Development of a specific AFLP-based SCAR marker for Chinese Race 34MKG of Puccinia graminis f. sp. tritici.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a fungus that causes the devastating fungalwheat stem rust disease in wheat production. Rapid identification of the physiological races of Pgt are very importance for the prevention of wheat stem rust. In this paper we developed a molecular method to identify the most prevalent race of Pgt, as a supplement for traditionally used host-specific methods. Amplified fragment length polymorphism (AFLP) was employed as a means of analyzing DNA polymorphisms in six common physiological races of Pgt in China and Ug99. In total, 64 pairs of primers were used for AFLP screening of race-specific molecular markers. One primer pair-namely, E7/M7 (5'-GACTGCGTACCAATTCG G-3'/5'-GATGAGTCCTGAGTAACGG-3')-yielded a unique band for the race 34MKG that was purified and cloned into the pGEM-T vector for sequencing. We then designed a new primer pairs (sequence-characterized amplified region marker) to amplify the 171-bp fragment and confirmed that the marker was highly specific for 34MKG. These results provide a new tool for monitoring different races of Pgt for improved control of wheat stem rust in China.

Characterization of Wheat Monogenic Lines with Known Sr Genes and Wheat Lines with Resistance to the Ug99 Race Group for Resistance to Prevalent Races of Puccinia graminis f. sp. tritici in China.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is one of the most serious fungal diseases in wheat production, seriously threatening the global supply of wheat and endangering food security. The present study was conducted to evaluate wheat monogenic lines with known Sr genes to the most prevalent P. graminis f. sp. tritici races in China. In addition, wheat lines introduced from the International Maize and Wheat improvement Center (CIMMYT) with resistance to the Ug99 race group were also evaluated with the prevalent Chinese P. graminis f. sp. tritici races. The monogenic lines containing Sr9e, Sr21, Sr26, Sr31, Sr33, Sr35, Sr37, Sr38, Sr47, and SrTt3 were effective against races 21C3CTTTM, 34C0MRGSM, and 34C3MTGQM at both seedling and adult-plant stages. In contrast, monogenic lines containing Sr6, Sr7b, Sr8a, Sr9a, Sr9b, Sr9d, Sr9f, Sr9g, Sr13, Sr16, Sr18, Sr19, Sr20, Sr24, Sr28, Sr29, and Sr34 were highly susceptible to these races at both seedling and adult-plant stages. Lines with Sr5, Sr10, Sr13, Sr14, Sr15, Sr17, Sr21, Sr22, Sr23, Sr25, Sr27, Sr29, Sr30, Sr32, Sr36, and Sr39 were resistant to one or more of the tested races. Among the 123 CIMMYT lines, 38 (30.9%) showed varying levels of susceptibility to Chinese P. graminis f. sp. tritici races. The results should be useful for breeding wheat cultivars with resistance to stem rust.

Virulence structure and its genetic diversity analyses of Blumeria graminis f. sp. tritici isolates in China.

BACKGROUND: Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew severely affects yield security wheat production in China. Understanding the virulence structure and genetic variations of this pathogen is important for breeding wheat lines resistant to wheat powdery mildew. However, information related to genes controlling resistance remains elusive. This study analyzes the virulence structure and the genetic diversity of pathogenic Bgt populations isolated from northeastern (Liaoning, Heilongjiang) and northwestern (Gansu) China, two representative wheat producing areas, on 37 wheat cultivars each carrying a known powdery mildew resistance (Pm) gene. RESULTS: Bgt isolates from northeastern China show higher frequencies of virulence genes than populations from Gansu Province. Many of the known Pm genes failed to provide resistance in this study. However, Pm21 provided 100% resistance to all isolates from all three provinces, obtained during two consecutive years, while Pm13 provided 100% resistance in Gansu. Pm13, Pm16, Pm18, and Pm22 also showed partial resistance in northeastern China, while Pm16, Pm18, Pm22, Pm5 + 6 and Pm2 + 6 +? maintained some resistance in Gansu. Genetic diversity among populations in different regions was detected by cluster analyses using expressed sequence tag-simple sequence repeat (EST-SSR). When the genetic similarity coefficient is relatively high, populations from the same regional origin are mostly clustered into one group while populations from different regions exhibit large genetic differences. CONCLUSION: Pm21 remains the best choice for breeding programs to maintain resistance to Bgt. Only 58% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and frequency of virulence gene data.

