Ru-containing single crystal nickel base superalloy during creep at elevated temperature

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Available online at www.sciencedirect.com

ScienceDirect

Development and identification of a dwarf wheatLeymus mollis double substitution line with resistance to yellow rust and Fusarium head blight Jixin Zhaoa , Yang Liua , Xueni Chengb , Yuhui Panga,c , Jiachuang Lia , Zhenqi Sud , Jun Wua , Qunhui Yanga , Guihua Baie,⁎, Xinhong Chena,⁎ a

Shaanxi Key Laboratory of Genetic Engineering for Plant Breeding, College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China b College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China c College of Agriculture, Henan University of Science and Technology, Luoyang 471023, Henan, China d Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang 050035, Hebei, China e USDA, Hard Winter Wheat Genetics Research Unit, 4008 Throckmorton Hall, Manhattan, KS 66506, USA

AR TIC LE I N FO

ABS TR ACT

Article history:

Leymus mollis (Trin.) Pilger (2n = 4x = 28, NsNsXmXm,), a wild relative of common wheat,

Received 28 June 2018

possesses many potentially valuable traits for genetic improvement of wheat, including

Received in revised

strong, short stems, long spikes with numerous spikelets, tolerance to drought and cold

form 4 November 2018

stresses, and resistance to many fungal and bacterial diseases. In the present study, a

Accepted 24 November 2018

wheat–L. mollis double substitution line DM96 was selected from a F6 progeny of a cross

Available online 30 January 2019

between M842-16 (an octoploid Tritileymus line) and D4286 (a Triticum durum line) using genomic in situ hybridization (GISH), simple sequence repeat (SSR) markers, and expressed

Keywords:

sequence tagged sequence site (EST-STS) markers. Chromosome analysis at mitosis and

Disease resistance

meiosis showed that DM96 had a chromosome constitution of 2n = 42 = 21II. GISH analysis

Double substitution line

indicated that DM96 carried 38 chromosomes from wheat and two homologous pairs of Ns

Dwarfing

chromosomes from L. mollis. Fluorescent in situ hybridization (FISH) showed that

Triticum aestivum

chromosomes 2Ns and 3Ns from L. mollis had replaced wheat chromosomes 2D and 3D in DM96, which was confirmed by SSR and STS markers. The newly developed substitution line DM96 has shorter height, longer spikes and more kernels than its parents and showed high resistance to stripe rust and Fusarium head blight (FHB). Thus, this line is a new bridge material for the production of useful translocation lines for wheat genetic research and genetic improvement of wheat yield and disease resistance in breeding programs. © 2019 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⁎ Corresponding authors. E-mail addresses: [email protected], (G. Bai), [email protected]. (X. Chen). Peer review under responsibility of Crop Science Society of China and Institute of Crop Science, CAAS.

https://doi.org/10.1016/j.cj.2018.11.012 2214-5141 © 2019 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

