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Article

Characterization of a Wheat-Dasypyrum breviaristatum Chromosome Addition and Its Derived Progenies Carrying Novel Dasypyrum-Specific Gliadin Genes

by
Chengzhi Jiang
1,
Wenxi Jiang
1,
Min Liu
1,
Hongjin Wang
1,
Ennian Yang
2,
Zujun Yang
1,* and
Guangrong Li
1,*
1
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
2
Crop Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(7), 1673; https://doi.org/10.3390/agronomy12071673
Submission received: 2 June 2022 / Revised: 8 July 2022 / Accepted: 12 July 2022 / Published: 13 July 2022
(This article belongs to the Special Issue Utilizing Genetic Resources for Agronomic Traits Improvement)
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Abstract

:
The construction of the 28-chromosome karyotype of Dasypyrum breviaristatum was undertaken using multicolor non-denaturing fluorescent in situ hybridization (ND-FISH) and Oligo-FISH painting protocols. A novel wheat-D. breviaristatum line D2138 contained 44 chromosomes including a pair of D. breviaristatum 6VbS.2VbL translocation chromosomes. Individual F2 and F3 progenies of a cross between D2138 with wheat lines CM62, MY11 and JM22, respectively, were characterized using ND-FISH and molecular markers. A relatively high chromosome alteration rate within wheat and D. breviaristatum 6VbS and 2VbL was observed in the three progeny populations, suggesting that chromosome 6VbS.2VbL has a gametocidal-like gene. The different types of translocation and deletion lines allowed localization of D. breviaristatum-specific gliadin coding genes on sub-telomeric regions of 6VbS by PCR and acid polyacrylamide gel electrophoresis analysis. The positive effect of the D. breviaristatum 6VbS on agronomic and quality characters was also demonstrated. The new wheat-D. breviaristatum 6VbS and 2VbL translocation lines will be useful as novel germplasm for breeding purposes.

1. Introduction

The genus Dasypyrum (or Haynaldia) consists of only two species, the annual 2× Dasypyrum villosum and the perennial 2× and 4× D. breviaristatum [1]. The genome symbols of 2× D. villosum and D. breviaristatum were assigned to V and Vb, respectively [2]. Baum et al. [3] suggested the genome constitution of 4× D. breviaristatum as VVVbVb. The Dasypyrum species carried agronomically important genes for multiple disease resistance, end-use grain quality and drought tolerance [4]. The chromatin of D. villosum has been introgressed to wheat for at least six decades, and several genes for resistance to foliar diseases have been successfully transferred to different wheat background [5,6,7,8]. Above all, the 6VS.6AL translocation chromosome carrying the Pm21 gene conferring powdery mildew resistance is now an indispensable component of wheat cultivars in China. Over 40 commercial varieties carrying the 6VS.6AL have been cultivated on large areas [9] and the Pm21 gene itself from 6VS.6AL has been cloned [10,11,12,13]. In addition, the research has also been conducted with the aim of transferring useful genes from D. breviaristatum into wheat. A wheat-D. breviaristatum partial amphiploid [14] and several wheat-D. breviaristatum addition, substitution and translocation lines with novel agronomic traits have been developed and identified by conventional cytogenetic methods [15,16,17,18,19,20].
Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) are the most efficient techniques to detect alien chromosomes or segments in common wheat background [21]. A low-cost and high throughput non-denaturing FISH (ND-FISH) technology is powerful for identifying a large number of plants from wheat-alien hybridization [22], and the ND-FISH probes suitable for chromosome recognition of different wild Triticeae species were also developed [23,24,25]. Moreover, the Oligo-FISH painting systems using the bulked pools of 40–50 bp lengths of single-copy sequences have successfully enabled the assignment of chromosomes to specific linkage groups in Triticeae species [26], and hence are potentially powerful for the detection of Dasypyrum chromatin in diversified wheat backgrounds.
In the present study, we precisely identified D. breviaristatum chromosomes using ND-FISH and Oligo-FISH painting by multiple probes. A wheat-D. breviaristatum line D2138 and the F2-F3 progenies of D2138 with three wheat lines were characterized by ND-FISH and molecular markers. Thus, the objectives of this study were to (1) reveal the chromosome variation of the D2138 derivatives; (2) identify the chromosome deletion and translocation lines for physical mapping of gene(s) on 6VbS; (3) assess the agronomic and quality traits of new wheat-D. breviaristatum derivatives for breeding purposes.

