed higher GI value (>0.40), and only about 7 lines had lower GI value (<0.20). On the other hand, among 60 RILs with Vp-1Bf, over 50 lines showed lower GI value (<0.40), and only about 7 lines had higher GI value (>0.60). The result of composite interval mapping revealed that a major QTL for seed dormancy was flanked by Xwmc446 and Vp1 on 3BL, which was proximal to Vp1 (7.6–8.4 cM). The locus could explain 24.6–40.7% of total phenotypic variation across three crop seasons. The above results could confirm that the novel allele had a striking association with high seed dormancy, and may be useful for improving PHS tolerance of white-grained wheat.     

Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.     

Genetic variation of Vp1 in Sichuan wheat accessions and its association with pre-harvest sprouting response

Pre-harvest sprouting (PHS) in bread wheat is a major abiotic constraint reducing yield and influencing the production of high quality grain. In China both spring and winter wheat regions are affected by PHS. Sichuan lies in southwest China, where the most of rainfall occurs during April to September when wheat is harvested. The present investigation was conducted to identify the allelic variability of Vp1, a gene that plays a role in maintenance and induction of dormancy, among Sichuan landraces and recent cultivars with different dormancy levels and to find potential sources of PHS resistance for breeding. Sichuan landrace and cultivar wheat accessions had a wide range of dormancy levels. The average germination index (GI) of Sichuan landrace accessions was 0.232, whereas at 0.674 it was much higher for cultivars. The different dormancy levels between landraces and cultivars indicated that pre-harvest sprouting resistance might have been neglected in recent Sichuan wheat breeding programs. The average GI of white grained accessions was higher than for red grained accessions. Particular Vp-1B gene fragments were specific in landraces or cultivars and in white or red grained accessions. The results indicated that Vp-1B markers could be used to distinguish cultivars and landraces. Significant relationships between certain Vp-1B allelesand GI of Sichuan wheat accessions were shown by Spearman’s rank correlation analysis.     

Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8'-hydroxylase). Wheat TaABA8'OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8'OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8'-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.     

Nucleotide Variation in the NCED3 Region of Arabidopsis thaliana and its Association Study with Abscisic Acid Content under Drought Stress

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with ANOVA analysis suggested that the 274th site (P←→S) and the 327th site (P←→R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.     

Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.