Xia Zhang ,Caihong Cui ,Yinguang Bao ,Honggang Wang ,Xingfeng Li ,*

a State Key Laboratory of Crop Biology,Shandong Agricultural University,Tai’an 271018,Shandong,China

b Tai’an Subcenter of the National Wheat Improvement Center,Agronomy College,Shandong Agricultural University,Tai’an 271018,Shandong,China

ABSTRACT Thinopyrum intermedium has been used as a resource for improving resistance to biotic and abiotic stresses and yield potential in common wheat.Wheat line SN304 was derived from a cross between common wheat cultivar Yannong 15 and Th.intermedium.Genomic in situ hybridization (GISH) produced no hybridization signal in SN304 using Th.intermedium genomic DNA as a probe,but fluorescence in situ hybridization (FISH) using oligonucleotides AFA-3,AFA-4,pAs1-1,pAs1-3,pAs1-4,pAs1-6,pSc119.2-1,and (GAA)10 as probes detected hybridization signals on chromosomes 2A,7A,2B,3B,6B,and 7B in SN304 that differed from Yannong 15.Results of specific markers also indicated that there were Th.intermedium chromatin introgressions on different chromosomes in SN304.In a hydroponic culture experiment,SN304 not only produced more biomass and higher stem and leaf dry weight but also accumulated more phosphorus than Yannong 15 under phosphorus-deficiency stress.Moreover,SN304 produced a lower pH and released more organic acids,especially oxalic acid,than Yannong 15,which suggests that SN304 exudates enabled more absorbance of P than Yannong 15 under comparable conditions.The results indicate that SN304 is a wheat-Th.intermedium introgression line with tolerance to phosphorus-deficiency stress.

Keywords:In situ hybridization Phosphorus deficiency Thinopyrum intermedium Triticum aestivum Wide crosses

1.Introduction

Thinopyrum intermedium(Host) Barkworth and D.R.Dewey(2n=6x=42) has many useful traits and is a valuable reservoir of genes for wheat improvement worldwide [1,2].Various studies on the genomic composition ofTh.intermediumundertaken for decades have proposed different genomic formulae;EeEeEbEbStSt and JJJsJsStSt are the most commonly accepted [3–7].Further progress in this aspect has occurred recently.The genomic composition of JrJrJvsJvsStSt was proposed using EST-SSR markers developed from the St,Eband Eegenome progenitors,cytogenetic analysis and array-based SNP genotyping of wheat-Th.intermediumintrogression lines[8,9].Th.intermediumreadily crosses with common wheat and has proven to be a valuable resource for improvement of resistance to diseases,tolerance to abiotic stresses,and quality-related traits [10].ManyTh.intermediumderivatives have been reported,including addition lines,substitution lines,translocation lines and cryptic translocation lines [11–17].Some potentially important disease resistance genes were introgressed into wheat fromTh.intermedium,includingLr38(leaf rust resistance),Sr44(stem rust resistance),Pm40andPm43(powdery mildew resistance),Bdv2andBdv3(Barley yellow dwarf virusresistance),andWsm1(Wheat steak mosaic virusresistance)[18–23].However,the transfer and utilization of resistance to abiotic stresses inTh.intermedium,such as tolerance to low phosphorous,have rarely been studied.

Phosphorus (P) is one of the most limiting macronutrients for crop productivity.P deficiency is a common phenomenon in agricultural soils worldwide.Although the total P in the soil can be high,it is often not present in an available form.Accumulated(surplus) P in agricultural soils is suggested to be sufficient to sustain crop yields worldwide for approximately 100 years.However,few unfertilized soils release phosphorus fast enough to meet the phosphorus requirements of crops [24].Plant species display different levels of adaptation to low phosphorus,and various physiological or morphological root characteristics can affect phosphorus acquisition [25–27].However,it is not clear how growing roots respond to low phosphorus availability.Although applications of phosphatic fertilizers make the soil a potentially enormous phosphorus pool,the efficiency of P fertilizer use is frequently low.Root exudates such as citrate and phytate contributed to improved phosphorus acquisition efficiency in tobacco (Nicotiana tabacumL.) when both exudates are produced in P-deficient soil [28].Furthermore,grain phosphorus and phytate contents are important for early growth and quality of wheat (Triticum aestivumL.),some grain traits including number of productive tillers,spikelets per spike,grain weight,harvest index and grain P concentration and uptake,have differences under P deficiency [29].So P deficiency stress could affect grain yield,and it plays a role in screening phosphorus tolerant genotypes in wheat improvement.

