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

CHEN Hong-xin, HAN Hai-ming, Ll Qing-feng, , ZHANG Jin-peng, LU Yu-qing, YANG Xin-ming, Ll Xiu-quan, LlU Wei-hua, Ll Li-hui

1 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

2 College of Agronomy, Northwest A&F University, Yangling 712100, P.R.China

Abstract 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 2n=22II 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 7D was absent in derivative line II-13 while II-23 lacked chromosomes 4B and 7A based on SSR analysis combining fluorescence in situ hybridization (FISH)analysis with pAs1 and pSc119.2 as probes. Chromosomes 2P and 7P were detected in both II-13 and II-23. Another pair of P genome chromosomes in II-23 was determined to be 4P based on expressed-sequences tags-sequence tagged sites(EST-STS) markers specific to A. cristatum and FISH with probes pAcTRT1 and pAcpCR2. Overall, these results suggest that II-13 was a 7P (7D) substitution line with one pair of additional 2P chromosomes and II-23 was a multiple 4P (4B), 7P(7A) substitution line with one pair of additional 2P 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.

Keywords: common wheat, Agropyron cristatum, derivatives, in situ hybridization, molecular markers

1. lntroduction

Common wheat (Triticum aestivumL.) is a globally important crop and a vital source of protein, vitamins, and minerals,accounting for 20% of the calories consumed by humans(Tester and Langridge 2010). As a tertiary gene pool of wheat, wild relatives possess many genes that can be used for genetic improvement of wheat. Addition lines of wheat and wild relatives play an irreplaceable role as the bridge for transferring superior foreign genes into common wheat.Breeders and geneticists have successfully established many disomic addition lines between wheat and its relatives,e.g.,Haynaldia villosa,Agropyron cristatum,Aegilops biuncialis,Hordeum californicum,Thinopyrum elongatum,Rye, and used these materials to transfer alien genes intoT.aestivum(Chenet al. 1994; Liet al. 1997; Schneideret al.2005; Konget al. 2008; Fuet al. 2012; Nguyenet al. 2015).

As a way of transferring exogenous substances, addition lines are used to create translocation lines and introgression lines which have more potential uses. Seed storage protein genes derived from two translocation lines were obtained through ionizing radiation of the mature female gametes of Chinese Spring (CS)-Dasypyrum villosum1V#4 disomic addition line;Glu-V1andGli-V1/Glu-V3loci were mapped to the region of FL 0.50–1.00 of 1V#4S ofD.villosum(Zhanget al. 2014). Three wheat-Leymus racemosusdisomic addition (DA) lines DA5Lr#1, DA7Lr#1, and DALr.7 resistant to Fusarium Head Blight (FHB) were used to develop wheat-L.racemosustranslocation lines through irradiation and gametocidal gene-induced chromosome breakage. From this, a total of nine wheat-alien translocation lines with wheat scab resistance were identified (Chenet al. 2005). Puet al. (2015) applied60Co-γ irradiation to Chinese Spring-Thinopyrum bessarabicumchromosome 4J disomic addition line, identified its progenies by molecular and cytological methods, and determined that chromosome 4J was physically divided into 24 segmental blocks and the blue-grained geneBaThbwas further mapped into the region corresponding to block 4JL-11. Li Zet al. (2016)successfully identified three 1RS.1BL translocation lines that expressed high resistance to severalPuccinia striiformisf. sp.Triticipathotypes. Kumaret al. (2017) selected a translocation line with improved dough strength from among wheat-Thinopyrum elongatumderivatives.

Relatives of wheat grow under natural conditions, and mainly exist in the same population, and thus there is a high genetic diversity between different varieties of the same subspecies and even between different individuals of the same variety. Two varieties ofSecale cereale- Imperial and King II were hybridized with Chinese Spring, leading to two series of disomic addition lines; addition lines belonging to the same homologous group behaved differently (Alkhimovaet al. 1999).Plants inAgropyronare cross pollinated plants andAgropyroncristatum(2n=4x=28, PPPP)is the most common species. Liet al. (1997) obtained a series of stable, disomic addition lines, such as 1P addition line II-3-1a (Panet al. 2017), 2P addition line II-9-3 (Li H Het al.2016), 4P addition lines II-21-2 and II-21-6 (Liuet al. 2010),and 7P addition line II-5-1 (Luet al. 2016). In addition,4844-12, 5113, 5114, 5106, II-26, and II-29-2iwere all wheat-A.cristatumchromosome 6P disomic addition lines.However, these lines could be divided into four different types after clustering analysis (Hanet al. 2014). Although these lines possessing the same basic genome P, structural rearrangement and mutation happened on chromosomes.Therefore, creating multiple addition lines belonging to the same homologous group of the same species not only enriches wheat genetic resources, but also provides a reference for the study of evolutionary processes.

