Physiological and Transcriptome Analysis Illuminates the Molecular Mechanisms of the Drought Resistance Improved by Alginate Oligosaccharides in Triticum aestivum L.

Alginate oligosaccharides(AOS)enhance drought resistance in wheat(Triticum aestivum L.),but the definite mechanisms remain largely unknown.The physiological and transcriptome responses of wheat seedlings treated with AOS were analyzed under drought stress simulated with polyethylene glycol-6000.The results showed that AOS promoted the growth of wheat seedlings and reduced oxidative damage by improving peroxidase and superoxide dismutase activities under drought stress.A total of 10,064 and 15,208 differentially expressed unigenes(DEGs)obtained from the AOS treatment and control samples at 24 and 72 h after dehydration,respectively,were mainly enriched in the biosynthesis of secondary metabolites(phenylpropanoid biosynthesis,flavonoid biosynthesis),carbohydrate metabolism(starch and sucrose metabolism,carbon fixation in photosynthetic organisms),lipid metabolism(fatty acid elongation,biosynthesis of unsaturated fatty acids,alpha-linolenic acid metabolism,cutin,suberine and wax biosynthesis),and signaling transduction pathways.The up-regulated genes were related to,for example,chlorophyll a-b binding protein,amylosynthease,phosphotransferase,peroxidase,phenylalanine ammonia lyase,flavone synthase,glutathione synthetase.Signaling molecules(including MAPK,plant hormones,H_(2)O_(2) and calcium)and transcription factors(mainly including NAC,MYB,MYB-related,WRKY,bZIP family members)were involved in the AOS-induced wheat drought resistance.The results obtained in this study help underpin the mechanisms of wheat drought resistance improved by AOS,and provides a theoretical basis for the application of AOS as an environmentally sustainable biological method to improve drought resistance in agriculture.

QTL Mapping for Drought Tolerance at Stages of Germination and Seedling in Wheat (Triticum aestivum L.) Using a DH Population

Drought is a major constraint in many wheat( Triticum aestivum L.) production regions. Quantitative trait loci (QTLs) conditioning drought tolerance at stages of germination and seedling in wheat were identified in a double haploid (DH) population derived from the cross, Hanxuan10×Lumai14, using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Interval mapping analysis revealed that QTLs for drought tolerance at germination stage were located on chromosomes 1B, 2B, 5A, 6B, 7A and 7B, respectively, and the most effective QTL was mapped on chromosome 2B, explaining 27.2% of phenotypic variance. The QTLs for drought tolerance at seedling stage were located on 1B, 3B and 7B, respectively, and the most effective QTL was mapped on chromosome 3B, explaining 21.6% of phenotypic variance. Their positions were different from those of QTLs conferring drought tolerance at germination stage, indicating that drought tolerance at germination stage and seedling stage was controlled by different loci. Most of the identified QTLs explained 18% or more of phenotypic variance for drought tolerance at germination and seedling stage, and would be useful in future for marker assisted selection programs and cultivar improvement.

Growth traits and nitrogen assimilation-associated physiological parameters of wheat(Triticum aestivum L.) under low and high N conditions

In this study, 14 wheat cultivars with contrasting yield and N use efficiency （NUE） were used to investigate the agronomic and NUE-related traits, and the N assimilation-associated enzyme activities under low and high N conditions. Under deficient-N, the cultivars with high N uptake efficiency （UpE） and high N utilization efficiency （UtE） exhibited higher plant biomass, yields, and N contents than those with medium and low NUEs. The high UpE cultivars accumulated more N than other NUE type cultivars. Under sufficient-N, the tested cultivars showed similar patterns in biomass, yield, and N content to those under deficient-N, but the varietal variations in above traits were smaller. In addition, the high UpE cultivars displayed much more of root biomass and larger of root length, surface area, and volume than other NUE type cultivars, indicating that the root morphological traits under N deprivation are closely associated with the plant biomass through its improvement of the N acquisition. The high UtE cultivars showed higher activities of nitrate reductase （NR）, nitrite reductase （NIR）, and gluta- mine synthetase （GS） at stages of seediling, heading and filling than other NUE type cultivars under both low and high N conditions. Moreover, the high UpE and UtE cultivars also displayed higher photosynthetic rate under deficient-N than the medium and low NUE cultivars. Together, our results indicated that the tested wheat cultivars possess dramatically genetic variations in biomass, yield, and NUE. The root morphological traits and the N assimilation enzymatic acitivities play critical roles in regulating N accumulation and internal N translocation under the N-starvation stress, respectively. They can be used as morphological and biochemical references for evaluation of UpE and UtE in wheat.