We have developed many octoploid amphiploid lines,addition lines,and substitution lines from wheat×Th.intermediumcrosses[30–32].Here,we report the development of wheat-Th.intermediumintrogression line SN304 with tolerance to phosphorus deficiency.Genomicin situhybridization (GISH),fluorescencein situhybridization (FISH) and molecular marker analysis were conducted to determine the genomic composition of SN304.

2.Materials and methods

2.1.Plant materials

Xiaoyan 54 (XY54,control) and Yannong 15 (YN15) are winter wheat cultivars with different degrees of P efficiency.XY54 has a large root biomass,and its strong acidification ability is the main mechanism that allows it to acquire more P in P-deficient calcareous soil [33].Line SN304 was selected from a BC2F7progeny of a cross between YN15 andTh.intermedium.

2.2.Genome in situ hybridization (GISH)

Fresh root tip cells (RTC) collected from germinating seeds were treated with 1.0 MPa nitrous oxide(N2O)for 2 h and then immersed in 90%glacial acetic acid.Slides of RTC were prepared using the procedure described by Cui et al.[34].Th.intermediumDNA was labeled with 488-5-dUTP by nick translation following the manufacturer’s instructions (Invitrogen,Loughborough,East Midlands,UK).Sheared genomic DNA of YN15 was used as blocking DNA.The hybridization mixture was prepared as described by He et al.[35].GISH signals were detected with the fluorescein-conjugated anti-digoxigenin antibody,and the slides were mounted with a thin layer of Vectrashield antifade solution containing 4′,6-diamidino-2-phenylindole (DAPI).Fluorescence signals were viewed and photographed with a fluorescence microscope(Olympus BX-61,Tokyo)equipped with a CCD(DSRi1,Nikon,Japan) camera.

2.3.Fluorescence in situ hybridization (FISH)

FISH was performed with eight probes,including AFA-3,AFA-4,pAs1-1,pAs1-3,pAs1-4,pAs1-6,pSc119.2-1,and (GAA)10[36].Oligonucleotides(synthesized by Sangon Biotech,Shanghai,China)pSc119.2-1 and (GAA)10were labeled with 5-FAM (5-carboxyfluorescein),and the others were labeled with 5-TAMRA(5-carboxytetramethylrhodamine).Probe labeling,hybridization,and signal detection were identical to the GISH protocol.

2.4.Molecular marker analyses

DNA was extracted from fresh leaves of YN15,SN304,andTh.intermediumusing the SDS-phenol method [37].Molecular markers including SSR,EST-SSR,and STS markers,covering all wheat chromosomes were obtained from the GrainGenes database(http://wheat.pw.usda.gov).We also used a specific primer pair that was synthesized based on the published pLeUCD2 DNA repeat sequence (PLe2),which can detectTh.intermediumchromatin in wheat derivatives.The primers 2P1(5′-ACAATCTGAAAATCTG GACA-3′)and 2P2(5′-TCATATTGAGACTCCTATAA-3′)were derived from the repetitive DNA sequence pLeU-CD2[38].Polymerase chain reaction(PCR)was performed in a thermal cycler(Bio-Rad 9600,Hercules,CA,USA)in a reaction mixture(10μL)containing 1μL template DNA(100 ngμL-1of concentration),2μL primer,2μL ddH2O and 5μL 2×Power Taq PCR MasterMix(BioTeke Corporation,Wuxi,Jiangsu,China).Amplification of DNA was performed using a touchdown PCR protocol as detailed by Hao et al.[39].The amplified products were separated in 8% nondenaturing polyacrylamide gels and 1%agarose electrophoresis,then developed and photographed.