2. Materials and Methods

2.1. Plant Materials

D. breviaristatum accession PI 564517 was obtained from the National Small Grains Collection at Aberdeen, Idaho, USA. The wheat-D. breviaristatum partial amphiploid TDH-2 (genome AABBVbVb) was as described by Yang et al. [14]. Lines TDV-1 (AABBVV), D11-5 (2Vb substitution, Li et al. [16]), D2176 (1Vb addition, Wang et al. [20]), D2150 (5Vb addition, Zhang et al. [18]), D2139 (7Vb addition, Li et al. [17]) and D2532 (3VbS.2VbL, Yu et al. [25]) were maintained in our lab at the University of Electronic Science and Technology of China. Line Pm97034 was obtained from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and line 92R137 was provided by the Cytogenetics Institute, Nanjing Agricultural University. Line D2138 was obtained from a BC1F4 generation of the crosses between wheat cultivar ‘Mianyang 11’ (MY11) and TDH-2. The F2 to F4 lines from D2138 crossed to MY11, Chuanmai62 (CM62) and Jimai22 (JM22) were used to analyze the chromosomal variation and agronomic traits.

2.2. Fluorescence In Situ Hybridization (FISH)

The root tips chromosome preparation protocol followed the procedure of Han et al. [27]. The synthetic oligonucleotides Oligo-B11, Oligo-pDb12H, Oligo-k288, Oligo-D, Oligo-pSc119.2 and Oligo-pTa535 [22,23,25] were either 5′ end-labelled with 6-carboxyfluorescein (6-FAM) for green or 6-carboxytetramethylrhodamine (TAMRA) for red signals. The non-denaturing FISH (ND-FISH) by the synthesized probes was described by Yu et al. [25]. After the oligo-based FISH, the sequential FISH with bulk painting with oligos was conducted following the description by Li and Yang [28]. The pictures of FISH results under Olympus BX-53 microscope were taken by a DP-70 CCD camera.

2.3. Molecular Marker Analysis

DNA was extracted from young leaves of D. breviaristatum, TDH-2, D2138, and derived lines were extracted [29]. The CINAU (Cytogenetics Institute, Nanjing Agricultural University, Nanjing, China) primers [30] for the physical location in specific chromosomes were obtained by searching the database of Wheat Genome Assembly ref. v1.0. The PCR protocol and the amplified products were separated by 8% PAGE gel as described by Hu et al. [31].

2.4. Gliadin Electrophoresis and Gene Sequences Analysis

Acid polyacrylamide gel electrophoresis (APAGE) for seeds gliadin separation were described by Yang et al. [14]. The AS-PCR primers referred Li et al. [32] were used for mapping and cloning of Dasypyrum specific α-gliadin genes, The alignment for the resulted α-gliadin genes was subjected to phylogenetic analysis by using the new version of the MEGA software [33]. The presence or absence of T cell stimulatory epitopes, the innate peptide p31–43 and the 33-mer peptide for gluten sensitive persons were detected from the ammino acid sequences of α-gliadin genes as reported [32,34,35].

2.5. Agronomic Traits and Grain Quality Observation

The agronomic traits observations were collected from two field replications at the Xindu Experimental Station, Chengdu, China during the 2019–2021 seasons. The ten seedlings after FISH examination were grown on each replicate row of 1.5 m long, with a 25 cm row spaced. About 10–30 individuals with presence or absence of alien chromosome constitution from different progenies of the three populations were measured for comparison. The protein content, wet gluten content, Zeleny sedimentation value, water absorption and grain hardness of whole grains from the harvested plants were determined using the near infrared spectroscopy DA7250 (Perten, Hägersten, Sweden) according to the manufacturers’ instructions. The software SPSS 20 (IBM, Armonk, NY, USA) was used for statistical analysis of the measured data.

3. Results

3.1. Karyotyping of Dasypyrum breviaristatum Revealed by Sequential Oligo-Fish Painting

The 4× D. breviaristatum is a species maintaining high levels of cross-pollination. Hence, it is difficult to distinguish each of the chromosome pairs and determine the linkage groups of the chromosomes based on conventional ND-FISH with probes Oligo-pTa535 and Oligo-pSc119.2. In the present study, we were able to establish a standard karyotype of 4× D. breviaristatum by sequential ND-FISH and Oligo-FISH painting by oligo pools of Synt1 to Synt7. The mitotic metaphase chromosomes of the D. breviaristatum accession PI564517 were firstly hybridized with probes Oligo-pSc119.2, Oligo-pTa535 (Figure 1a), and the 28 D. breviaristatum chromosomes could be easily distinguished by the FISH hybridization patterns. By using the sequential Oligo-FISH painting with Synt1 to Synt7, the D. breviaristatum chromosomes were assigned according to linkage groups one to seven for each four chromosomes (Figure 1). The results suggest that at least three groups of D. breviaristatum chromosomes have undergone significant structural change to be detected by FISH. As shown in Figure 1, the comparison of FISH patterns of each four copies of the Vb chromosomes indicated that the hybridization sites of Oligo-pSc119.2 on the telomeric and sub-telomeric regions of both arms or one arm were high polymorphic, while the FISH patterns of Oligo-pTa535 appeared stable among different copies of the chromosomes. Only the four copies of chromosome 4Vb showed similar FISH patterns.
Li et al. [26] established the karyotype of the seven pairs of homologous D. breviaristatum chromosomes in line TDH-2 using FISH by probes Oligo-pTa535, Oligo-pSc119.2 and Synt1 to Synt7. The FISH karyotypes of D. breviaristatum in wild species and those in the TDH-2 background showed that D. breviaristatum chromosomes 1Vb to 7Vb [26] of TDH-2 were mostly like chromosomes 1Vb-III, 2Vb-III, 3Vb-I, 4Vb-I, 5Vb-II, 6Vb-I and 7Vb-IV of D. breviaristatum PI564517 (Figure 1e), respectively. The results indicated that individual Dasypyrum chromosome pairs, when transferred from the 4× grass (VbVbVbVb) to the wheat-D. breviaristatum partial amphiploid TDH-2 (AABBVbVb), may become stable, which allows reliable identification of these D. breviaristatum chromosomes in subsequent wheat background.