Our laboratory has created and obtained a series of wheat-A.cristatumderivatives and within them, lines II-13 and II-23 had 44 chromosomes but were not disomic addition lines, because they possessed more than two P chromosomes. Limited research has focused on multiple P chromosomes in a wheat background. Using II-13 and II-23 as materials, the aims of the present study were to: (1)analyze the chromosomal constitution of wheat-A.cristatumderivatives II-13 and II-23; and (2) develop new wheat-A.cristatumdisomic addition and substitution lines.

2. Materials and methods

2.1. Plant materials

Wheat-A.cristatumderivatives II-13 (2n=44) and II-23(2n=44), developed by hybridization betweenA.cristatumaccession Z559 (2n=4x=28, PPPP, from Xinjiang, China)andTriticum aestivumcv. Fukuhokomugi (2n=6x=42,AABBDD, Fukuho for short), were provided by the Center of Crop Germplasm Resources Research in the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences(CAAS), Beijing, China.

2.2. Chromosome preparation

The seeds were soaked in water in Petri dishes with double moistened filter papers at 24°C for 24 h. Roots were sampled when they were approximately 1.5–2.0 cm long, and excised root tips were treated with nitrous oxide gas for 2 h. Treated root tips were fixed in 90% acetic acid for 8 min and stored in 70% ethanol at 20°C until use (Cuadradoet al. 2000).Young spikes containing meiotic divisions in the pollen mother cells (PMCs) were collected and fixed in Carnoy’s fixative(ethanol:acetic acid=3:1, v/v). Cytological observations were conducted using a BX51 Olympus Phase Contrast Microscope (Olympus Corp., Tokyo, Japan) and images were taken with a digital camera. The slides were fixed using ultraviolet light in a TL-2000 Ultraviolet Translinker (Japan)for one minute when needed for GISH detection.

2.3. GlSH and FlSH detection

Genomicin situhybridization (GISH) and fluorescencein situhybridization (FISH) were conducted to detect alien chromosomes in common wheat background. Total genomic DNA ofA.cristatumand Fukuhowere extracted from fresh leaves by the modified CTAB method (Gill 1991).The purity and concentration of DNA were measured using a spectrophotometer. The blocker DNA was prepared by sonicating the Fukuho genomic DNA in a supersonic cleaner(model LED-50, Jiangsu, China) for 5 min. All probes were labeled by nick translation.

GISH and FISH were performed on the root tip cells with a commonly used method according to Katoet al.(2004) and Hanet al. (2006). Cytological images were observed under a Nikon Eclipse E600 (Japan) fluorescence microscope. Images were captured with a CCD camera(Diagnostic Instruments, Inc., Sterling Heights, MI, USA),and processed with Adobe Photoshop to adjust brightness and contrast when required.

2.4. Molecular marker analysis

To determine the genomic composition of the wheat-A.cristatumderivatives, a total of 76 wheat SSR markers physically or genetically mapped on all wheat chromosomes were chosen. In addition, 53 EST-STS markers specific to wheat-A.cristatumdisomic addition lines were used in the study according to the publishedA.cristatumtranscriptome sequences (Zhanget al. 2015, 2017). All wheat SSR primers were obtained from the Graingenes website (http://wheat.pw.usda.gov/GG2/index.shtml). The reaction mixture contained 10 mmol L–1Tris-HCl (pH 8.3), 50 mmol L–1KCl,3.0 mmol L–1MgCl2, 5.0 mmol L–1of each dNTP, 5.0 mmol L–1of each primer, 60 ng of genomic DNA, and 1 U ofTaqpolymerase. DNA amplification was performed in a PTC-200 thermocycler (MJ Research, Watertown, MA, USA),which was programmed for 5 min at 94°C; then 38 cycles of 1 min at 94°C, 1 min at 50–61°C, and 1 min at 72°C; and 10 min at 72°C for a final extension. The PCR products were separated on 8% polyacrylamide non-denaturing gels and were visualized following silver-staining.

