Transcriptomic and metabolomic analysis provides insights into lignin biosynthesis and accumulation and differences in lodging resistance in hybrid wheat

The use of hybrid wheat is one way to improve the yield in the future.However,greater plant heights increase lodging risk to some extent.In this study,two hybrid combinations with differences in lodging resistance were used to analyze the stem-related traits during the filling stage,and to investigate the mechanism of the difference in lodging resistance by analyzing lignin synthesis of the basal second internode(BSI).The stem-related traits such as the breaking strength,stem pole substantial degree(SPSD),and rind penetration strength(RPS),as well as the lignin content of the lodging-resistant combination(LRC),were significantly higher than those of the lodgingsensitive combination(LSC).The phenylpropanoid biosynthesis pathway was significantly and simultaneously enriched according to the transcriptomics and metabolomics analysis at the later filling stage.A total of 35 critical regulatory genes involved in the phenylpropanoid pathway were identified.Moreover,42%of the identified genes were significantly and differentially expressed at the later grain-filling stage between the two combinations,among which more than 80%were strongly up-regulated at that stage in the LRC compared with LSC.On the contrary,the LRC displayed lower contents of lignin intermediate metabolites than the LSC.These results suggested that the key to the lodging resistance formation of LRC is largely the higher lignin synthesis at the later grain-filling stage.Finally,breeding strategies for synergistically improving plant height and lodging resistance of hybrid wheat were put forward by comparing the LRC with the conventional wheat applied in large areas.

Breeding and Cultivation Technique of Recessive Genic Male Sterility Hybrid Wheat New Variety Xinan 112

Xinan 112 is the hybrid combination（11S12） by wheat recessive genic male sterility line 2011Z1 （08L5070） and restoring line K152-2, particpating in the regional test of 2011-2012, 2012-2013 and production test of 2013-2014 in Chongqing. The results showed that 3 years average yield was 4 167.5 kg/hm2, which was increased 10.5% compared with CK Yumai 7, 1 000-grain weight was 45.8 g, which was 1.7 g heavier than the control; grain number per ear was 39.3, more than 1.6 grains compared with CK. The results of 2 years quality determination were： bulk density of 811 g/L, falling number of 353 s, crude protein of 15.15%, wet gluten of 31.2%, water adsorption of 62.9 ml/100 g, formation time of 5.5 min, stable time of 6.5 min, softening degree of 90 F.U., and powder quality coefficient of 96 mm. Therefore, Xinan 112 is a high yield and high protein gluten wheat variety and is suitable for cultivated in Chongqing and climate contion similar areas.

Grain Sink Strength Related to Carbon Staying in the Leaves of Hybrid Wheat XN901

XN901 is a K-type three-line hybrid wheat with a high yield potential, and its leaves and stem remaining green during grain maturation, suggesting much assimilate stay in leaves and stem. The grain water content, grain volume, carbohydrate content, and enzyme activity of sucose metabolism in the grain, as well as source-sink relationship were studied in order to investigate the physiological reason of the assimilate remaining in leaves and stem at the late stage. The results showed that the hybrid grains had more water and soluble sugar, higher activities of acid invertase and sucrose synthase at the early stage that led to a faster expansion growth, greater grain volume and faster starch synthesis at the early to mid stage of grain development. Also it had a longer period for actively filling. As a result, the grain weight and yield of the hybrid were increased by 14 and 15% respectively compared to that of Shaan 229. Additionally, the biomass of XN901 was 41.7% more than that of control, but its harvest index was 9% lower than Shaan 229. However, its lower activity of sucrose synthase indicated a lower sink activity at the late stage, resulting in a slow rate of filling and starch synthesis. Also, the hybrid wheat XN901 had a large source-sink ratio. It is the main reason for much assimilate remaining in the straw at the late stage and lower harvest index. Strengthening the sink activity and raising the harvest index should be the key means of improving the yield of hybrid wheat.

Research and application of hybrid wheat in China

Hybrid wheat is recognized as a preferred approach to improve wheat yield,and it will be a competition focus in high-tech seed industry in the future. We have made a breakthrough for the first time in creation of two-line hybrid wheat system,which reaches the world leading level in wheat research and has laid an important foundation for the future direction of the world wheat research. Similar to hybrid rice,the innovation of two-line hybrid wheat system is another achievement in science and technology. The application of hybrid wheat in China will greatly increase the food production,and make a great significance to food production and food security. This paper introduces the development process and major breakthrough of hybrid wheat in China,and the main bottleneck and countermeasures in the application of hybrid wheat.