3.2. Characterization of Wheat-Dasypyrum breviaristatum Addition Line D2138

Line D2138 with 2n = 44 was obtained from a BC1F4 generation of the cross between TDH-2 and ‘Mianyang 11’ (MY11) wheat. Sequential ND-FISH using probes Oligo-pSc119.2, Oligo-pTa535 was performed to characterize the chromosome constitution of D2138 (Figure 2). The ND-FISH results showed a pair of chromosomes in D2138 with both faint and strong Oligo-pSc119.2 hybridization sites at the telomeric region of both arms, and clear hybridization signals of Oligo-pTa535 in sub-telomeric and centromeric regions (Figure 2a). The FISH pattern of the chromosomes was not identical to a complete D. breviaristatum chromosome (Figure 1e). We thus concluded that D2138 carried a rearranged D. breviaristatum chromosome.
The Oligo-FISH painting method was used to reveal the constitution of chromosomes in D2138. The probe Synt2 produced strong green signals on the chromosome pairs of 2A, 2B and 2D (Figure 2b), and the probe Synt6 generated clear red signals on the chromosome 6A, 6B and 6D (Figure 2b) along their chromosome entire lengths, respectively. The D. breviaristatum chromosome in D2138 revealed by Synt2 and Synt6 that the rearranged chromosome was in fact a translocation involving the 2Vb and 6Vb chromosomes arms. A comparison to the karyotype of seven pairs of D. breviaristatum chromosomes using Oligo-pTa535 and Oligo-pSc119.2 is illustrated in Figure 1. We confirmed that the D. breviaristatum chromosome translocation in D2138 is 6VbS.2VbL (Figure 2c,d).