2.5.Hydroponics and measurement of phosphate efficiency parameters

A hydroponics experiment consisting of three wheat genotypes(SN304,XY54,and YN15),three levels of P (10,50,and 200μmol L-1,referenced as P10,P50,and P200)and three replicates was established.Seeds of three wheat genotypes were germinated uniformly on plastic nets held by plastic pots.After one week,the seedlings were transferred into glass bottles(containing 400 mL nutrient solution)wrapped in tin foil.In addition to different phosphorus concentrations the nutrient solutions contained 1 mmol L-1NH4NO3,0.1 mmol L-1Fe-EDTA,1 mmol L-1MgSO4,0.7 mmol L-1K2SO4,1.5 mmol L-1CaCl2,0.1×10-3mmol L-1H3BO4,0.5 × 10-3mmol L-1(NH4)6Mo7O24,0.5 × 10-4mmol L-1CuSO4,and 0.1 × 10-3mmol L-1MnSO4.The pH of the solutions was adjusted to 5.8 using 1 mol L-1HCL and 1 mol L-1NaOH,and all the solutions included the microbial inhibitor thymol at 0.01 g L-1[40].The culture bottles were randomly placed in a glasshouse with a 14 h light and 10 h darkness photoperiod and temperature range of 15–25 °C.Their positions were randomly rotated weekly,and an air pump supplied air to the nutrient solutions on a continuous basis during the experiments.

The shoots and roots were harvested after growth in a glasshouse for 25 days.The roots were scanned by the LA-S plant root analytical system (Hangzhou Wanshen Detection Technology Co.,Ltd.,Hangzhou,China) to calculate root total length (TRL),surface area (RSA),volume (RV),and root-tip number (RTN).All roots (including the root mat) and plant tissues were oven-dried at 105 °C for 30 min and subsequently at 80°C.The plant samples were used to measure shoot and root dry weights and root:shoot ratio.The dried root and shoot samples were ground and digested with HNO3-HClO4(4:1) to determine total P contents using ICP optical emission (ICP-AES;Fisons-ARL Accuris,Ecublens,Switzerland).

The pH values of the culture solutions were measured using a pH meter (PHM62 standard pH meter,Copenhagen,Denmark)after 11,14,17 and 19 days of growth.At 15,20 and 25 days,5 mL samples of culture solution were taken from each of the three wheat genotypes and analyzed for root exudates using highperformance liquid chromatography (HPLC) (Agilent 1200,Waldbronn,Germany)with a 0.45 μm water membrane filter.The HPLC conditions were as follows:RP-ODS-C18 analytical column at 25 °C,test wavelength 214 nm,and mobile phase using H3PO4solution(pH 2.2)at a speed of 1.0 mL min-1[41].The types of root exudates of wheat were determined by comparison with the liquid chromatograms of the standard,and the root exudate concentrations were calculated according to standard curves.Oxalic acid,tartaric acid,malic acid,and citric acid standards of chromatographic purity were purchased from Sigma.

2.6.Statistical analysis

Data were analyzed using SPSS 20.0 (SPSS,Inc.,Chicago,IL,USA),and all analyses were performed in triplicate.Significant differences between means were tested by the least significant difference (LSD) atP<0.05.

3.Results

3.1.Cytogenetic characterization of SN304

Root tip chromosome analysis showed that the chromosome number of SN304 was 2n=42.GISH analysis was performed to examine the chromosome composition of SN304.No obvious fluorescence hybridization signal was detected(Fig.1a)indicating that SN304 either had no chromatin fromTh.intermediumor that such segments,if any,were too small to be detected by GISH.