TH E C ROP J O U R NA L 7 (2 0 1 9) 51 6 –5 2 6

1. Introduction Wheat-related species exhibit genetic diversity in agronomically important genes that can be used in modern breeding. Many wheat relatives from the Triticeae can be crossed with wheat, and alien chromosomes or chromosome segments carrying superior agronomic traits can be incorporated into wheat by distant hybridization and cytogenetic manipulation [1]. Wide hybridization allows individual alien chromosomes or chromosome segments to be transferred into wheat for developing new wheat germplasm lines or varieties with desirable exogenous genes [2,3]. Among wheat relatives, Leymus mollis (Trin.) Pilger, an allotetraploid (2n = 4x = 28, NsNsXmXm) species in the Triticeae tribe of family Poaceae. L. mollis has strong stems, long spikes with large numbers of spikelets, tolerance to abiotic stresses such as drought, cold, salt, and marginal soils, and resistance to fungal and bacterial diseases. It is an excellent germplasm resource for forage and crop improvement [4,5]. The species Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is the diploid donor of the Ns genome of L. mollis, but the source of the Xm genome has not been determined [6–8]. Wheat–Leymus amphidiploids generated by distant hybridization between wheat and L. mollis were firstly reported in the late 1960s [9]. Subsequently, several tall, perennial hybrids were obtained from crosses between common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) line Hpph carrying a 5BS/ 5RL translocation that permits homoeologous chromosome pairing, and Leymus species, L. arenarius (L.) Hochst (2n = 8x = 56, JJJJNNNN), L. racemosus (2n = 4x = 28, NsNsXmXm) and L. mollis [10]. Using embryo culture and colchicine treatment hybrids of a similar type were produced by crossing hexaploid wheat cultivar Sicco (carrying the crossability allele kr1 on chromosome 5B from cv. Chinese Spring) and tetraploid winter wheat (T. carthlicum Nevski in Kom, 2n = 4x = 28, AABB) cv. Dorgimicum to L. arenarius and L. mollis [11]. A powdery mildew resistant wheat-Leymus hybrid, AD 99, carrying the A and B genomes and one D-genome chromosome from wheat plus six chromosomes from L. mollis was selected [12]. We initiated distant hybridization between common wheat and L. mollis in the 1980s [13] and developed several octoploid partial amphiploid Tritileymus lines (2n = 56) using embryo rescue and colchicine treatment of the F1 hybrids from cross T. aestivum cv. 7182 × L. mollis [14]. Different lines of Tritileymus, such as M842 were identified using cytogenetic methods. Selected lines with different genome compositions, AABBDDNsNs and AABBDDXmXm, had many desirable agronomic traits such as long spikes with many florets, large seeds, and tolerance to cold and drought [15,16]. A translocation line, Shannong 0096 with resistance to stripe rust, was identified from the progeny of M842 × T. aestivum cv. Yannong 15 [17]. One wheat-L. mollis triple substitution line, 05DM6, was obtained from the progeny of durum wheat (T. durum Desf., 2n = 4x = 42, AABB) cv. Trs-372 × M842-12 [18]. Another triple substitution line, 10DM50, and a disomic substitution line, 10DM57, were selected from the progeny of M842 × durum line D4286 [19,20]. Disomic substitution line DM45 with a glutenin gene from L. mollis was identified from the progeny of 05DM6 × T. aestivum cv. 7182, in which the L. mollis

517

chromosome 1Ns replaced the wheat chromosome 1D [21]. Two chromosome addition lines, M11003-4-4-1-1 (a double disomic addition line) and M11003-4-3-8-13-15 (a double monosomic addition line) with resistance to stripe rust, were selected from the progeny of M842 × T. aestivum cv. 7182 [22,23]. A wheat-L. mollis double substitution line has not been reported. In this study, wheat-L. mollis double substitution line DM96 was obtained from a cross between octoploid Tritileymus M842-16 and durum line D4286. The objectives of this study were to (a) determine the chromosome pairing and genomic origin of transferred alien chromosome segments using cytogenetic methods, (b) examine the chromosome composition using FISH, SSR and EST-STS markers, and (c) evaluate the potential usefulness of the agronomic traits of this line for wheat improvement.

2. Materials and methods 2.1. Plant materials The plant materials used in this study include L. mollis (accession number: BM01), P. huashanica (accession number: 0503383, octoploid Tritileymus line M842-16 (2n = 56, AABBDDNsNs), bread wheat cultivars 7182 and Huixianhong, durum wheat line D4286, and one wheat-L. mollis double substitution line DM96. The parents of octoploid Tritileymus M842-16 were the wheat cv. 7182 and L. mollis accession BM01. Line DM96 was selected from the F6 progeny of octoploid Tritileymus M842-16 × durum line D4286. Wheat line 7182 was used as a control in the agronomic trait assessment as well as in DNA marker analysis. Wheat cv. Huixianhong was used as a susceptible control in yellow rust response assays. All materials are deposited in the Northwest A&F University College of Agronomy germplasm collection.

2.2. Mitotic and meiotic analyses Root tips from germinating seeds of substitution line DM96 collected at 1–2 cm in length were incubated in ice-water overnight and then fixed in Carnoy's fixative solution I (ethanol:acetic acid = 3:1, v/v). Young spikes were collected and fixed in Carnoy's fixative solution II (absolute ethanol: chloroform:glacial acetic acid = 6:3:1, v/v/v). The fixed root tips and anthers from the young spikes were squashed and stained with 1% acetocarmine on glass slides for cytological examination. After determination of chromosome numbers and pairing, coverslips were removed using liquid nitrogen, and then air dried in room temperature for GISH and FISH analyses.