Effects of reduced nitrogen and suitable soil moisture on wheat(Triticum aestivum L.) rhizosphere soil microbiological,biochemical properties and yield in the Huanghuai Plain,China

Soil management practices affect rhizosphere microorganisms and enzyme activities, which in turn influence soil ecosystem processes. The objective of this study was to explore the effects of different nitrogen application rates on wheat(Triticum aestivum L.) rhizosphere soil microorganisms and enzyme activities, and their temporal variations in relation to soil fertility under supplemental irrigation conditions in a fluvo-aquic region. For this, we established a split-plot experiment for two consecutive years(2014–2015 and 2015–2016) in the field with three levels of soil moisture: water deficit to no irrigation(W1), medium irrigation to(70±5)% of soil relative moisture after jointing stage(W2), and adequate irrigation to(80±5)% of soil relative moisture after jointing stage(W3);and three levels of nitrogen: 0 kg ha^–1(N1), 195 kg ha^–1(N2) and 270 kg ha^–1(N3). Results showed that irrigation and nitrogen application significantly increased rhizosphere microorganisms and enzyme activities. Soil microbiological properties showed different trends in response to N level;the highest values of bacteria, protease, catalase and phosphatase appeared in N2, while the highest levels of actinobacteria, fungi and urease were observed in N3. In addition, these items performed best under medium irrigation(W2) relative to W1 and W3;particularly the maximum microorganism(bacteria, actinobacteria and fungi) amounts appeared at W2, 5.37×10^7 and 6.35×10^7 CFUs g^–1 higher than those at W3 in 2014–2015 and 2015–2016, respectively;and these changes were similar in both growing seasons. Microbe-related parameters fluctuated over time but their seasonality did not hamper the irrigation and fertilization-induced effects. Further, the highest grain yields of 13 309.2 and 12 885.7 kg ha^–1 were both obtained at W2 N2 in 2014–2015 and 2015–2016, respectively. The selected properties, soil microorganisms and enzymes, were significantly correlated with wheat yield and proved to be valuable indicators of soil quality. These results clearly demonstrated that the combined treatment(W2 N2) significantly improved soil microbiological properties, soil fertility and wheat yield on the Huanghuai Plain, China.

TaMIR1119, a miRNA family member of wheat(Triticum aestivum),is essential in the regulation of plant drought tolerance

Through regulating target genes via the mechanisms of posttranscriptional cleavage or translational repression, plant miRNAs involve diverse biological processes associating with plant growth, development, and abiotic stress responses, in this study, we functionally characterized TaMIR1119, a miRNA family member of wheat （Triticum aestivum）, in regulating the drought adaptive response of plants. TaMIR1119 putatively targets six genes categorized into the functional classes of transcriptional regulation, RNA and biochemical metabolism, trafficking, and oxidative stress defense. Upon simulated drought stress, the TaMIR1119 transcripts abundance in roots was drastically altered, showing to be upregulated gradually within a 48-h drought regime andthat the drought-induced transcripts were gradually restored along with a 48-h recovery treatment. In contrast, most miRNA target genes displayed reverse expression patterns to TaMIR1119, exhibiting a downregulated expression pattern upon drought and whose reduced transcripts were re-elevated along with a normal recovery treatment. These expression analysis results indicated that TaMIR1119 responds to drought and regulates the target genes mainly through a cleavage mechanism. Under drought stress, the tobacco lines with TaMIR1119 overexpression behaved improved phenotypes,, showing increased plant biomass, photosynthetic parameters, osmolyte accumulation, and enhanced antioxidant enzyme （AE） activities relative to wild type. Three AE genes, NtFeSOD, NtCAT1;3, and NtSOD2,1, encoding superoxide dismutase （SOD）, catalase （CAT）, and peroxidase （POD） proteins, respectively, showed upregulated expression in TaMIR1119 overexpression lines, suggesting that they are involved in the regulation of AE activities and contribution to the improved cellular reactive oxygen species （ROS） homeostasis in drought-challenged transgenic lines. Our results indicate that TaMIR1119 plays critical roles in regulating plant drought tolerance through transcriptionally regulating the target genes that modulate osmolyte accumulation, photosynthetic function, and improve cellular ROS homeostasis of plants.