2.5. Evaluation of agronomic traits

II-13, II-23, and Fukuho were planted at the experimental farm in Beijing, China. All materials were planted in a randomized block arrangement with three repeats with spacing 30 cm apart and rows 2.0 m long with 20 grains per row. Plant height, spike length, spikelets per spike,grain number per spike, fertile tiller numbers, thousandgrain weight, length and width of flag leaves, and field incidence were evaluated on 20 randomly selected plants.The data were analyzed using Statistical Analysis System version 9.2 (SAS Institute Inc., Cary, NC, USA) adopting Duncan’s multiple range tests analysis of variance at α=0.05 significance level.

3. Results

3.1. lnvestigation of agronomic traits of ll-13 and ll-23

The basic agronomic traits of II-13 and II-23 are shown in Table 1 and Fig. 1. The average performance of II-13 individuals was as follows: average plant height was 67.3 cm, spike length was 7.7 cm, spikelets per spike was 19, grain number per spike was 40, and thousand-grain weight was 17.4 g. The plant type of II-23 was compact,spikelet number per spike was 22, grain number per spike was 43, and thousand-grain weight was 21.9 g. In addition,the flag leaves of two derivatives were narrower and shorter than those of Fukuho. Through two years of investigation,II-13 was moderately susceptible to powdery mildew while II-23 showed high resistance to powdery mildew with spots on the basal leaves.

3.2. Chromosome composition analysis of ll-13 and ll-23

Mitotic observations of root-tip cells showed that 27 of the 29 plants of II-13 had the chromosome number 2n=44(40W+4P) (Fig. 2-A), accounting for 93.1%. Based on mitotic analysis of II-23, 87% had 38 wheat chromosomes and sixA.cristatumchromosomes (Fig. 2-C). The meiotic analysis of PMCs at metaphase I showed that the chromosome configurations of II-13 and II-23 were both 2n=22II, and all alien chromosomes were paired as bivalents (Fig. 2-B and D). No univalents or multivalents were found at metaphase I. These results indicated thatthe two derivative lines had normal chromosomal behaviors and were cytogenetically stable.

Table 1 Evaluation of agronomic traits of II-13, II-23, and Fukuho1)

Fig. 1 Agronomic traits of II-13, II-23, and Fukuho. A, plant morphologies. B, evaluation of powdery mildew resistance.

3.3. FlSH identification for wheat chromosome composition in ll-13 and ll-23

To confirm wheat chromosome compositions, we performed FISH using P genomic DNA, clone pAs1, and pSc119.2 as probes. The results showed that a pair of 7D chromosomes was absent in II-13 (Fig. 3-A) while in II-23 we did not find chromosome 4B and 7A (Fig. 3-B).

3.4. SSR markers analysis for wheat chromosome composition in ll-13 and ll-23

Fig. 2 Genomic in situ hybridization (GISH) analysis of the root cells and pollen mother cells at meiotic metaphase I of II-13 and II-23. A and B, GISH analysis of the root cells and pollen mother cells at meiotic metaphase I of II-13, respectively. C and D,GISH analysis of the root cells and pollen mother cells at meiotic metaphase I of II-23, respectively. Agropyron cristatum genomic DNA was labelled with digoxigenin-11-dUTP red fluorescence and the wheat DNA was counterstained with DAPI (4’,6-diamidino-2-phenylindole) blue fluorescence. Arrows show the P chromosomes.

Fig. 3 Genomic in situ hybridization (GISH) analysis of the root of II-13 and II-23. A, II-13. B, II-23. For both, the repetitive sequence clone pSc119.2 was labelled with fluorescein-12-dUTP and visualized with green fluorescence. The repetitive sequence clone pAs1 and Agropyron cristatum genomic DNA were labelled with Texas red-5-dCTP and visualized with red fluorescence.Chromosomes were counterstained with DAPI (4’,6-diamidino-2-phenylindole) and visualized with blue fluorescence. P1, P2, and P3 were A. cristatum chromosomes and A, B, and D were wheat genomes.