Yield-related agronomic traits evaluation for hybrid wheat and relations of ethylene and polyamines biosynthesis to filling at the mid-grain filling stage

Because of the yield increase of 3.5-15%compared to conventional wheat,hybrid wheat is considered to be one of the main ways to greatly improve the wheat yield in the future.In this study,we performed a principal component analysis(PCA)on two-line hybrids wheat and their parents using the grain weight(GW),the length of spike(LS),the kernel number of spike(KSN),and spike number(SPN)as variables.The results showed that the variables could be classifed into three main factors,the weight factor(factor 1),the quantity factor 1(factor 2)and the quantity factor 2(factor 3),which accounted for 37.1,22.6 and 18.5%,respectively of the total variance in different agronomic variables,suggesting that the GW is an important indicator for evaluating hybrid combinations,and the grain weight of restorer line(RGW)could be used as a reference for parents selection.The hybrid combination with a higher score in factor 1 direction and larger mid-parent heterosis(MPH)of the GW and its parents were used to carry out the analysis of grain fling,1-aminocylopropane-1-carboxylicacid(ACC)and polyamine synthesis related genes.The results suggested that the GW of superior grain was significantly higher than that of inferior grains in BS1453xJS1(H)and its parents.Both grain types showed a weight of H between BS 1453(M)and JS1(R),and a larger MPH,which may be caused by their differences in the active fling stage and the grain fling rate.The ADP-glucose pyrophosphorylase(AGPase),granule bound starch synthase I(GBSS/),starch synthaseⅡ(SSS),and starch branching enzyme-Ⅰ(SBE-1)expression levels of H were intermediated between M and R,which might be closely related to MPH formation of the GW.Compared with R and H,the GW of M at maturity was the lowest.The expression levels of spermidine synthase 2(Spd2),ornithine decarboxylase(ODC)and S adenosylmethionine decarboxylase(SAMDC)had significantly positive correlations with the grain flig rate(1=0.77,0.51,0.59"),suggesting their major roles in the grain flling and heterosis formation.These provide a theoretical basis for improving the GW of photo-thermo-sensitive male sterile lines(PTSMSL)by changing the endogenous polyamine synthesis in commercial applications.

Effects of Different Row Ratios of Male and Female Parents and Application Amount of Nitrogen Fertilizer on Hybrid Wheat Seed Production and Its Component Factors

In order to screen suitable high hybrid wheat seed production technology,the split-plot experiment design was adopted and study was carried out about the effects of the different row ratios of male and female parents and application amount of nitrogen fertilizer on hybrid wheat seed production and its component factors. The results showed that the seed production increased with the increase in the number of female parent row. When the row ratio of male and female parents was 2 ∶ 6,the seed production was 3 683. 8 kg/ha; when the application amount of nitrogen fertilization was 50 kg/ha,the seed production was 3 649. 4 kg/ha; the interaction between the row ratio of male and female parents and the application amount of nitrogen fertilizer indicated that when the row ratio of male and female parents was 2∶ 6 and the application amount of nitrogen fertilizer was 300 kg/ha,the seed production reached the highest( 4160. 6 kg/ha). The row ratio of male and female parents and application amount of nitrogen fertilizer had significant effect on the component factors of seed production,including the number of grains per spike,spike weight and setting percentage. When the row ratio of male and female parents was 2∶ 5,the number of grains per spike,spike weight and setting percentage were the highest at 26. 7 grains,1. 12 g,and 62. 6% respectively; when application amount of nitrogen fertilizer was 450 kg/ha,the number of grains per spike,spike weight and setting percentage were the highest at 26. 0 grains,1. 08 g,and59. 2% respectively; the interaction of row ratio of male and female parents and application amount of nitrogen fertilizer had significant effect on the number of grains per spike,spike weight and setting percentage; when the row ratio of male and female parents was 2∶ 5 and the application amount of nitrogen fertilizer was 300 kg/ha,the number of grains per spike,spike weight and setting percentage were the highest at 29. 6grains,1. 24 g,and 71. 6% respectively. The number of grains per spike is the largest component factor for seed production. Increasing the number of grains per spike can increase the seed production. According to the effects of row ratio of male and female parents and application amount of nitrogen fertilizer on the component factors of seed production,the optimal condition was 2∶ 5-2∶ 6 for row ratio of male and female parents and 300-450 kg/ha for application amount of nitrogen fertilizer.