Multiple probes,including oligonucleotides AFA-3,AFA-4,pAs1-1,pAs1-3,pAs1-4,pAs1-6,pSc119.2-1,and (GAA)10,were used as probes to analyze the karyotypes of SN304 and YN15 by FISH (Fig.1b and c).Chromosomes 2A,7A,2B,3B,6B,and 7B of SN304 differed from those of YN15 (Fig.1d).A specific green hybridization signal on chromosome 2AL and one stronger green hybridization signal on 7AS were observed in SN304,but not in YN15.Compared to YN15,a green hybridization signal was missing in the subterminal region of chromosome 2BL in SN304.The signals labeled on chromosomes 3BS and 6BL were enhanced profoundly,and a red hybridization signal was missing on 3BL,whereas two green signals were missing on 6BL in SN304 compared to YN15.On chromosome 7BL,there was a green signal in SN304 instead of a red signal in YN15 in the same place,and a green signal was missing on the subterminal region of 7BL in SN304.These FISH results indicate that various structural changes had occurred in SN304 chromosomes,these might cause by the introgressions of chromatin fromTh.intermediumor the wheat chromosome rearrangement during the process of selfing.

3.2.Molecular characterization of the putative introgression line SN304

Molecular markers were used to determine the homoeologous relationships of the alien chromosomes.To confirm whether the introgressed chromatin was derived fromTh.intermedium,SSR,EST-SSR,and STS markers were used to detect SN304,YN15,andTh.intermediumsequences.Among the 2584 molecular markers tested,68 markers amplified unique bands fromTh.intermediumin SN304.One specific primer pair derived fromTh.intermediumwas also used in the analysis[38].The results showed that primer 2P1-2P2 amplified a DNA fragment specific toTh.intermedium.This indicated that SN304 had a specific fragment ofTh.intermedium(Fig.2).Furthermore,the physical locations of a few polymorphic markers corresponded to chromosomal regions where there were differing FISH signals between SN304 and YN15 (Fig.S1).

3.3.Characteristics of SN304 and YN15 under different phosphorus levels

Biomass production and other traits were assessed after 25 days of growth in hydroponic culture under three P concentrations(Table S1;Fig.3).With increased P concentration,shoot dry weight(SDW)of the three genotypes at the P50 level were higher than the P10 and P200 levels(P<0.05).This indicated that both the lower and higher P concentrations inhibited plant growth.Line SN304 produced 2.84%,16.7%,and 17.7%more SDWthan YN15 at all three P concentrations,respectively(Table S1;Fig.3a).The SDW of SN304 was also higher than that of the P-efficient genotype XY54 at different P levels(Table S1;Fig.3a).However,SN304 had lower root dry weight(RDW)than the other two wheat genotypes at the P10 and P50 levels(Table S1;Fig.3b).Thus,SN304 also had the lowest root:shoot ratio(Table S1;Fig.3d).Plant dry weight(PDW)of SN304 was higher than that of YN15(13.8%and 15.3%at the P50 and P200 levels,respectively)(Table S1;Fig.3c).These results indicated that SN304 developed more biomass than YN15,and was more tolerant to phosphorous deficiency.The root:shoot ratios of the three wheat genotypes decreased with increasing phosphorus concentration.Line SN304 had the least root mass but produced more shoot dry weight than the other two genotypes.These results indicated that root growth was not the most important factor for plant growth.

Fig.1.GISH and FISH analyses of SN304 and YN15.GISH of a mitotic metaphase cell of SN304(a)using 488-5-dUTP labeled genomic DNA of Th.intermedium as probe and the genomic DNA of YN15 for blocking.FISH of the mitotic metaphase cells of SN304(b)and YN15(c).The red color indicates signals by AFA-3,AFA-4,pAs1-1,pAs1-3,pAs1-4,and pAs1-6,and the green color shows signals by pSc119.2-1 and(GAA)10.Karyotypes of SN304(left)and YN15(right)based on FISH(d).The yellow arrows indicate regions with differing signals between SN304 and YN15.