2.3. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) Genomic DNA of P. huashanica was used as the probe in GISH analysis. DNA was extracted from fresh leaves using a cetyltrimethyl ammonium bromide protocol and labeled with a DIG-nick-translation mix (Roche, Germany) according to the manufacturer's instructions. In situ hybridization was

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performed following Schwarzacher et al. [24] and Han et al. [25] with slight modifications. The probe was detected using anti-digoxigenin-FITC and chromosomes were counterstained with propidium iodide (PI). Fluorescent signals were examined using an Olympus BX60 fluorescence microscope and photographed with a Pixera Penguin 150CL CCD camera (Pixera Corporation, Santa Clara, CA, USA). For FISH analysis, clone pAs1 was labeled by nick translation with digoxigenin11-dUTP and used as the probe. FISH was performed as described by Mukai et al. [26] and Schneider et al. [27] with slight modifications. After FISH, the slides were washed with 2× saline sodium citrate (SSC) for 5 min, fixed with 4% paraformaldehyde for 10 min at room temperature, and used for sequential GISH performed by the above protocol for GISH.

2.4. DNA marker analysis Three hundred and eighty four pairs of chromosome-specific SSR primers from different sources [28–33] were selected to characterize the chromosome composition of DM96 (Table S1). Those SSR were evenly distributed across the 21 wheat chromosomes. SSR analysis followed Liu et al. [34] and PCR products were separated on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The SSR data were scored using GeneMarker version 1.97 (Soft Genetics LLC, State College, PA, USA). SSR fragment sizes were compared between wheat cv. 7182 and the substitution line DM96 to determine the sources of chromosomes in DM96. A similarity ratio was calculated for each chromosome as the ratio between the number of primer pairs that successfully amplified the same size of PCR products in the chromosomes of DM96 and 7182 to the number of primer pairs that amplified PCR only in 7182. Sixty-one EST-STS primers were used to identify the alien chromosomes in DM96. Those EST-STS markers were evenly located on all seven wheat homoeologous groups (Table S2) and were obtained from the Wheat Haplotype Polymorphisms website (http://wheat.pw. usda.gov/), except for primers MWG, MAG and BCD affiliated with homoeologous group 6 developed by Wu et al. [35]. PCR and electrophoresis procedures of EST-STS markers followed Du et al. [36].

2.6. Evaluation of disease reaction During the 2016 and 2017 field growing seasons, the double substitution line DM96 and its parents were evaluated for reaction of adult plants to the fungal pathogens Puccinia striiformis f. sp. tritici (Pst) and Fusarium graminearum Schwabe [teleomorph Gibberellazeae (Schw.) Petch.], which cause yellow rust and Fusarium head blight (FHB), respectively. Both yellow rust and FHB evaluations were conducted in the Experimental Farm of Northwest A&F University. The experiments were arranged in randomized complete block design with two replications. For yellow rust evaluation, mixed Pst races (CYR32, CYR33, and Hybrid 46 provided by the College of Plant Protection, Northwest A&F University) were used to induce infection by evenly dusting urediospores over leaves of susceptible spreader rows following Ma et al. [37]. Infection types (IT) were recorded three weeks after inoculation using a 0–4 rating scale, where 0 refers to immunity with no visible symptoms, 0; refers to highly resistant reactions showing necrotic flecks without uredinia, 1 refers to resistant uredinia with distinct necrosis, 2 refers to moderately resistant responses involving small to medium-sized uredinia with chlorosis and necrosis, 3 refers to moderate susceptibility showing moderate-sized sporulating uredinia, and 4 refers to large sporulating uredinia without chlorosis or necrosis [38]. FHB resistance to disease spread within a spike (Type II resistance) was evaluated following single floret inoculation as described by Bai et al. [39,40]. F. graminearum strain PH1 was provided by the College of Plant Protection, Northwest A&F University). Five to 6 randomly selected wheat spikes were inoculated by injecting 10 μL of a conidial spore suspension (100 spores μL−1) into the floral cavity between the lemma and palea of a single floret in the middle of a spike using a syringe. Inoculated spikes were covered with a plastic bag for 48 h to maintain high humidity. Infected spikelets and total spikelets per spike were counted at 15 d after inoculation [41]. A reaction index (RI at a 1–5 scale) and the infected spikelet rate (ISR%) were calculated following Yang et al. [42].