Molecular Characterization and Expression Analysis of TaZFP15, a C_2H_2-Type Zinc Finger Transcription Factor Gene in Wheat (Triticum aestivum L.)

Based on sequencing of part clones in a root subtractive cDNA library, an expressed sequence tag （EST） sharing high similarity to a rice C2H2 zinc finger transcription factor （ZFP15） was obtained in wheat. Through bioinformatics approach, the wheat C2H2-type ZFP gene referred to TaZFP15 has been identified and characterized. As a full-length cDNA of 670 bp, TaZFP15 has an open reading frame of 408 bp and encodes a 135-aa polypeptide. TaZFP15 contains two C2H2 zinc finger domains and each one has a conserved motif QALGGH. The typical L-box, generally identified in the C2H2 type transcription factors, has also been found in TaZFP15. Phylogenetic analysis suggested that TaZFP15 shares high similarities with rice ZFP15 （GenBank accession no. AY286473）, maize ZFP （GenBank accession no. NM_001159094） and a subset of other zinc-finger transcription factor genes in plant species. The expression of TaZFP15 was up-regulated by starved-Pi stress, showing a pattern to be gradually elevated along with the progression of the Pi-stress in a 23-h treatment regime. Similarly, the transcripts of TaZFP15 in roots were also induced by nitrogen deficiency, and abiotic stresses of drought and salinity. No responses of TaZFP15 were detected in roots to nutrition deficiencies of P, Zn, and Ca, and the external treatment of abscisic acid （ABA）. TaZFP15 could be specifically amplified in genome A, B, and D, and without variability in the sequences, suggesting that TaZFP15 has multi-copies in the homologous hexaploid species. Transgenic analysis in tobacco revealed that up-regulation of TaZFP15 could significantly improve plant dry mass accumulation via increasing the plant phosphorus acquisition capacity under Pi-deficiency condition. The results suggested that TaZFP15 is involved in mediation of signal transductions of diverse external stresses.

The Relationship Between Developmental Accumulation of Leaf Soluble Proteins and Vernalization Response of Wheat(Triticum aestivum L.em. Thell)

The relationship between vernalization requirement and quantitative and qualitative changes in total leaf soluble proteins were determined in one spring （cv. Kohdasht） and two winter （cvs. Sardari and Norstar） cultivars of wheat （Triticum aestivum L.） exposed to 4℃. Plants were sampled on days 2, 14, 21 and 35 of exposure to 4℃. The final leaf number （FLN） was determined throughout the vernalization periods （0, 7, 14, 24, and 35 d） at 4℃. The final leaf number decreased until days 24 and 35 in Sardari and Norstar eultivars, respectively, indicating the vernalization saturation at these times. No clear changes were detected in the final leaf number of Kohdash cultivar, verifying no vernalization requirement for this spring wheat cultivar. Comparing with control, clear cold-induced 2-fold increases in proteins quantity occurred after 48 h following the 4℃-treatment in the leaves of the both winter wheat cultivars but, such response was not detected in the spring cultivar. However, the electrophoretic protein patterns showed between-cultivar and between-temperature treatment differences. With increasing exposure time to 4℃, the winter cultivars tended to produce more HMW polypeptides than the spring cultivar. Similar proteins were induced in both Sardari and Norstar winter wheat cultivars, however, the long vernalization requirement in Norstar resulted in high level and longer duration of expression of cold-induced proteins compared to Sardari with a short vernalization requirement. These observations indicate that vernalization response regulates the expression of low temperature （LT） tolerance proteins and determines the duration of expression of LT- induced proteins.