Influences of Root Zone Restriction and Nitrogen Nutrition on Hybrid Wheat Growth

To study the physiological effects of small root zonel plants of a hybrid wheat variety (Triticum aestivnmL.cv. Meiyou 4) were grown in small pots (1 litre) or large pots (8 litre) with 1ow nitrogen (50 mg kg-1 soil)and high nitrogen (200 mg kg-1 soil). Restricting root zone decreased dry weight of plants at the stages ofstem elongation and flowering, compared to those of control plants grown in the large pots (P<0.01). Sprayingof 6-benzylaminopurine (50 μmol L-1) increased dry weight of plants and chlorop hyll concentration in leaves.Restriction of root zone decreased the concentrations of total nitrogen, chlorophyll and soluble protein in thefiag leaf and acce1erated senescence of the leaves. Supply of high nitrogen delayed senescence of the fiag leafThe results suggested that the shortage of nutrients, especially nitrogen deficiency was the primary reasonfor the decreased growth of plant in the treatment of root zone restriction.

The Growth Analysis in the Kernel Filling Process of K-Type Hybrid 901 and Its Parent Wheat

Using Matlab software, the grain filling process of hybrid wheat 901 and its parents was fitted by Richards equation W = A/（1 ＋ Be^-kt in order to study the characteristics of grain filling of the hybrid. The active grain growth period of the hybrid was 6 d longer than that of Shaan 229, and its final grain weight （43.7 g/1 000 grains） was higher than that of Shaan229 （36.3 g/1 000 grains）. N values of 901 and R205 were both less than 1, and their grain growth was faster in the early filling stage, while slower in the middle-late stage. N value of Shaan 229 was 〉1~ and its growth was slower in the early stage and faster in the middle stage. The period of early stage of 901 was shorter and of middle-late stage was longer. The situation of Shaan 229 was totally reversed. For parents, the father plant R205 was similar to hybrid wheat 901, whereas its mother plant K3314A similar to Shaan 229. It has been found that Richards equation was more suitable for fitting the grain filling process of wheat than Logistic equation.

The contribution of distant hybridization with decaploid Agropyron elongatum to wheat improvement in China

Wheat is a staple food crop in the world as well as in China. Because of the progress of wheat breeding and other agricultural ＂sci-technologies, the wheat grain yield per unit area has increased more than five folds from 1952 to 2006 in China. The first part of this article briefly reviews the history of wheat breeding in China. Second, the establishment of ＂Triticum aestivum-Agropyron＂ distant hybridization system and its contribution to wheat production and breeding in China are summarized. Finally, the future challenges of wheat breeding are discussed, which include how to increase the utilization efficiencies of water, soil nutrient and light energy through breeding. As an example, our research progress on how to increase light use efficiency in wheat through breeding is introduced and discussed.

Studies on the Utilization Potentiality of the Nucleo-Cytoplasmic Hybrid in Wheat

A series of comparative studies was carried out on the genetic effects of 25 alien cytoplasms of wheat on the growth potential, heading stage, fertility, resistance against diseases, important agronomic traits and its heterosis of 125 nuclei-cytoplasmic hybrids of wheat. The results indicated that there were clearly effects of alien cytoplasms on some characteristics, but the nucleus still exerted main effect on other characteristics. The effect of interactions between nucleus and cytoplasm was comparative obvious in some combination. Consequently, when we utilize the effects of alien cytoplasms, we should pay full attention to the facts such as the characteristic to be improved, the effects of cytoplasm, nucleus, the nucleus-cytoplasm interactions on that characteristics. From the preliminary studies, we believed that the cytoplasmic types of M0, S1, Sv, D2, D and B, and the nucleo-cytoplasmic hybrids of (Ae. sharonensis)-B174, (Ae. squarrosa)-352-35 (Ae. cylindrica)-352-35, (Ae. cylindria)-E EN-1, (Ae. cylindrica)-NPFP, and (Ae. speltoides)-352-35 would have some utilization potentiality in cultivar improvement.