3.3. Transmission of D. breviaristatum 6VbS and 2VbL in D2138 and Wheat Hybrids

A study was undertaken to determine the transmission of the D. breviaristatum 6VbS.2VbL of D2138 in different wheat backgrounds. D2138 was crossed as the female with the wheat cultivars MY11, JM22 and CM62 and the individual progeny plants were identified by ND-FISH. The standard karyotype of parent lines MY11, JM22 and CM62 by ND-FISH with probes Oligo-pSc119.2 and Oligo-pTa535 is shown in Figure S1. Lines MY11 and JM22 had a normal wheat karyotype, while CM62 contained the reciprocal chromosome translocations of 5BS.7BS and 5BL.7BL. The three lines showed a slight FISH polymorphism for the distribution of Oligo-pSc119.2 signals on chromosomes 5A, 2B, 6B and 3D. The distinct FISH karyotype of the three lines can be effectively used for the identification of any chromosome rearrangements in the D2138 derived progenies.
A total of 818 individual F2 plants including 270, 286, 262 plants from the hybrids of CM62, JM22 and MY11 with D2138, respectively, were screened by ND-FISH using multiple probes Oligo-pDb12H, Oligo-B11, Oligo-k288, Oligo-D and Oligo-pSc119.2 + Oligo-pTa535. The chromosome numbers of the 818 plants were analyzed (Table S1). Up to 132 plants (16%) had 43 chromosomes, while telocentric chromosomes were observed in 24% plants. Based on the comparison of the karyotype of D2138 and the other wheat parental lines, different types of chromosome variations of D. breviaristatum and wheat chromosomes were demonstrated (Table 1, Figure S2). Among the 818 plants, only six plants had two 6VbS.2VbL chromosomes, and 154 plants (about 18%) contained monosomic 6VbS.2VbL chromosome, indicating the low transmission rates of 6VbS.2VbL. Sixty plants carried a 6VbS telosome and 81 plants had a 2VbL telosome. A total of 17 and eight plants contained iso-telosomic 6VbS.6VbS and 2VbL.2VbL, respectively, indicating the breakage of 6VbS.2VbL, and the subsequent re-fusion of the telosomic 6VbS and 2VbL arms. Among the three hybrid combinations, the transmission rate of 6VbS in population D2138 × CM62 (48 plants) was higher than that involving crosses with MY11 (23 plants) and JM22 (12 plants), while a lower frequency of 2VbL transmission rate was observed in D2138 × CM62 progenies. Meanwhile, FISH patterns of the 6VbS.2VbL chromosome revealed about 11 types of showing clear deficiencies occurring on both 6VbS and 2VbL arms (Figure 3a). In total, of all the F2 plants studied, nine chromosome arms had translocated to 6VbS, and four chromosome arms translocated to 2VbL were also observed (Figure 3b). These wheat-D. breviaristatum translocation chromosomes with complicated translocations were observed in about 1.2% plants (Figure 3b).
FISH revealed the presence of aneuploidy and chromosome rearrangements in the introgression lines. About 26.9% (220 of 818) of plants showed chromosome variation in the F2 generations, 119 plants were aneuploids, while others had deletions or translocations among wheat chromosomes (Figure 3). Most of the wheat chromosomal aberrations were observed in the D2138 derived plants lacking either one or both 6VbS.2VbL chromosomes (Figure S2). Among the different groups of cross combinations, the F2 plants of D2138 × CM62 showed lowest chromosomal variations for wheat with 12 types of structural alterations. However, about 30–38 types of deletions and translocations were noticed in progeny from the crosses of D2138 with MY11 and JM22. The results clearly show that different rates of chromosome alteration, possibly induced by monosomic 6VbS.2VbL, occurred in the different wheat background. Five types of B-B and six types of B-D chromosomes translocations were frequently observed (Figure 3c). The deletions or telomeric wheat chromosomes were detected in B-genome chromosomes (17 types) involving 1B-7B, while A-genome chromosomes (eight types) and D-genome chromosomes (two types) were lower frequency (Figure S3).
We observed that in progeny of a monosomic 6VbS addition line from the cross D2138 × CM62, the plants with disomic 6VbS reached 36.6% (34 of 93), whereas only one plant with monosomic 6VbS was found. About 33 out of 34 plants with disomic 6VbS occurred in the background of homozygous 5BS.7BS and 5BL.7BL chromosomes. The present analysis detected a high frequency of chromosomal instability in the offspring of monosomic 6VbS plant, while the progenies derived from plants carrying disomic 6VbS were cytologically stable.

3.4. Identification and Physical Location of 6VbS.2VbL Chromosome Deletions

Lines D76, D64, D54 and D23 containing four types of the homozygous 6VbS.2VbL deletions were recovered in F3 progenies from hybrids of D2138 and wheat (Figure 4). D76 contained 42 chromosomes with a pair of broken 6VbS.2VbL at the deletion breakpoint in FL0.5 length of 2VbL (designated 6VbS.2VbL-1). D64 contained the 6VbS.2VbL with the deletion breakpoint located closed to the centromeric region at FL0.2 length of 2VbL (designated 6VbS.2VbL-2). D54 contained the broken 6VbS.2VbL with the breakpoint on 6VbS located at the Oligo-pTa535 signal of sub-telomeric site (designated 6VbS.2VbL-3). D23 contained the 6VbS.2VbL chromosomes deletion breakpoint located closed to the centromeric region at FL0.15 length of 6VbS (designated 6VbS.2VbL-4). As indicated in Figure 4, all four types of 6VbS.2VbL deletion lines were confirmed by the sequential FISH with the Dasypyrum-specific probe Oligo-pDb12H. All four 6VbS.2VbL deletion lines were transferred into the next generation after self-pollination for further mapping studies to locate molecular markers and agronomically important gene(s).
Zhang et al. [30] designed CINAU primers from D. villosum 6V genomic DNA sequences. Wang et al. [19,20] reported that the CINAU markers were useful for targeting D. breviaristatum 2Vb chromosome-specific regions. A total of 75 CINAU markers previously mapped onto the short arms of group 6 were tested for D. breviaristatum 6VbS amplification, compared to its wheat parent MY11. A total of 36 pairs of primers produced identical bands from 6VbS of D2138 and 6VS of 92R137 and Pm97034, 39 markers showed polymorphic amplification between 6VbS and 6VS from these lines. The results revealed that extensive sequence divergence may have accumulated among the Dasypyrum species in their evolutionary process. Four markers located on the telomeric region were absent in deletion line 6VbS.2VbL-3 in D54, and 16 markers showed no amplification for 6VbS.2VbL-4 in line D23 (Figure 4). The DNA sequences of all the CINAU primers were compared using the BLAST algorithm to the wheat genome database reference version of IWGSC WGA v1.0. The locations of linkage group 6 CINAU markers showed considerably match with the FISH patterns in 6VbS deletions, while those on 2A, 2B and 2D match 2VbL, respectively (Figure 4). Comparing the physical locations of those CINAU markers on 6D, the breakage point is estimated to be located at 25Mb on 6VbS.2VbL-3 in D54 and at 170Mb on 6VbS.2VbL-4 in D23, respectively. These markers allow tracing the transmission of the specific 6VbS chromatin in wheat background by PCR methods.