Related to ABI3/VP1-Like 1 (RAVL1) regulates brassinosteroid-mediated activation of AMT1;2 in rice (Oryza sativa).

The promotive effects of brassinosteroids (BRs) on plant growth and development have been widely investigated; however, it is not known whether BRs directly affect nutrient uptake. Here, we explored the possibility of a direct relationship between BRs and ammonium uptake via AMT1-type genes in rice (Oryza sativa). BR treatment increased the expression of AMT1;1 and AMT1;2, whereas in the mutant d61-1, which is defective in the BR-receptor gene BRI1, BR-dependent expression of these genes was suppressed. We then employed Related to ABI3/VP1-Like 1 (RAVL1), which is involved in BR homeostasis, to investigate BR-mediated AMT1 expression and its effect on NH4+ uptake in rice roots. AMT1;2 expression was lower in the ravl1 mutant, but higher in the RAVL1-overexpressing lines. EMSA and ChIP analyses showed that RAVL1 activates the expression of AMT1;2 by directly binding to E-box motifs in its promoter. Moreover, 15NH4+ uptake, cellular ammonium contents, and root responses to methyl-ammonium strongly depended on RAVL1 levels. Analysing AMT1;2 expression levels in different crosses between BRI1 and RAVL1 mutant and overexpression lines indicated that RAVL1 acts downstream of BRI1 in the regulation of AMT1;2. Thus, the present study shows how BRs may be involved in the transcriptional regulation of nutrient transporters to modulate their uptake capacity.

Genome-wide identification of the SWEET gene family in wheat.

The SWEET (sugars will eventually be exported transporter) family is a newly characterized group of sugar transporters. In plants, the key roles of SWEETs in phloem transport, nectar secretion, pollen nutrition, stress tolerance, and plant-pathogen interactions have been identified. SWEET family genes have been characterized in many plant species, but a comprehensive analysis of SWEET members has not yet been performed in wheat. Here, 59 wheat SWEETs (hereafter TaSWEETs) were identified through homology searches. Analyses of phylogenetic relationships, numbers of transmembrane helices (TMHs), gene structures, and motifs showed that TaSWEETs carrying 3-7 TMHs could be classified into four clades with 10 different types of motifs. Examination of the expression patterns of 18 SWEET genes revealed that a few are tissue-specific while most are ubiquitously expressed. In addition, the stem rust-mediated expression patterns of SWEET genes were monitored using a stem rust-susceptible cultivar, 'Little Club' (LC). The resulting data showed that the expression of five out of the 18 SWEETs tested was induced following inoculation. In conclusion, we provide the first comprehensive analysis of the wheat SWEET gene family. Information regarding the phylogenetic relationships, gene structures, and expression profiles of SWEET genes in different tissues and following stem rust disease inoculation will be useful in identifying the potential roles of SWEETs in specific developmental and pathogenic processes.

Race and virulence characterization of Puccinia graminis f. sp. tritici in China.

Wheat stem rust was once the most destructive plant disease, but it has been largely controlled. However, to prevent future problems, the ongoing development of resistant wheat varieties requires knowledge of the changing virulence patterns for Pgt virulence of the fungus that causes wheat stem rust and the detection of new races. Surveys were conducted from 2013-2014 to determine the races of the Pgt present in China. Low levels of stem rust infections were found in China during this investigation and 11 Puccinia graminis f. sp. tritici (Pgt) samples were obtained. In addition, 22 Pgt samples collected from the alternate host (Berberis) were obtained and have been reported for the first time. Fifty-three isolates were obtained from all samples. Four race groups, including 13 physiological races, were identified. They included the most prevalent races, 34C3MTGQM and 34C6MRGQM, with 13.2% predominance, followed by 34C0MRGQM at 11.3%. Six new races that were virulent against the resistance genes, Sr5 + Sr11, were found for the first time in China, namely 34C0MRGQM, 34C3MTGQM, 34C3MKGQM, 34C3MKGSM, 34C6MTGSM, and 34C6MRGQM, with a predominance of 11.3, 13.2, 9.4, 9.4, 1.9, and 13.2%, respectively. Most of the genes studied were ineffective against one or more of the tested isolates, except Sr9e, Sr21, Sr26, Sr31, Sr33, Sr38, Sr47, and SrTt3. Genes Sr35, SrTmp, Sr30, Sr37, Sr17, and Sr36 were effective in 92.5, 86.8, 84.9, 84.9, 79.3, and 77.4% of the tested isolates, respectively. In contrast, all of the isolates were virulent against Sr6, Sr7b, Sr9a, Sr9b, Sr9d, Sr9g, and SrMcN. Our results indicate that remarkable differences exist among the categories of the races in this study (i.e., their known virulence gene spectra) and the Pgt races reported previously. In addition, the sexual cycle of Pgt may contribute to its diversity in China.

Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Province in China.

Wheat stem rust, caused by Puccinia granimis f. sp. tritici, severely affects wheat production, but it has been effectively controlled in China since the 1970s. However, the appearance and spread of wheat stem rust races Ug99 (TTKSK, virulence to Sr31), TKTTF (virulence to SrTmp) and TTTTF (virulence to the cultivars carrying Sr9e and Sr13) have received attention. It is important to clarify the effectiveness of resistance genes in a timely manner, especially for the purpose of using new resistance genes in wheat cultivars for durable-resistance. However, little is known about the stem rust resistance genes present in widely used wheat cultivars from Gansu. This study aimed to determine the resistance level at the seedling stage of the main wheat cultivars in Gansu Province. A secondary objective was to assess the prevalence of Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 using molecular markers. The results of the present study indicated that 38 (50.7%) wheat varieties displayed resistance to all the tested races of Puccinia graminis f. sp. tritici. The molecular marker analysis showed that 13 out of 75 major wheat cultivars likely carried Sr2; 25 wheat cultivars likely carried Sr31; and nine wheat cultivars likely carried Sr38. No cultivar was found to have Sr25 and Sr26, as expected. Surprisingly, no wheat cultivars carried Sr24. The wheat lines with known stem rust resistance genes could be used as donor parent for further breeding programs.

Seedling Resistance to Stem Rust and Molecular Marker Analysis of Resistance Genes in Wheat Cultivars of Yunnan, China.

Stem rust is one of the most potentially harmful wheat diseases, but has been effectively controlled in China since 1970s. However, the interest in breeding wheat with durable resistance to stem rust has been renewed with the emergence of Ug99 (TTKSK) virulent to the widely used resistance gene Sr31, and by which the wheat stem rust was controlled for 40 years in wheat production area worldwide. Yunnan Province, located on the Southwest border of China, is one of the main wheat growing regions, playing a pivotal role in the wheat stem rust epidemic in China. This study investigated the levels of resistance in key wheat cultivars (lines) of Yunnan Province. In addition, the existence of Sr25, Sr26, Sr28, Sr31, Sr32, and Sr38 genes in 119 wheat cultivars was assessed using specific DNA markers. The results indicated that 77 (64.7%) tested wheat varieties showed different levels of resistance to all the tested races of Puccinia graminis f. sp. tritici. Using molecular markers, we identified the resistance gene Sr31 in 43 samples; Sr38 in 10 samples; Sr28 in 12 samples, and one sample which was resistant against Ug99 (avirulent to Sr32). No Sr25 or Sr26 (effective against Ug99) was identified in any cultivars tested. Furthermore, 5 out of 119 cultivars tested carried both Sr31 and Sr38 and eight contained both Sr31 and Sr28. The results enable the development of appropriate strategies to breed varieties resistant to stem rust.

Transposon Ds-Mediated Insertional Mutagenesis in Rice (Oryza sativa).

Rice (Oryza sativa) is the most important consumed staple food for a large and diverse population worldwide. Since databases of genomic sequences became available, functional genomics and genetic manipulations have been widely practiced in rice research communities. Insertional mutants are the most common genetic materials utilized to analyze gene function. To mutagenize rice genomes, we exploited the transpositional activity of an Activator/Dissociation (Ac/Ds) system in rice. To mobilize Ds in rice genomes, a maize Ac cDNA was expressed under the CaMV35S promoter, and a gene trap Ds was utilized to detect expression of host genes via the reporter gene GUS. Conventional transposon-mediated gene-tagging systems rely on genetic crossing and selection markers. Furthermore, the activities of transposases have to be monitored. By taking advantage of the fact that Ds becomes highly active during tissue culture, a plant regeneration system employing tissue culture was employed to generate a large Ds transposant population in rice. This system overcomes the requirement for markers and the monitoring of Ac activity. In the regenerated populations, more than 70% of the plant lines contained independent Ds insertions and 12% expressed GUS at seedling stages. This protocol describes the method for producing a Ds-mediated insertional population via tissue culture regeneration systems. © 2016 by John Wiley & Sons, Inc.