Detection of Allelic Variation in Chinese Wheat (Triticum aestivum L.) Germplasm with Drought ToleranceUsing SSR Markers

Allelic variation in two domestic wheat landraces, Pingyaobaimai and Mazhamai, two cornerstone breeding materials and their derived cultivars with drought tolerance was detected by SSR (simple sequence repeat) markers. The clustering of 25 accessions showed that the similarity between Pingyaobaimai and Yandal817, the latter was developed from the former, was 0.71, the highest one of all accessions, but the similarities were very low between these two accessions and other accessions including their derived cultivars. A similar situation was revealed between Mazhamai and its derived cultivars. Pingyaobaimai and its three derived cultivars shared three alleles at loci Xgwm526, Xgwm538 and Xgwm126 on chromosome arms 2BL, 4BL and 5AL, respectively. There were six shared alleles in Mazhamai and its derived cultivars, in order of Xgwm157, Xgwm126, Xgwm212, Xgwm626, Xgwm471 and Xgwm44 on chromosome arms 2DL, 5AL, 5DL, 6BL, 7AS and 7DC, respectively. Only one shared allele was detected between the pedigrees of Pingyaobaimai and Mazhamai. The difference of shared alleles in two cornerstone breeding materials and their derived cultivars revealed the diversity in Chinese wheat germplasm with drought tolerance and the complication in genetic basis of drought tolerance in wheat.

Effects of Auxins and Media on Callus Induction of Chinese Spring Wheat(Triticum aestivum L.)

The effects of auxins and media on callus induction from the mature and immature embryos of Chinese spring wheat （Triticum aestivum L.） varieties were investigated. It was found that genotype, medium, auxin source and concentration had the significant effects on the induction of embryogenic callus, explants germination and the increment of callus fresh weight. For immature embryos cultured on MS medium, 2 mg L^-1 of 2, 4-D was optimal, and the highest frequency of embryogenic callus （33.50%） was observed. For the mature embryos on N6 medium, 4 mg L^-1 of 2, 4-D was optimal. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. These results indicated that 2, 4, 5-T was superior to 2, 4-D and NAA in the culture of immature embryos. This is the first report about the effect of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail.

Conditional and unconditional QTLs mapping of gluten strength in common wheat (Triticum aestivum L.)

Dissecting the genetic relationships among gluten-related traits is important for high quality wheat breeding. Quantita- tive trait loci （QTLs） analysis for gluten strength, as measured by sedimentation volume （SV） and gluten index （GI）, was performed using the QTLNetwork 2.0 software. Recombinant inbred lines （RILs） derived from the winter wheat varieties Shannong 01-35xGaocheng 9411 were used for the study. A total of seven additive QTLs for gluten strength were identi- fied using an unconditional analysis. QGi1D-13 and QSv1D-14 were detected through unconditional and conditional QTLs mapping, which explained 9.15-45.08% of the phenotypic variation. QTLs only identified under conditional QTL mapping were located in three marker intervals： WPT-3743-GLU-D1 （1D）, WPT-7001-WMC258 （1B）, and WPT-8682-WPT-5562 （1B）. Six pairs of epistatic QTLs distributed nine chromosomes were identified. Of these, two main effect QTLs （QGi1D-13 and QSvlD-14） and 12 pairs of epistatic QTLs were involved in interactions with the environment. The results indicated that chromosomes 1B and 1D are important for the improvement of gluten strength in common wheat. The combination of conditional and unconditional QTLs mapping could be useful for a better understanding of the interdependence of different traits at the QTL molecular level.