3.5. Characterization of New Wheat-D. breviaristatum Translocation Lines

We set out to recover plants homozygous for the wheat-2VbL and wheat-6VbS translocations from the F4 generation of hybrids between D2138 and wheat. Translocations involved 6VbS, including 6BS.6VbS and 3DS.6VbS were also obtained in 11–25% of plants (Figure 5a–d). Homozygous wheat-D. breviaristatum 2AS.2VbL and 2DS.2VbL translocations with stable transmission were observed (Figure S4). These wheat-2VbL translocation chromosomes displayed higher transmission rates than those translocations with 6VbS involving non-homologous chromosomes in the latter selfed progenies.

3.6. Sequences of α-Gliadin Genes Located on 6VbS

The acid polyacrylamide gel electrophoresis (APAGE) produced distinctive bands in the α/β, γ and ω zones of storage gliadin proteins from seeds of wheat-D. breviaristatum derivatives and wheat lines using Chinese Spring (CS) as a control (Figure 6a). We found that the CS-D. breviaristatum partial amphiploids TDH-2 and D2138 displayed identical specific bands in α/β zone, but these were absent in wheat parents MY11 and JM22. The F3 lines from the cross between D2138 and MY11 were also studied by APAGE. Results showed that the lines with 6VbS-1, 6VbS-2 also carried the specific gliadin products, and the lines F3–5 and F3–7 were missing those bands. Presumably the D. breviaristatum 6VbS chromosome may be responsible for distinct Vb specific gliadin bands with electrophoretic mobility of α-gliadin regions.
Li et al. [32] produced three SCAR-PCR markers to targeting the Dasypyrum specific α-gliadin genes. In the present study, the α-gliadin gene specific primers were used to screen the wheat-D. breviaristatum derivatives with different Vb chromosomes introgressed (Figure S5). We found that the line D2138 with 6VbS displayed a specific amplification, while the 2Vb containing line D11-5 and other wheat-D. breviaristatum derivatives were absent of amplification. It was then possible to determine locations of the D. breviaristatum α-gliadins on 6VbS of D2138. We further mapped the PCR product of D. breviaristatum α-gliadin genes on the 6VbS.2VbL deletion chromosomes and found that the line carrying 6VbS.2VbL-1 to 6VbS.2VbL-3 was positive for the PCR product, while the line carrying 6VbS.2VbL-4 did not show any PCR amplification (Figure S5). The results indicated that the D. breviaristatum α-gliadin genes are located between FL0.10-0.80 on chromosome 6VbS, with a presumed physical location on about the 20Mb to 170Mb region of 6VbS.
The positive PCR products specifically targeting the D. breviaristatum α-gliadin genes on 6VbS from D2138 were cloned for sequencing. Out of the 14 D. breviaristatum α-gliadin genes sequences (PJ1-14) ranging from 884bp to 984bp, three sequences PJ1, PJ10 and PJ12 with complete ORF of 297, 328 and 328 amino acids, respectively. After searching for the innate p31–43 and the 33-mer peptides in the amino acid sequences, none of the toxic epitops variants for gluten sensitive persons were detected in these 6Vb derived α-gliadin genes. The remaining 11 sequences were pseudogenes due to the in-frame stop codon that occurred at non-repetitive domains at C-terminal domains. The present 6VbS derived α-gliadin sequences and the reported D. villosum and D. breviaristatum gene sequences were used to construct a phylogenetic tree (Figure 6b). The result indicated that the D. villosum and D. breviaristatum α-gliadin gene sequences were clearly separated as two clusters, and the present 6VbS gliadin genes and 4× D. breviaristatum gliadin sequences were clustered in one group.