3. Results 3.1. Identification of alien chromosomes in DM96 using GISH

2.5. Evaluation of morphological traits of DM96 DM96 and its parents, M842-16 and D4286, as well as its earlier progenitor 7182 were planted at the Experimental Farm of Northwest A&F University in the 2016 and 2017 growing seasons. Ten random plants per genotype were evaluated for morphological traits including plant height, number of spikes per plant, spike length, number of spikelets per spike, number of kernels per spike, self-fertility, and 1000-kernel weight. The kernel characters (length, width, and area) were measured by scanning 50 randomly selected kernels per sample and analyzing the images using a Digimizer software (https:// www.digimizer.com/download.php). Heading date was recorded at 50% spike emergence. The mean trait values for each genotype over both years were used for analysis of variance (ANOVA). Duncan's multiple range test was used to determine differences among accessions.

DM96 is an F6 dwarf line with long spikes and was selected from the progenies of octoploid Tritileymus M842-16 × durum line D4286. All 10 DM96 plants had a chromosome number of 2n = 42 (Fig. 1A). Sequential genomic in situ hybridization (GISH) analysis of randomly selected root tip cells using total P. huashanica genomic DNA as probe showed that root tip mitotic cells had four differentially fluorescing chromosomes (Fig. 1B), suggesting that DM96 was a wheat-L. mollis substitution line carrying 38 wheat chromosomes plus four Ns chromosomes from L. mollis.

3.2. Meiotic analysis of DM96 Fifty five pollen mother cells (PMCs) of DM96 were examined at meiotic metaphase I; examined, 46 (83.6%) had 21 bivalents with an averages 17.89 ring bivalents, 2.87 rod bivalents and

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A

B

Fig. 1 – Cytogenetic analysis of DM96 at mitosis. (A) Chromosomes (2n = 42) in root tip cells. (B) Sequential GISH at the same cell at metaphase using the total DNA from Psathyrostachys huashanica as probe to show yellow-green fluorescent hybridization signals from four foreign chromosomes.

0.47 univalents. Multivalent associations were not observed (Fig. 2A, Table 1). GISH analysis using P. huashanica genomic DNA as a probe showed that PMCs at metaphase I had two bivalents with strong hybridization signals (Fig. 2B). These bivalents were most likely formed by two pairs of Ns chromosomes from L. mollis, substituting for two pairs of wheat chromosomes. Those results confirmed that DM96 is a wheat-L. mollis double disomic substitution line.

3.3. Sequential FISH/GISH analyses of DM96 Comparison of FISH patterns of DM96 and wheat cv. Chinese Spring and others [26,27,43] (Fig. 3A) using clone pAs1 as the probe identified wheat chromosomes 1D, 4D, 5D, 6D, and 7D, but not 2D and 3D (Fig. 3B). Sequential GISH using total genomic DNA of P. huashanica as probe demonstrated that the two pairs of chromosomes had the fluorescent signals characteristic of the Ns genome, indicating that they were chromosomes 2Ns and 3Ns (Fig. 3C).

3.4. DNA marker analysis of DM96

A

B Fig. 2 – Cytogenetic analysis of DM96 at meiosis. (A) Chromosome pairing at metaphase I in the pollen mother cells showing 21 bivalents. (B) GISH image using the total DNA from P. huashanica as the probe to show yellow-green fluorescent hybridization signals from two ring bivalents.

Among the 384 primers, 306 (79.69%) and 294 (76.56%) amplified bands in the wheat cv. 7182 and DM96, respectively (Tables 2 and S1). Among amplified primers those from wheat chromosomes 2D and 3D had much lower marker similarity ratios (16.67% for 2D and 27.78% for 3D) between 7182 and DM96 than those from the other wheat chromosomes (46.67%–100.00%, Table 2). This confirmed that DM96 does not carry wheat chromosomes 2D and 3D. EST-STS marker analysis showed that five pairs of primers (CD452803, BE404332, BQ160526, BQ169707, and BE444851) from group 2 (Fig. 4A) and four pairs of primers (BF200774, BF291730, BF429203, and BM137713) from group 3 of L. mollis (Fig. 4B)

Table 1 – Chromosome pairing at metaphase I in pollen mother cells of DM96. Line

Number of observed cells

Chromosomes pairing I

II Rings

DM96 55

0.47 (0–4)

17.89 (18–21)

Rods 2.87 (0–4)

Total 20.76 (19–21)