Allelic Variation in Loci for Adaptive Response and Its Effect on Agronomical Traits in Chinese Wheat(Triticum aestivum L.)

Heading date was an important trait that decided the adaptation of wheat to environments. It was modiifed by genes involved in vernalization response, photoperiod response and development rate. In this study, four loci Xgwm261, Xgwm219, Xbarc23 and Ppd-D1 which were previously reported related to heading time were analyzed based on three groups of wheat including landraces （L）, varieties bred before 1983 （B82） and after 1983 （A83） collected from Chinese wheat growing areas. Generally, heading date of landrace was longer than that of varieties. Signiifcant differences in the heading time existed within the groups, which implied that diversiifcation selection was much helpful for adaptation in each wheat zone. Photoperiod insensitive allele Ppd-D1a was the ifrst choice for both landrace and modern varieties, which promoted the heading date about four days earlier than that of sensitive allele Ppd-D1b. The three SSR loci had different characters in the three groups. Predominant allele combination for each zone was predicted for wheat group L and A83, which made great contribution to advantageous traits. Xgwm219 was found to be signiifcantly associated with heading date in Yellow and Huai River Winter Wheat Zone （Zone II） and spike length in Middle and lower Yangtze Valley Winter Wheat Zone （Zone III）, which implied functional diversiifcation for adaption. Variation for earliness genes provided here will be helpful for whet breeding in future climatic change.

Expression Pattern Analysis of Zinc Finger Protein Genes in Wheat(Triticum aestivum L.) Under Phosphorus Deprivation

Zinc finger protein（ZFP） genes comprise a large and diverse gene family, and are involved in biotic and abiotic stress responses in plants. In this study, a total of 126 ZFP genes classified into various types in wheat were characterized and subjected to expression pattern analysis under inorganic phosphate（Pi） deprivation. The wheat ZFP genes and their corresponding GenBank numbers were obtained from the information of a 4×44K wheat gene expression microarray chip. They were confirmed by sequence similarity analysis and named based on their homologs in Brachypodium distachyon or Oriza sativa. Expression analysis based on the microarray chip revealed that these ZFP genes are categorized into 11 classes according to their gene expression patterns in a 24-h of Pi deprivation regime. Among them, ten genes were differentially up-regulated, ten genes differentially downregulated, and two genes both differentially up- and down-regulated by Pi deprivation. The differentially up- or down-regulated genes exhibited significantly more or less transcripts at one, two, or all of the checking time points（1, 6, and 24 h） of Pi stress in comparison with those of normal growth, respectively. The both differentially up- and down-regulated genes exhibited contrasting expression patterns, of these, TaWRKY70;5 showed significantly up-regulated at 1 and 6 h and down-regulated at 24 h whereas TaAN1AN20-8;2 displayed significantly upregulated at 1 h and downregulated at 6 h under deprivation Pi condition. Real time PCR analysis confirmed the expression patterns of the differentially expressed genes obtained by the microarray chip. Our results indicate that numerous ZFP genes in wheat respond to Pi deprivation and have provided further insight into the molecular basis that plants respond to Pi deprivation mediated by the ZFP gene family.

Cloning of TaCYP707A1 Gene that Encodes ABA 8′-Hydroxylase in Common Wheat (Triticum aestivum L.)

The plant hormone abscisic acid （ABA） regulates many important physiological and developmental processes in plants. The objective of this study was to clone the ABA 8′-hydroxylase gene in common wheat. In the present study, we used the eDNA sequence of barley HvCYP707A1 gene （GenBank accession no. AB239299） as a probe for BLAST search against the common wheat （Triticum aestivum L.） EST database in GenBank. All wheat ESTs sharing high similarity with the reference gene were subjected to contig assembly. Primers were designed based on the constructed contigs to clone the wheat CYP707A1 gene, designated as TaCYP707A1. The genomic DNA sequence of TaCYPTO7A1 gene comprised five exons and four introns, with a size of 2225 bp. The corresponding cDNA sequence of TaCYP707A1 was 1737 bp, containing an open reading frame （ORF） of 1431 bp, a 42-bp 5′-untranslated region （UTR） and a 264-bp 3′UTR, with 94.9% of identical sequences to HvCYP707A1 gene （AB239299）. The neighbor joining tree indicated that the deduced amino acid sequences of TaCYP707A1 gene was highly similar to those of barley and rice. The TaCYP707A1 gene was located on chromosome 6BL using a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 6BS. These results will be of high importance in understanding of molecular mechanism of ABA catabolism.