3.7. Plant Agronomic Traits and Grain Quality Observation

The agronomic traits were measured on plants of the wheat-D. breviaristatum derivatives lines D2138 and F2 progenies from the crosses between D2138 and wheat parents MY11, CM62 and JM22 grown in the field during 2019 and 2021 seasons (Table 2). Both the plant height and tiller number per plant were significantly increased in D2138 and the derived progenies with 6VbS. This suggests that 6VbS may carry gene(s) for increasing the plant height and enhanced tiller number per plant in the wheat background (Figure S6). In addition, spikes of the disomic 6VbS addition line have supernumerary florets, which give more compact spikes than those of CM62. It is thus clearly shown that the 6VbS introgression lines may have great potential to yield improvement.
Grain protein content (GPC), wet gluten content (WGC), Zeleny sedimentation value (ZEL), water absorption (ABS), and grain hardiness values (GH) were assessed for D2138, its derived progenies and their wheat parents MY11, CM62 and JM22 (Table 2). Line D2138 showed an increased GPC, WGC, ZEL, GH values, indicating higher dough strength compared to the wheat lines. Plants carrying disomic 6VbS in the progenies derived from crosses between D2138 and MY11, JM22 and CM62 displayed increased protein contents and grain hardness compared with plants without 6VbS. The results indicated that the 6VbS may have potentially positive effects on improving the quality of wheat.

4. Discussion

The molecular and cytogenetic evidence have suggested the distinct chromosomal divergence between D. villosum and D. breviaristatum species [3,4,36,37]. Recently, Yu et al. [25] constructed a standard karyotype of 1Vb–7Vb chromosomes through ND-FISH identification of the wheat-D. breviaristatum partial amphiploid TDH-2. In the present study, we firstly produced the standard FISH karyotype of four copies of 1Vb–7Vb chromosomes of wild 4× D. breviaristatum based on the sequential ND-FISH and Oligo-FISH painting technique (Figure 1). The heterogeneity for heterochromatin content of each D. breviaristatum chromosome is thought to play an important role in evolutionary divergence between diploid and tetraploid Dasypyrum species, which may be related to the adaptation of their environments for the open-pollinated species [38,39]. Meanwhile, the large number of documented interspecific and intraspecific chromosome variations and significant genomic diversification among different Dasypyrum accessions will benefit the incorporation of novel diversity for future wheat improvement using wild Dasypyrum resources by wide hybridization.
The monosomic alien addition and substitution lines are considered to be linking bridges for the desirable genes transfer from wild species through chromosomal manipulation [40,41]. A single alien chromosome added to the wheat genome could induce a few structures alterations of wheat chromosomes [42,43]. Taking advantage of fast multicolor FISH methods and high-resolution karyotyping of D. breviaristatum chromosomes by present study and Yu et al. [25], we precisely identified the 6VbS.2VbL in D2138 and discovered different types of chromosomal changes from D2138 derived progenies. Wang et al. [19] identified the translocations involved 2VbL including T6DS.6DL-2VbL, T7BS.7BL-2VbL, and T2BL.2VbL from the crosses of the 2Vb(2D) line D11-4 and wheat. The present study developed new lines with homozygous compensating translocations 2AS.VbL and 2DS.2VbL, which will be useful for revealing the effect of 2VbL for breeding purposes.
The alien chromosomes carrying gametocidal (Gc) genes causing a high frequency of chromosome breakage and the chromosomal mutations were frequently detected in the progenies of wheat plants [44,45,46]. Gametocidal genes have been located mostly on homoeologous groups 2, 3, 4 chromosomes of diploid and polyploid Aegilops species [45,47]. It is also observed that a group 6 chromosome of Ae. speltoides carries Gc-like genes [39,40]. In the present study, 818 plants were detected in the F2 generation from the crosses between D2138 with 6VbS.2VbL and three wheat lines. We revealed that 11.9% plants contained translocation and deletion chromosomes. The structural changes in wheat and D. breviaristatum chromosomes may be associated with transmission of 6VbS.2VbL. Moreover, most of the wheat chromosomal aberrations were observed in the D2138 derived plants lacking either one or both 6VbS.2VbL, suggesting that chromosome 6VbS.2VbL has a Gc-like gene. The chromosomal aberrations occurred at higher frequencies in plants lacking 6VbS (28%), but also were detected at lower frequencies (8%) in plants with mono- or disomic 6VbS. We also detected a high frequency of chromosomal instability in the offspring of monosomic 6VbS plants, whereas progenies derived from disomic 6VbS plants became cytologically stable. It is likely that the gametocidal action may possibly be located on 6VbS instead of on 2VbL. The Gc-like genes induced the breakage of chromosomes, and the broken chromosomes may fuse with other broken chromosomes, which would further generate complicated translocations or aberrations [46]. The presence of Gc-like gene in 6VbS may cause enough chromosomal variation from progenies of hybrids, which is potentially useful of wheat-alien transfer. Our observation for D2138-derived progenies indicated that the distribution ratio of fracture sites in deletion lines involved the wheat chromosomes from B group > A group > D group. The chromosome rearrangement frequently occurred in the repetitive sequences-rich regions, in which the B- genome has a high percentage of heterochromatin and tandem repeats contents [24]. The self-pollination of the materials to retain the deletion of chromosomes or the wheat-6VbS or 2VbL translocation lines is recommended for further physical mapping of interesting genes on target region.
Shewry et al. [48] reported that the D. villosum chromosomes 4V and 6V carried Gli-V2 and Gli-V3, respectively. The α-gliadin genes of D. villosum have been mapped onto the short arm of chromosome 6V. Li et al. [32] produced three SCAR-PCR markers to target the α-gliadin gene sequences for D. villosum and D. breviaristatum. In the present study, we further located the D. breviaristatum-specific gliadin genes on telomeric region of 6VbS by APAGE and PCR markers. Moreover, the present study produced 6VbS derived α-gliadin sequences that lacked innate p31–43 and 33-mer peptides for gluten sensitive persons. We also screened a wide set of Dasypyrum α-gliadin sequences [32] and found that none of these sequences had the 33-mer peptide. It may provide probabilities to select less toxic germplasm for gluten sensitive persons or celiac disease patients by introgression of 6VbS derived α-gliadin genes. The polygenetic analysis confirmed that the D. villosum and D. breviaristatum contained a high diversity and percentage of pseudogenes for the α-gliadin family on 6VS or 6VbS. De Pace et al. [49] investigated the grain quality effect of wheat-D. villosum introgression lines and found that the protein content was significantly improved. Wang et al. [9] revealed that the 6VS.6AL translocation has no risk of reducing the grain end-use quality. Vaccino et al. [50] also reported that chromatin from D. villosum 6VS improves protein and micronutrient content by small and large-scale tests in CS background [9]. We revealed that the wheat-6VbS introgression lines bestow positive effects on grain protein contents and hardness in different wheat backgrounds (Table 2), which is due to the introduction of additional multi-copies of gliadin genes from 6VbS. The gliadins frequently present in the monomer or polymer fraction may have positive effects on the physical properties of wheat flour dough [48,50]. It is possible that some gliadins do contribute positively to different end-use quality, but the overall amount and composition of gliadins from alien species may need to be examined according to specific wheat backgrounds.