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C

A 1D

6D

7D

4D 1D

7D 4D

5D

5D

6D

B Fig. 3 – Sequential FISH and GISH analyses on root tip cells of DM96. (A) Mitotic chromosomes (2n = 42) in root tip cells. (B) FISH pattern using clone-pAs1 as probe. The yellow pAs1-specific fluorescent signals identify chromosomes from the D genome; 2D and 3D are missing, but are replaced by two pairs of unknown chromosomes with bands similar to 2D and 3D bands (arrows). (C) Sequential GISH on the same metaphase I using the total genomic DNA of P. huashanica as the probe showing fluorescent signals (arrows) covering the two pairs of unknown chromosomes.

amplified common bands between DM96, L. mollis, and P. huashanica that have Ns chromosomes, but those bands were not amplified in wheat line 7182 and durum wheat D4286. In contrast, the bands amplified by the EST-STS primers from chromosome groups 1, 4, 5, 6, and 7 of L. mollis and P. huashanica were not detected in DM96. These results confirmed that DM96 carries chromosomes 2Ns and 3Ns from L. mollis.

3.5. Morphological traits of DM96 DM96 was 54.73 cm tall, significantly shorter than its parents (Fig. 5A, Table 3). Spike length (12.15 cm) and number of spikelets per spike (21.14) of DM96 were similar to those of octoploid Tritileymus M842-16, but significantly higher than parents 7182 and D4286 (Fig. 5B, Table 3). DM96 had a significantly lower number of kernels per spike than that any of the three parents, with the lowest self-fertility among the four genotypes evaluated. Number of spikes per plant of

DM96 was slightly higher than all three parents, but the difference was not significant. DM96 had red kernels that were not fully filled (Fig. 5C). Kernel length and size of DM96 were similar to those of M842-16, but significantly higher than those of parent 7182 although the kernel width was not significantly different among the four genotypes (Table 3). The 1000-kernel weight of DM96 was the lowest among the four genotypes due to late-maturity that caused some shriveling. Overall, the double substitution line DM96 showed significant improvement in several yield traits including reduced plant height, longer spike length, increased number of spikelets per spike, and improved kernel length.

3.6. Disease reactions of DM96 DM96 showed a lower FHB reaction index (RI = 1.73) and infected spike ratio (ISR = 8.03%) than its parents, D4286 and 7182 (Fig. 6A, Table 3), but similar RI (1.98) and ISR (7.91%) to those of the parent M842-16, indicating that DM96 inherited

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Table 2 – Comparison of SSR banding patterns between common wheat cv. 7182 and the substitution line DM96. Chromosome Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7

1A 1B 1D 2A 2B 2D 3A 3B 3D 4A 4B 4D 5A 5B 5D 6A 6B 6D 7A 7B 7D

No. of markers 15 16 15 23 17 14 16 34 21 17 17 21 23 16 23 11 17 17 16 15 20 384

Total

No. amplified in 7182 11 13 12 19 14 12 10 28 18 13 15 17 16 13 21 10 11 15 11 11 16 306

No. amplified in DM96 8 14 14 20 15 6 13 30 11 14 16 17 17 11 21 10 16 9 11 9 12 294

6 7 12 13 9 2 5 17 6 10 12 14 12 8 15 7 9 7 7 7 11

FHB resistance from M842-16. DM96, and M842-16 were also significantly more resistant to yellow rust with infection type 0, whereas the durum parent D4286 and the wheat parent

A

M 1 2 3 4 5

M 1 2 3 4 5

No. with the same product size in 7182 Similarity and DM96 ratio (%) 54.55 53.85 100.00 68.42 64.29 16.67 50.00 60.71 27.78 76.92 80.00 82.35 75.00 61.54 71.43 70.00 81.82 46.67 63.64 63.64 68.75

7182 had infection types of 1 and 2, respectively (Fig. 6B, Table 3). The susceptible control Huixianhong had the infection type 4. These results indicate that DM96 carries yellow rust

M 1 2 3 4 5

M 1 2 3 4 5

M 1 2 3 4 5

BE444851

2000 bp

750 bp 500 bp

B

CD452803

BE404332

BQ160526

BQ169707

M 1 2 3 4 5

M 1 2 3 4 5

M 1 2 3 4 5

M 1 2 3 4 5

2000 bp

750 bp 500 bp

BF200774

BF291730

BF429203

BM137713

Fig. 4 – EST-STS analysis of DM96. EST-STS markers derived from homologous groups 2 (A) and 3 (B) amplified target bands in samples containing Ns chromosomes (DM96, L. mollis, and P. huashanica). M, DNA ladder; Lane 1, common wheat 7182; Lane 2, durum line D4286; Lane 3, DM96; Lane 4, L. mollis; Lane 5, P. huashanica. Arrows show the target bands.