Mapping QTLs for stomatal density and size under drought stress in wheat(Triticum aestivum L.)

Stomatal density and size affect plant water use efficiency, photosynthsis rate and yield. The objective of this study was to gain insights into the variation and genetic basis of stomatal density and size during grain filling under drought stress（DS） and well-watered（WW） conditions. The doubled haploid population derived from a cross of wheat cultivars Hanxuan 10（H10）, a female parent, and Lumai 14（L14）, a male parent, was used for phenotyping at the heading, flowering, and mid- and late grain filling stages along with established amplified fragment length polymorphism（AFLP） and simple sequence repeat（SSR） markers. The stomatal density of doubled haploid（DH） lines was gradually increased, while the stomatal lengths and widths were gradually decreased during grain filling stage. Twenty additive QTLs and 19 pairs of epistatic QTLs for the 3 traits were identified under DS. The other 20 QTLs and 25 pairs epistatic QTLs were obtained under WW. Most QTLs made more than 10% contributions to the total phenotypic variations at one growth stage under DS or WW. Furthermore, QTLs for stomatal density near Xwmc74 and Xgwm291 located on chromosome 5A were tightly linked to previously reported QTLs regulating total number of spikelets per spike, number of sterile spikelets per spike and proportion of fertile spikelets per spike. Qsw-2D-1 was detected across stages, and was in the same marker region as a major QTL for plant height, QPH.cgb-2D.1. These indicate that these QTLs on chromosomes 5A and 2D are involved in regulating these agronomic traits and are valuable for molecular breeding.

TaGW2L,a GW2-like RING finger E3 ligase,positively regulates heading date in common wheat(Triticum aestivum L.)

RING finger E3 ligases play an important role in regulating plant growth and development by mediating substrate degradation.In this study,we identified TaGW2L,encoding a Grain width and weight2(GW2)-like RING finger E3 ligase,as a novel positive regulator of heading date in wheat(Triticum aestivum L.).TaGW2L exhibited high amino acid sequence similarities with TaGW2 homoeologs,particularly in the conserved RING finger domain.Expression analysis indicated that TaGW2L was constitutively expressed in various wheat tissues.TaGW2L showed transactivation activity in yeast and could interact with the ubiquitin-conjugating enzymes E2_(s).An in vitro ubiquitination assay verified that TaGW2L possessed a similar E3 ligase activity to TaGW2.Overexpression of the TaGW2L-7A homoeolog in wheat led to a significantly earlier heading date under both natural conditions and long-day conditions.Transcriptome analysis revealed that multiple known genes positively regulating wheat heading were significantly upregulated in the TaGW2L-7A-overexpression plants compared with the wild-type control.Together,our findings shed light on the role of TaGW2L in wheat heading date and provide potential applications of TaGW2L for the adaptation improvement of crops.

QTL for grain yield and yield parameters in winter wheat(Triticum aestivum L.)

Field trials with a set of 108 doubled haploid lines(DHs) derived from a cross between the Chinese winter wheat cvs.CA9613 and H1488 were run at Beijing(China).Phenotypic data were recorded for major agronomic yield traits,i.e.grain weight per ear,grain number per ear and thousand grain weight(Tgw) in two field trials at Beijing.Based on the phenotypic data and a genetic map comprising 168 SSR markers,an analysis of quantitative trait loci(QTL) was carried out for yield and yield parameters using the composite interval mapping(CIM) approach.A total of 14 QTL were detected for these traits across two environments.Four of these QTL located on chromosomes 1A and 2B,respectively,exhibited pleiotropic effects.Loci showing pleiotropic effects will be very useful for understanding the homeologous relationships of QTL and designing an appropriate marker-assisted selection programme by multi-trait selection in order to accumulate("pyramide") favorable alleles at different loci.