Fig.2.Amplified with genome-specific primers 2P1-2P2.M,marker(2 kb ladder);1,Th.intermedium;2,SN304;3,YN15.

3.4.Comparison of phosphorus uptake of wheat genotypes at different phosphorus concentrations

Analysis of plant P uptake was evaluated by LSD.Significant differences (P<0.05) in plant P uptake were observed for all three wheat genotypes subjected to different treatments.As the phosphorus concentration increased from P10 to P50,phosphorus uptake increased by 3.42,2.31,and 2.29 fold,respectively.Similarly,phosphorus uptake increased by 1.07,1.25,and 1.37 fold when phosphorus concentration increased from P10 to P50,respectively.Besides,plant P uptake by SN304 improved by 6.0%,5.7%,and 18.9% compared to YN15,respectively.The shoot P uptake of SN304 was higher than that of YN15 at 10.2%,6.7%,and 20.2%.However,root P uptake of SN304 was less than that of YN15,specifically,lower by 31.6%,11.7%,and 4.4%.In the P10 treatment,SN304 tended to have higher shoot P uptake than XY54,but SN304 had less shoot P uptake than XY54 in the P50 and P200 treatments.Because SN304 had higher plant P uptake and lower root dry weight than YN15 in all P treatments,SN304 had a higher P uptake efficiency than YN15,with increases of 38.9%,12.8% and 21.0% (Table S1;Fig.3e–h).

3.5.Physiological and biochemical reactions of low phosphorus tolerance under hydroponic conditions

3.5.1.Rhizospheric pH

We examined the rhizospheric acidification of the three wheat genotypes as low pH likely promotes the dissolution of phosphorus.Multiple comparisons were analyzed by LSD to identify significant differences atP<0.05.The initial pH of the nutrient solution was 5.8,and the pH values in the media for all three genotypes decreased significantly at all P levels after 11 days of growth(Fig.4).The rhizosphere pH of SN304 decreased more in P10 than in P50 and P200,indicating that there was a stronger ability to acidify the rhizospheric environment at low phosphorus.Furthermore,with all three P treatments,SN304 always produced a lower pH value than YN15 or XY54.Thus,the root system of SN304 was more capable of secreting acid or releasing proton than that of YN15.

3.5.2.Oxalic acid secretion

The composition of root exudates was determined by HPLC after 15,20 and 25 days of growth(Figs.5 and 6).The time and value of the first and the second peaks were consistent with the standard samples of oxalic acid and tartaric acid,respectively.According to the standard curve and formula/function,we obtained the concentration of root exudates (Fig.6).Low phosphorus stress promoted the secretion of organic acids,which is also an important mechanism of adapting to low-P stress.In the P10 treatment,the oxalic acid exudates of all wheat genotypes were higher than for the P50 and P200 genotypes,indicating that low phosphorus induced wheat to secrete more oxalic acid.In addition,SN304 exuded more oxalic acid during the three test periods than did YN15 and XY54 in all three P treatments but especially at the P10 level(Fig.6).In conclusion,SN304 had a comparatively stronger ability to secrete oxalic acid,thereby allowing dissolution of more phosphorus.

Fig.3.Biomass and P uptake of three wheat genotypes at different phosphorus levels.P uptake efficiency=P uptake in plant/root dry weight Means with different lowercase letters are significantly different (LSD, P <0.05).P10,P50,and P200 refer to P concentrations of 10,50,and 200 μmol L-1.

Fig.4.Variation in pH values in the growth media of three wheat genotypes.

Fig.5.HPLC model chromatogram for standard sample.

Fig.6.Oxalate secretion from different wheat genotypes.Culture times:(a)15 days;(b)20 days;(c)25 days.P10,P50,and P200 refer to applied P concentrations of 10,50,and 200 μmol L-1.Means with different lowercase letters are significantly different (LSD, P <0.05).