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resistance gene(s), but it is still unknown if the gene was from L. mollis or the durum parent.

4. Discussion Wide hybridization has been used to transfer chromosomes or chromosome segments into adapted wheat genotypes and thereby expands the wheat gene pool [3,12]. Alien substitution lines are a first step in producing alien translocation lines [44]. Many germplasm lines have been developed from wide crosses between wheat and L. mollis [14–23]. The present study is a first report of a double substitution line (DM96) containing two pairs of Ns chromosomes from L. mollis. This line exhibited normal chromosome pairing in PMCs, and therefore, should be cytogenetically stable. It also has several desirable agronomic traits including reduced plant height and resistance to wheat FHB and stripe rust, which are important traits of wheat improvement. The repeat sequence pAs1 (a D-genome-specific clone) has be used as a cytological landmark for identification of chromosomes in the D genome [26,27,45]. The FISH pattern obtained using this sequence permits identification of all Dgenome chromosomes although some weak hybridization might occur in some chromosomes of A and B genomes [45,46]. In this study, the double substitution line DM96 was selected from progeny of octoploid Tritileymus M84216 × durum line D4286, and chromosomes in A and B genomes of octoploid Tritileymus should pair with A and B

C

chromosomes from durum. In genome D, the FISH banding pattern using pAs1 as the probe identified chromosome pairs 1D, 4D, 5D, 6D, and 7D, but not chromosomes 2D and 3D in DM96, indicating that DM96 carries two Ns chromosome pairs from octoploid Tritileymus substituting for wheat chromosomes 2D and 3D. Sequential GISH of DM96 using genomic DNA of P. huashanica as probe identified two pairs of Ns chromosomes from L. mollis. Comparison of the pAs1 banding patterns of wheat 2D and 3D with the two pairs of Ns chromosomes from L. mollis suggested that the chromosome pair with pAs1 hybrization at the terminal ends of the long arms, and sub-termini and termini of the short arms is more likely to be chromosome 3Ns (Fig. 3B, dovetail arrows), and the pair with pAs1 hybridization at the terminal ends of the short arms and the sub-termini of the long arms is chromosome 2Ns (Fig. 3B, triangular arrows). Based on the pAs1 banding patterns of wheat chromosomes 2D and 3D [27,43,47], the putative pAs1 FISH pattern and idiogram of L. mollis' chromosomes 2Ns and 3Ns can be used as genome-specific markers to identify the 2Ns and 3Ns chromosomes in wheat backgrounds (Fig. 7). The use of DNA markers to identify alien chromosomes has made a significant contribution to chromosome engineering and plant breeding programs. SSR and EST-STS markers are ideal tools for testing the authenticity wheat genetic stocks of and for identifying chromosome addition lines, substitution lines, and translocation lines [38,48–51]. This method is particularly important for genetic analysis of alien chromatins from wheat relatives that lack diagnostic DNA

7182

DM96

B 7182 7182

DM96 DM96

A Fig. 5 – Images of DM96 and its parent 7182 showing mature plant phenotypes (A), spikes (B), and kernels (C).

–

1.73 8.03

4.90 52.63

4.78 63.22

1.98 7.91

ISR (%) RI

4

0

–

206

23.16 ± 0.05A – –

3.83 ± 0.05a

–

8.42 ± 0.10A

–

–

± 2.7 21.14 ± 1.07A

17.80 ± 0.84B

10.40 ± 0.55B 12.15 ± 1.07A –

D

±

± 18.50 ± 1.22B

±

±

±

IT, infection type; RI, reaction index; ISR, infected spike ratio; −, no data recorded. Capital and small letters indicate significant differences at P = 0.01 and P = 0.05, respectively.