5. Conclusions

In summary, the distinctive FISH patterns between D. breviaristatum and D. villosum chromosomes were observed clearly by using probes of different repetitive sequences and the single-copy oligo pools. This allowed identification of individual Dasypyrum chromosomes of the wild species and also transferring them into a wheat background. The heterozygous FISH patterns of tetraploid D. breviaristatum chromosomes confirmed a high level of genetic diversity within this species that may be exploited for the ongoing transfer of D. breviaristatum chromatin to wheat. D. breviaristatum-specific cytogenetic and molecular markers can be used to trace the chromosomes or chromosome segments of Dasypyrum introgressed to wheat. We identified wheat-D. breviaristatum chromosome 6VbS and 2VbL derivatives with novel agronomic and quality characters that will be potentially useful for wheat breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12071673/s1, Figure S1: ND-FISH of mitotic metaphase of wheat parents CM62, MY11, and JM22 by probes Oligo-pTa535 (red) + Oligo-pSc119.2 (green); Figure S2: The distribution of different types of chromosome variation of F2 plants derived from D2138 and three wheat hybrids; Figure S3: The wheat chromosome variation from the F2 progenies between D2138 and three wheat lines; Figure S4: FISH of wheat-D. breviaristatum 2VbL translocation lines D310 and D334 from F4 progenies of D2138 and MY11 hybrids; Figure S5: PCR amplification of D. breviaristatum-specific gliadin gene marker on wheat-D. breviaristatum introgression lines; Figure S6: The adult plant morphology of the lines D2138, JM22, and their F2 plants; Table S1: Chromosome number variation of F2 plants derived from hybrids between D2138 and three wheat lines.

Author Contributions

Z.Y. and G.L. designed the research; C.J., W.J., M.L. and H.W. performed experiments; Z.Y., E.Y. and C.J. analyzed the data; Z.Y. and G.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31971886), and the International Cooperation Project (2022YFH0012) of the Science and Technology Department of Sichuan, China.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available on request. Data are contained within the article.