Although SN304 had a smaller root system and lower acid phosphatase the results showed that it generated the lowest pH,secreted higher levels of oxalic acid,produced more biomass and had higher P uptake efficiency than YN15 under conditions of phosphorus deficiency.Therefore,introgression line SN304 is more tolerant to phosphorus deficiency.

4.Discussion

Wild relatives of common wheat have been widely used as sources of potentially useful traits for wheat improvement.Th.intermediumis a source of quality-related traits,resistance to diseases and pests,and tolerance to abiotic stresses[10,42].A number of genes have been transferred into wheat in the form of wheat-Th.intermediumpartial amphiploids,chromosome addition,substitution,and translocation lines,including some putative cryptic translocations[43,44].However,no report is available about tolerance to phosphate deficiency genes introduced fromTh.intermedium.

GISH has proven to be an effective method to identify alien chromosomes.However,it seems unable to detect small segments of alien introgressions[45–47].Here,we provide evidence that SN304 contains introgressed chromatin regions fromTh.intermediumby FISH and molecular markers;however,these introgressions were apparently too small to be detected by the GISH methods used in the study.Despite the difference in the FISHbanding patterns of chromosomes between SN304 and YN15,we cannot rule out the possibility of chromosome recombination and structural rearrangement within wheat itself during distant hybridization,or caused by outcrossing during the intervening generations.

Wheat-Th.intermediumintrogression line SN304 was tolerant to phosphorus deficiency.Positive linkages were observed between QTL for TN(tiller number),SDW(shoot dry weight)and SPU(shoot P uptake)on chromosomes 4B,5A,5D and 7B[48].We also located a major QTL for P utilization efficiency(PUE)flanked by markersXgdm68andXgwm156on chromosome 5A.Furthermore,a major QTL for grain phosphorus use efficiency on chromosome 2B and several QTL for root characteristics and phosphorus deficiency stress response were detected on chromosome 7B[49].FISH results indicated that SN304 might contain introgressed chromatin regions on chromosomes 2A,7A,2B,3B,6B,and 7B.However,the relationship of phosphorus use efficiency of SN304 and the putative introgressed alien chromatin is unclear and requires more work for confirmation.

Root exudates play an important role in improving soil phosphorus availability,and most of the exudates are organic acids,including citric acid,oxalic acid,tartaric acid,and malic acid.The present results showed that the root exudates of SN304 under hydroponic conditions consisted mainly of oxalic acid.It was concluded that organic acids cause an increase in P mobilization and P uptake in wheat,but this response is highly specific to each organic acid[50].Also,it was proposed that the exudation of organic acids and organic anions increases the resin-P content,suggesting that organic anions rather than rhizosphere acidification are more important for P solubility and availability for plant growth[51].There is evidence that low-phosphorus growth conditions in rice significantly increased the number of productive tillers,root biomass,root-shoot ratio,root bleeding rate,and root acid phosphatase activity.Therefore,tolerance of low-phosphorus levels was closely associated with root growth[52].Under low-P conditions,SN304 had higher levels of oxalic acid exudates and greater absorption capacity for insoluble inorganic phosphorus than YN15,showing that SN304 possessed a stronger ability to activate insoluble forms of P.Nevertheless,the correlation between high oxalic acid exudate levels and P-deficiency tolerance is worthy of further study.We inferred that SN304 adapted to phosphorus insufficiency due to its stronger root system than YN15,which is likely related to alien chromatin introgressed fromTh.intermedium.

CRediT authorship contribution statement

Xia Zhangperformed experiments and prepared the manuscript.Caihong CuiandYinguang Baoperformed parts of experiments.Honggang Wangperformed parts of experiments and revised the manuscript.Xingfeng Lidesigned the experimental program and prepared the manuscript.All authors reviewed and approved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Key Research and Development Program of China(2016YFD0102000),National Natural Science Foundation of China(31671675),and Natural Science Foundation of Shandong Province (ZR2015CM034 and ZR2016CM30).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.08.014.