Genetic Diversity of Recurrent Selection Populations with Ms2 Gene Assessed by Gliadins in Common Wheat (Triticum aestivum L.)

The male-sterile lines with Ms2 gene were highly evaluated in recurrent selection in wheat （Triticum aestivum L.）. Three populations C6 （population after six cycles of selection）, C7 （population after seven cycles of selection）, and C8 （population after eight cycles of selection） were constructed through recurrent selection with 12 parental materials （P）. Acid polyacrymide gel electrophoresis （A-PAGE） analysis was used to identify gliadin patterns and evaluate the genetic diversity in 12 parents and three populations. A total of 63 bands were identified, of which 17 polymorphic bands and 7 unique bands were present in populations and seven polymorphic bands and four unique bands were present in parents. The number of polymorphic and unique bands decreased gradually from C6 to C8, especially for to- and y-gliadins. The genetic distances in C6, C7, and C8 were calculated. The distributions of genetic distance were different in three recurrent selection populations. From C6 to C8, the genetic distance was 0.2687, 0.2652 and 0.1987, respectively. Statistically significant differences were detected between C7 and C8 with the T value of 37.9718. The result of cluster analysis based on genetic similarity matrix of three populations fitted well to those of principle coordinates analysis （PCoA）. Compared with 12 parents, almost all individuals of three populations are new genotypes. Most of the individuals from C6 and C7 could be divided into two groups, while most individuals of C8 were in one cluster. In conclusion, the results indicated that the genetic diversity was decreased severely according to the information revealed by A-PAGE, although some variations could be created in the recurrent selection. It was necessary to introduce diverse germplasm based on the genetic database of recurrent population to maintain and improve the breeding efficiency in the further program.

Combining Phytate/Ca^(2+) Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat (Triticum aestivum L.) Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase （Rubisco） present within plant leaf tissues. In the present study, total proteins were extracted from wheat （Triticum aestivum L.） leaves by a conventional trichloroacetic acid （TCA）/acetone method and a protocol first developed in this work. Phytate/Ca2＋ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis （2-DE）. The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as （758±4） protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.

Comparison of Two MicroRNA Quantification Methods for Assaying MicroRNA Expression Profiles in Wheat(Triticum aestivum L.)

Two microRNA （miRNA） quantification methods, namely, poly（A） reverse transcription （RT）-quantitative real-time polymerase chain reaction （qPCR） and stem-loop RT-qPCR, have been developed for quantifying miRNA expression. In the present study, five miRNAs, including miR166, miR167, miR168, miR159, and miR396, with different sequence frequencies, were selected as targets to compare their expression profiles in five wheat tissues by applying the two methods and deep sequencing. The study aimed to determine a simple, reliable and high-throughput method for detecting miRNA expressions in wheat tissues. Results showed that the miRNA expression profiles determined by poly（A） RT-qPCR were more consistent with those obtained by deep sequencing. Further analysis indicated that the correlation coefficients of the data obtained by poly（A） RT-qPCR and deep sequencing （0.739, P≤0.01） were higher than those obtained by stem-loop RT-qPCR and deep sequencing （0.535, P≤0.01）. The protocol used for poly（A） RT-qPCR is simpler than that for stem-loop RT-qPCR. Thus, poly（A） RT-qPCR was a more suitable high-throughput assay for detecting miRNA expression profiles. To the best of our knowledge, this study was the first to compare these two miRNA quantification methods. We also provided useful information for quantifying miRNA in wheat or other plant species.

Expression pattern and function analyses of the MADS thranscription factor genes in wheat(Triticum aestivum L.) under phosphorusstarvation condition

supported by the National Natural Science Foundation of China （31201674 and 31371618）;the National Transgenic Major Program, China （2011ZX08008）