Genomic DNA was isolated f-rom the wheat-A.cristatumderivatives and both parents Fukuho andA.cristatumaccession Z559. To confirm the genomic composition of these two derivatives, we tested 76 SSR primer pairs located on wheat ABD genome. The information of SSR markers is summarized in Table 2, and the amplification patterns of six markers are shown in Fig. 4 as examples. Of these,SSR markerswmc94andwmc634, located on chromosome 7D, did not amplify specific polymorphic DNA bands like in Fukuho while the remaining primers were positive, indicating that 7D was absent in II-13. Expected products of SSR markers specific to chromosome 4B (wmc511andwmc310)and 7A (gwm260andwmc596) were not detected in II-23,whereas products were obtained for the primers specific to other chromosomes. Therefore, we deduced that 4B and 7A were absent in II-23.

3.5. EST-STS marker analysis of P chromosomes in ll-13 and ll-23

EST-STS markers gave specific products for II-13 and II-23.Thirteen markers amplified specific bands in II-13 and II-23,of which nine markers were distributed on 2P and four markers on 7P. Moreover, seven STS markers assigned for 4P produced unique amplicons in II-23. The specific bands inA.cristatumproduced by the EST-STS primer pairs for homologous groups 1, 3, 5, and 6, were not detected in the two derivatives or in Fukuho. These results indicated that derivative line II-13 might contain the homologous chromosomes of groups 2 and 7 of the P genome fromA.cristatum, i.e., 2P, 7P, and II-23 might possess chromosomes 2P, 4P, and 7P (Fig. 5).

3.6. FlSH identification of P chromosomes in ll-13 and ll-23

In order to verify the EST-STS marker analysis, we used dispersed repeat sequence pAcTRT1 and pAcpCR2 (Hanet al. 2017) as probes to detect P chromosomes. According to the signal distribution in chromosomes and chromosome arm ratio (long/short arm), experimental results revealed that the two pairs of alien chromosomes in II-13 were 2P and 7P (Fig. 6-A). Meanwhile, three pairs of exogenous chromosomes in II-23 were proved to be 2P, 4P, and 7P(Fig. 6-B). These results were consistent with the results of molecular markers.

3.7. Acquiring alien addition lines and substitution lines

By continuous backcrosses and selfcrosses, some novel alien disomic addition lines were obtained among the progeny of II-13 (or II-23) and recurrent parent Fukuho(Table 3). The plants 2-57 (Fig. 7-A) and 2-72 (Fig. 7-B)were wheat-A.cristatum2P addition lines; the plants 4-7,4-11, and 4-12 (Fig. 7-C–E) were wheat-A.cristatum4P addition lines; and the plant 7-7 (Fig. 7-F) was a wheat-A.cristatum7P addition line. In addition, five plants were identified as wheat-A.cristatumsubstitution lines, including 4P substitution lines (4-6, 4-8) (Fig. 7-G and H) and 7P substitution lines (7-49, 7-64, 7-65) (Fig. 7-I–K); 7-64 and 7-65 were 7D (7P) substitution lines.

Table 2 The primer sequences of wheat SSR markers

Fig. 4 Amplification patterns of wheat SSR markers. M, marker; lane 1, Agropyron cristatum cv. Z559; lane 2, Triticum aestivum cv. Fukuho; lane 3, II-13; lane 4, II-23. The arrows indicate wheat specific bands.

Fig. 5 Amplification patterns of Agropyron cristatum EST-STS markers. M, marker; lane 1, Agropyron cristatum cv. Z559; lane 2,Triticum aestivum cv. Fukuho; lane 3, II-13; lane 4, II-23. The arrows indicate P chromosomes specific bands.

Fig. 6 Fluorescence in situ hybridization (FISH) detection of the root cells of II-13 and II-23. A, II-13. B, II-23. The repetitive sequence of Agropyron cristatum pAcTRT1 and pAcpCR2 were used as probes; pAcpCR2 were labeled with Texas red-5-dCTP,pAcTRT1 were labeled with fluorescein-12-dUTP, showing green and red signals, respectively. The chromosome image at the bottom were P chromosomes in II-13 and II-23.