Synthesis of a Wheat/Maize Hybrid CENH3 Gene, the Genetic Transformation of Wheat, Its Chromosomal Localization and Effects on Chromosome Behaviors in Wheat/Maize Somatic Hybrids

Centromere-specific histone H3 (CENH3) replaces the canonical histone H3 in nucleosomes of functional centromeres, and plays important roles in faithful chromosome segregation during cell division. CENH3 is also important in the recognition of alien centromeres and determines the accommodation or elimination of alien chromosomes in interspecific or intergenic hybridization. In this study, a maize full length CENH3 with a yellow fluorescent protein (YFP) tag at C-terminus (ZmCENH3-YFP) and a synthetic hybrid wmCENH3 with the N-terminus from wheat CENH3 and the histone fold domain (HFD) from maize tagged with a red fluorescent protein (RFP) at the C-terminus (wmCENH3-RFP) were transformed to wheat by biolistics transformation. Transgenic wheat plants with both ZmCNEH3-YFP and wmCENH3-RFP genes were identified by PCR. The expression of ZmCENH3-YFP was not observed, while the expression of wmCENH3-RFP could be detected by RT-PCR, direct fluorescence microscopy, and immunostaining with anti-RFP antibody. The expressed wmCENH3-RFP was localized to nuclei as dotted patterns, indicating its targeting to wheat centromeres. Somatic hybridization was performed between wmCENH3-RFP transgenic wheat and transgenic maize that expressed a ZmCENH3-YFP gene to investigate chromosome behaviors in somatic hybrids. Cytological and FISH analyses of somatic hybrid cells showed the formation of micronuclei and lagging chromatin in both somatic hybridizations with or without the wmCENH3-RFP transgene, indicating that ectopically expressed wmCENH3 could not overcome chromosome elimination in wheat/maize somatic hybrids. Immunostaining of wmCENH3-RFP and ZmCENH3-YFP in early stage somatic hybrid cells indicated that both wmCENH3-RFP and ZmCENH3-YFP proteins were expressed, but their binding patterns changed from the commonly observed dotted patterns to diffused ones, suggesting that the inactivation of CENH3 might be a factor for chromosome elimination in wheat/maize somatic hybridization.

Programmed cell death, antioxidant response and oxidative stress in wheat f lag leaves induced by chemical hybridization agent SQ-1

Male sterility induced by a chemical hybridization agent （CHA） is an important tool for utilizing crop heterosis. Leaves, especially the flag leaves, as CHA initial recipients play a decisive role in inducing male sterility. To investigate effects of different treatment times of CHA-SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in thistudy. CHA induced programmed cell death （PCD） as shown in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling （TUNEL） and DNA laddering analysis. In the early phase, CHA-SQ-1 trig- gered organelle changes arid PCD in wheat leaves accompanied by excess production of reactive oxygen species （O2- and H202） and down-regulation of the activities of superoxide dismutase （SOD）, catalase （CAT） and guaiacol peroxidase （POD）. Meanwhile, leaf cell DNAs showed ladder-like patterns on agarose gel, indicating that CHA-SQ-1 led to the activation of the responsible endonuclease. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-l-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative/antioxidative balance and ultimately developed into mature green leaves. These results provide explanation of the relations between PCD and anther abortion and practical application of CHA for hybrid breeding.

Comparative Proteomic Analysis of Wheat (Triticum aestivum L.) Hybrid Necrosis

Hybrid necrosis is the gradual premature death of leaves or plants in certain Fj hybrids of wheat （Triticum aestivum L.）. Comparison of protein expression in necrotic and normal wheat leaves showed that the abundance of 33 proteins was changed significantly, and 24 of these proteins were identified. These proteins were involved in plant growth and development, antioxidation, photosynthesis and carbon assimilation, amino acid and protein biosynthesis, cytological signal transduction, DNA and RNA modification, protein transport, folding and assembly according to their functions. The down-regulation of uroporphyrinogen decarboxylase and the up-regulation of lipoxygenases in necrotic leaves may be related to the oxidative stress in the necrotic cells. The heat shock proteins may play the cytoprotective role. The differential expression of photosynthesis and carbon assimilation related proteins indicated chlorophyll biosynthesis and chloroplast development were inhibited and might finally cause the gradual chlorosis and cell death in necrotic leaves. The results of this study give a comprehensive picture of the post-transcriptional response to necrosis in hybrid wheat leaves and serve as a platform for further characterization of gene function and regulation in wheat hybrid necrosis.