–

37.62 ± 4.49B

3.86 ± 0.07a 7.04 ± 0.09C 40.70 ± 4.33A ±

±

2

1 ±

42.62 ± 2.96A

7.80 ± 0.09B

3.66 ± 0.06a

24.32 ± 197 0.05A 20.34 ± 199 0.05A 19.23 ± 0.06B 190 3.89 ± 0.06a 8.88 ± 0.10A 44.78 ± 2.04A ±

65.17 7.18C 73.86 2.31B 88.55 2.47A 55.13 5.06D ±

65.57 3.94B 54.50 3.21C 73.20 4.15A 37.29 21.43 ± 1.39A

12.64 ± 0.85A 7.67 ± 0.52C

6.43 ± 2.70a 7.67 ± 2.16a 6.60 ± 1.14a 7.71 ± 3.55a – ±

94.43 2.44A D4286 75.00 4.10C 7182 83.60 0.89B DM96 54.73 4.03D Huixianhong – M842-16

Selffertility (%) Kernels per spike Spikelets per spike Spike length (cm) Spikes per plant Plant height (cm) Materials

Table 3 – Comparison of agronomic traits between DM96 and its relatives.

1000-Kernel weight (g)

Kernel length (mm)

Kernel width (mm)

Heading Kernel date area (mm2)

0

Yellow rust reaction (IT)

Fusarium head blight resistance

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523

markers [50,51]. Using such markers, the chromosome composition of two wheat-L. mollis addition lines and two wheat-P. huashanica chromosome 2Ns/2D substitution lines were identified [22,23,36,52]. In our previous study, two disomic substitution lines, 10DM57 and DM45, and two triple substitution lines, 05DM6 and 10DM50, were identified using both SSR and ESTSTS markers [18–21]. In the present study, 384 pairs of SSRs from all 21 wheat chromosomes and 61 EST-STSs from seven wheat homoeologous groups were used to determine the chromosome constitution and composition of the double substitution line DM96. The results from both marker and FISH analyses showed that DM96 carries chromosomes 2Ns and 3Ns from L. mollis replacing wheat chromosomes 2D and 3D. Plant height is an important agronomic trait of wheat. Dwarfing genes have a strong effect on wheat yield components, and most of these genes are associated with improved harvest index and grain number [53–55]. More than 20 semi-dwarfing genes (Rht) have been reported and some of them have been widely used to reduce plant height in wheat, and to improve lodging resistance. In some cases, those genes also convert more photosynthate to grain yield [56,57]. In this study, DM96 containing 2Ns and 3Ns chromosomes of L. mollis showed significantly reduced plant height compared to its parents, apparently without sacrificing beneficial spike and kernel characteristics. The reduced height gene was most likely contributed by L. mollis [14,16], and might represent a new gene for reduced height. Yellow (stripe) rust and FHB are destructive wheat diseases worldwide. Epidemics of these diseases can result in significant economic losses for wheat growers in terms of yield and quality [58,59]. About 80 named yellow rust resistance genes plus many additional QTL and seven FHB resistance genes (Fhb1–Fhb7) have been named in wheat [60,61]. Introgression of resistance genes from wild relatives to wheat has been recognized as a useful and environmentally safe approach to minimize economic losses due to these diseases [62,63]. L. mollis is an important perennial Triticeae species with considerable potential for wheat improvement and some wheat-L. mollis partial amphiploids, substitution lines and translocation lines have resistance to bacterial and fungal diseases [14,16,17]. In this study, the double substitution line DM96 containing L. mollis chromosomes 2Ns and 3Ns showed high resistance to yellow rust and FHB. The resistance genes involved likely originated from L. mollis [14,16,17]. The double substitution line DM96 has excellent agronomic features of long spikes, more kernels and resistance to FHB and yellow rust, and can be used as an intermediate parent in genetic engineering to transfer genes conferring these beneficial traits to wheat chromosomes.

Conflict of interest Authors declare that there are no conflicts of interest.

Acknowledgments This research was supported by the National Natural Science Foundation of China (31571650, 31771785), Basic Research Projects of the Natural Science Foundation of Shaanxi

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A

B

1

2

3

4

CK

1

2

3

4

Fig. 6 – Disease reactions of DM96 and its parents. (A) Spikes infected with Fusarium graminearum. (B) Leaves infected with Puccinia striiformis. 1, Triticum aestivum cv. 7182; 2, double substitution line DM96; 3, T. durum line D4286; 4, octoploid Tritileymus M842-16; CK, T. aestivum cv. Huixianhong.

2Ns

3Ns

Fig. 7 – pAs1 FISH pattern and idiogram of chromosome 2Ns and 3Ns from L. mollis.

Province (2015JM3095), and Tang Zhongying Breeding Funding Project at the Northwest A&F University.

Appendix A. Supplementary data Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.11.012.

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