4. Discussion

4.1. The genetic stability of multiple P chromosomes in wheat background

Table 3 Different types of addition and substitution lines from backcross progenies of II-13 and II-23

Fig. 7 Genomic in situ hybridization (GISH) identification of addition/substitution lines. A–K, 2-57, 2-72, 4-7, 4-11, 4-12, 7-7, 4-6,4-8, 7-49, 7-64, and 7-65.

The cytogenetic stability of alien substitution lines, addition lines, and translocation lines is of theoretical and practical significance for their application in genetics and plant breeding. The maintenance of such lines requires regular meiotic pairing and recombination in the subsequent selfing progenies (Cifuentes and Benavente 2009). According to the chromosome pairing observation in this study, all chromosomes from wheat andA.cristatumdisplayed regular meiosis. Plant type and chromosome composition of derivatives II-13 and II-23 were consistent for two years.II-13 and II-23 were also cytologically stable. By the detection ofin situhybridization and molecular markers,wheat chromosome 7D was replaced by chromosome 7P in II-13, while in II-23 wheat chromosomes 4B and 7A were substituted by 4P and 7P, respectively. Comparative genome mapping across cereal species has shown that homologous chromosomes are generally similar and conserved in terms of their gene content and gene order. The substitution phenomena of II-13 and II-23 happened spontaneously and these two derivative lines were genetically stable. According to the principle of homologous chromosome substitution compensation, imported chromosomes fromA.cristatumcompensated for some functions of homologous replaced wheat chromosomes in wheat background to some extent.Thus, more than 60% of II-13 and II-23 plants survived and produced seed under normal field conditions.

4.2. The theoretical and practical guidance of creating new materials

Chromosome structural rearrangement has been increasingly studied in recent years. Rearrangements in multiple genera belonging to Triticeae, such asElynus,Elytrigia, andLeymushave been identified (Kishiiet al.2004; Wanget al. 2010; Huet al. 2012; McArthuret al.2012). These rearrangements could be used to explore the evolutionary and cytogenetic relationship among wheat and its relatives.Agropyronis an important genus that is related to wheat and it possesses the basic genome P,with some variation in chromosomal structure. Thus, the genetic effects of genes located on different P chromosomes in wheat background can be studied by creating a great diversity of addition and substitution lines belonging to the same homologous groups.

According to previous studies (Li Q Fet al. 2016; Liet al. 2017), wheat-A.cristatum2P disomic addition line II-9-3 showed high resistance to powdery mildew. In our study, both II-13 and II-23 contained chromosome 2P, but showed susceptibility and resistance responses to powdery mildew, respectively. The P chromatins of these three lines came from the sameA.cristatumvariety, but they may not come from the same pollen. This suggests that there is a high genetic diversity inA.cristatumvarieties, even in a singleA.cristatumplant. It is also possible that different group P chromosomes interacted with each other under wheat background so that II-13 and II-23 showed different responses to powdery mildew.

Our laboratory has obtained a series of disomic addition lines by distant hybridization between common wheat andA.cristatum. Few reports have focused on substitution lines expect for chromosome 6P (Wuet al. 2006). Thus, our study expanded the basic materials to some extent by screening out five new disomic substitution lines. Subsequent research focusing on their genetic stability and desirable traits (like high yield and quality) will not only broaden the genetic diversity of wheat-A.cristatumresources, but also provide a foundation for creating more valuable materials and transferring more useful genes into wheat.

5. Conclusion

We identified wheat-A.cristatumderivatives II-13 and II-23 usingin situhybridization (GISH and FISH) and molecular marker analysis (SSR and EST-STS) and the chromosome compositions were that II-13 was a 7P (7D) substitution line with one pair of additional 2P chromosomes and II-23 was a multiple 4P (4B), 7P (7A) substitution line with one pair of additional 2P chromosomes. Moreover, we obtained six alien disomic addition lines and five alien disomic substitution lines by backcrossing. To evaluate the advantages of new materials, more identification work should be considered in future attempts at genetic improvement.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFD0100102).