An Alternative Analysis on Nilsson-Ehle’s Hybridization Experiment in Wheat —Theory of Dual Multiple Factors and Three Normal Distributions on Quantitative Inheritance (Continuation)

In this paper, an analysis on Nilsson-Ehle’s hybridization experiment in wheat was done by means of self-fertilization method along with the pericarp inheritance. It showed that because seeds of wheat were coated with 2n tissues of mother’s body, the grain colors were determined by mother’s genotype. The color of the F1 grains in this experiment was old red. The phenotypes of the F2 grains were uniformly medium red and did not segregate. Grains in the F3 generation segregated in the ratio 6 red (varying intensities of red) to 1 white with a definite probability respectively. This is the consequence of additive effect of 3 gene pairs and belongs to discrete distribution rather than continuous distribution. Therefore, the multiple-gene hypothesis based on this experiment cannot solve the continuous variations in inheritance of quantitative characters.

Breaking wheat yield barriers requires integrated efforts in developing countries

Most yield progress obtained through the so called ＂Green Revolution＂, particularly in the irrigated areas of Asia, has reached a limit, and major resistance genes are quickly overcome by the appearance of new strains of disease causing organisms.New plant stresses due to a changing environment are difficult to breed for as quickly as the changes occur.There is consequently a continual need for new research programs and breeding strategies aimed at improving yield potential, abiotic stress tolerance and resistance to new, major pests and diseases.Recent advances in plant breeding encompass novel methods of expanding genetic variability and selecting for recombinants, including the development of synthetic hexaploid, hybrid and transgenic wheats.In addition, the use of molecular approaches such as quantitative trait locus（QTL） and association mapping may increase the possibility of directly selecting positive chromosomal regions linked with natural variation for grain yield and stress resistance.The present article reviews the potential contribution of these new approaches and tools to the improvement of wheat yield in farmer＇s fields, with a special emphasis on the Asian countries, which are major wheat producers, and contain the highest concentration of resource-poor wheat farmers.

Filtering for SNPs with high selective constraint augments mid-parent heterosis predictions in wheat(Triticum aestivum L.)

To extend the contemporary understanding into the grain yield heterosis of wheat, the current study investigated the contribution of deleterious alleles in shaping mid-parent heterosis(MPH). These alleles occur at low frequency in the genome and are often missed by automated genotyping platforms like SNP arrays. The deleterious alleles herein were detected using a quantitative measurement of evolutionary conservation based on the phylogeny of wheat and investigations were made to:(1) assess the benefit of including deleterious alleles into MPH prediction models and(2) understand the genetic underpinnings of deleterious SNPs for grain yield MPH using contrasting crosses viz. elite × elite(Exp. 1) and elite × plant genetic resources(PGR;Exp. 2). In our study, we found a lower allele frequency of moderately deleterious alleles in elites compared to PGRs. This highlights the role of purifying selection for the development of elite wheat cultivars. It was shown that deleterious alleles are informative for MPH prediction models: modelling their additive-by-additive effects in Exp. 1 and dominance as well as associated digenic epistatic effects in Exp. 2 significantly boosts prediction accuracies of MPH. Furthermore,heterotic-quantitative trait loci's underlying MPH was investigated and their properties were contrasted in the two crosses. Conclusively, it was proposed that incomplete dominance of deleterious alleles contributes to grain yield heterosis in elite crosses(Exp. 1).

Development of marker Ms2 gene with a blue seed in durum and common wheat

In order to marker dominant nuclear gene Ms2 with a blue grain, a 4E disomic addition line ＇xiaoyanlanli＇（2n=44, AABBDD＋4EII） as the male parent to pollinate with male-sterile plants of durum wheat, controlled by a dominant nuclear gene Ms2, and a durum wheat line 89-2343 with Ms2 and blue seed marker on the same addition chromosome was developed. The genotype 89-2343 was crossed and backcrossed with a common wheat genotype 7739-3 to produce male fertile plants with blue seeds （MFP-BS）. To combine the blue seed marker, dwarf male-sterile plants carrying RhtlO and Ms2 were fertilized by pollen from selected MFP-BS. At last, the combination of blue seed marker, Ms2 and RhtlO was successfully produced. The segregation ratio of male sterility, seed color as well as chromosome configurations of the combinations suggested that the blue seed marker, Ms2 and RhtlO were located on the same chromosome. Cytological analysis indicated that the male sterile wheat line with a blue seed marker was 43 in chromosome number, with an additional chromosome. The transmission rate for blue seed male-sterile plants was 22.1% in common. In addition, the potential value for blue marker sterile lines in wheat breeding and hybrid production is discussed.

Gene Expression Profiles of Response to Water Stress at the Jointing Stage in Wheat

Drought is one of the most adverse environmental factors that impact on plant growth and reduce crop yields. To decipher the molecular mechanism of drought tolerance, we constructed a cDNA library using suppression subtractive hybridization （SSH） and dissected the gene expression profiles in seedling and jointing wheat plants after stress with PEG-6000. A total of 2 046 ESTs from the jointing stage library （J-Lib） were allocated to 961 contigs. Among the ESTs, 265 uni-genes in 12 categories were identified on the basis of sequence similarities and functional classifications. Most were known to be involved in protection, directly or indirectly, against water stress. To determine differences in gene expression profiles for water stress responses at the jointing and seedling stages, data from the J-Lib were compared with those from a 2- leaf seedling library （S-Lib） constructed by the same method. Significant differences were observed between the two libraries for function-known genes; signal transduction genes were far more active at jointing than at the seedling stage.

Comparing two approaches for introgression of germplasm from Aegilops tauschii into common wheat

Allelic diversity in the wild grass Aegilops tauschii is vastly greater than that in the D genome of common wheat(Triticum aestivum), of which Ae. tauschii is the source. Since the 1980 s,there have been numerous efforts to harness a much larger share of Ae. tauschii^ extensive and highly variable gene pool for wheat improvement. Those efforts have followed two distinct approaches: production of amphiploids, known as "synthetic hexaploids," between T. turgidum and Ae. tauschii,and direct hybridization between 丁. aestiuum and Ae. tauschii;both approaches then involve backcrossing to 丁. aestiuum. Both synthetic hexaploid production and direct hybridization have led to the transfer of numerous new genes into common wheat that confer improvements in many traits. This work has led to release of improved cultivars in China, the United States, and many other countries. Each approach to D-genome improvement has advantages and disadvantages. For example, production of synthetic hexaploids can incorporate useful germplasm from both T. turgidum and Ae.tauschii, thereby enhancing the A, B, and D genomes; on the other hand, direct hybridization rapidly restores the recurrent parent's A and B genomes and avoids incorporation of genes with adverse effects on threshability, hybrid necrosis, vernalization response, milling and baking quality, and other traits, which are often transferred when T. turgidum is used as a parent. Choice of method will depend in part on the type of wheat being developed and the target environment. However, more extensive use of the so-far underexploited direct hybridization approach is especially warranted.

Identification and genetic analysis of multiple P chromosomes of Agropyron cristatum in the background of common wheat

Agropyron cristatum, a wild relative of common wheat(Triticum aestivum L.), provides many desirable genetic resources for wheat improvement, such as tolerance to cold, drought, and disease. To transfer and utilize these desirable genes, in this study, two wheat-A. cristatum derivatives II-13 and II-23 were identified and analyzed. We found that the number of root tip cell chromosomes was 44 in both II-13 and II-23, but there were four and six P genome chromosomes in II-13 and II-23, respectively, based on genomic in situ hybridization(GISH). The chromosome configurations of II-13 and II-23 were both 2 n=22 II by the meiotic analysis of pollen mother cells(PMCs) at metaphase I, indicating that there were two and three pairs of P chromosomes in II-13 and II-23, respectively. Notably, wheat chromosome 7 D was absent in derivative line II-13 while II-23 lacked chromosomes 4 B and 7 A based on SSR analysis combining fluorescence in situ hybridization(FISH) analysis with p As1 and p Sc119.2 as probes. Chromosomes 2 P and 7 P were detected in both II-13 and II-23. Another pair of P genome chromosomes in II-23 was determined to be 4 P based on expressed-sequences tags-sequence tagged sites(EST-STS) markers specific to A. cristatum and FISH with probes p Ac TRT1 and p Acp CR2. Overall, these results suggest that II-13 was a 7 P(7 D) substitution line with one pair of additional 2 P chromosomes and II-23 was a multiple 4 P(4 B), 7 P(7 A) substitution line with one pair of additional 2 P chromosomes. Moreover, we obtained six alien disomic addition lines and five alien disomic substitution lines by backcrossing. These new materials will allow desirable genes from A. cristatum to be used in common wheat.