Acknowledgments

We would like to thank I. Dundas at University of Adelaide, Australia, for reviewing the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Karyotyping of mitotic metaphase of D. breviaristatum by sequential ND-FISH and Oligo-FISH painting. The sequential ND-FISH by probes (a,c) Oligo-pTa535 (red) + Oligo-pSc119.2 (green), Oligo-FISH painting by probes (b) Synt7 (green) + Synt1 (red), (d) Synt6 (red) + Synt5 (green), respectively. (e) The karyotype of 28 D. breviaristatum chromosomes is shown.
Figure 1. Karyotyping of mitotic metaphase of D. breviaristatum by sequential ND-FISH and Oligo-FISH painting. The sequential ND-FISH by probes (a,c) Oligo-pTa535 (red) + Oligo-pSc119.2 (green), Oligo-FISH painting by probes (b) Synt7 (green) + Synt1 (red), (d) Synt6 (red) + Synt5 (green), respectively. (e) The karyotype of 28 D. breviaristatum chromosomes is shown.
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Figure 2. Karyotyping of mitotic metaphase of wheat-D. breviaristatum D2138 by sequential ND-FISH and Oligo-FISH painting. Sequential ND-FISH by probes (a) Oligo-pTa535 (red) + Oligo-pSc119.2 (green), and (b) Oligo-FISH by bulk painting with “Synt” probes Synt2 (green) + Synt6 (red), respectively. The karyotype (c) and diagram (d) of D. breviaristatum chromosomes 6VbS.2VbL are shown.
Figure 2. Karyotyping of mitotic metaphase of wheat-D. breviaristatum D2138 by sequential ND-FISH and Oligo-FISH painting. Sequential ND-FISH by probes (a) Oligo-pTa535 (red) + Oligo-pSc119.2 (green), and (b) Oligo-FISH by bulk painting with “Synt” probes Synt2 (green) + Synt6 (red), respectively. The karyotype (c) and diagram (d) of D. breviaristatum chromosomes 6VbS.2VbL are shown.
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Figure 3. The chromosome variations from the F2 progenies between D2138 and three wheat lines. The probes Oligo-pTa535 and Oligo-pSc119.2 were used for ND-FISH to show the different types of chromosome variations including the modified D. breviaristatum chromosomes (a), wheat-D. breviaristatum translocations (b) and wheat chromosome translocations (c).
Figure 3. The chromosome variations from the F2 progenies between D2138 and three wheat lines. The probes Oligo-pTa535 and Oligo-pSc119.2 were used for ND-FISH to show the different types of chromosome variations including the modified D. breviaristatum chromosomes (a), wheat-D. breviaristatum translocations (b) and wheat chromosome translocations (c).
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Figure 4. Physical location of D. breviaristatum 6VbS.2VbL variations by specific markers. Chromosomes on the left show the 6VbS.2VbL deletion chromosomes by FISH using probes Oligo-pSc119.2 (green) and Oligo-pTa535 (red). The diagram on the right shows the physical locations of CINAU markers on 6VbS.2VbL with the “+” represent amplification, while “−” represent absent of the D. breviaristatum-specific bands.
Figure 4. Physical location of D. breviaristatum 6VbS.2VbL variations by specific markers. Chromosomes on the left show the 6VbS.2VbL deletion chromosomes by FISH using probes Oligo-pSc119.2 (green) and Oligo-pTa535 (red). The diagram on the right shows the physical locations of CINAU markers on 6VbS.2VbL with the “+” represent amplification, while “−” represent absent of the D. breviaristatum-specific bands.
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Figure 5. FISH of wheat-D. breviaristatum 6VbS translocation lines D230 and D237 from F4 progenies of D2138 and MY11 hybrids. The probes Oligo-D + Oligo-pDb12H (a), Oligo-k288 + Oligo-pDb12H (c), Oligo-pSc119.2 + Oligo-pTa535 (b,d) are used for ND-FISH, respectively. Lines D230 (a,b), D237 (c,d) contained a pair of wheat-D. breviaristatum chromosome translocations 6VbS.3DS and 6VbS.6BS, respectively.
Figure 5. FISH of wheat-D. breviaristatum 6VbS translocation lines D230 and D237 from F4 progenies of D2138 and MY11 hybrids. The probes Oligo-D + Oligo-pDb12H (a), Oligo-k288 + Oligo-pDb12H (c), Oligo-pSc119.2 + Oligo-pTa535 (b,d) are used for ND-FISH, respectively. Lines D230 (a,b), D237 (c,d) contained a pair of wheat-D. breviaristatum chromosome translocations 6VbS.3DS and 6VbS.6BS, respectively.
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Figure 6. The separation of gliadin proteins and phylogenetic tree of D. breviaristatum-specific α-gliadin gene sequences. (a) The gliadin separated by APAGE gel with the arrow indicates the D. breviaristatum-specific band. (b) The Phylogenetic tree based on the coding sequences of the α-gliadin genes from Dasypyrum species was generated by MEGA11 using two γ-gliadin gene sequences as outgroup. The bootstrap values were obtained using 1000 replications.
Figure 6. The separation of gliadin proteins and phylogenetic tree of D. breviaristatum-specific α-gliadin gene sequences. (a) The gliadin separated by APAGE gel with the arrow indicates the D. breviaristatum-specific band. (b) The Phylogenetic tree based on the coding sequences of the α-gliadin genes from Dasypyrum species was generated by MEGA11 using two γ-gliadin gene sequences as outgroup. The bootstrap values were obtained using 1000 replications.
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Table
Table 1. Chromosome constitutions of F2 plants derived from D2138 and three wheat hybrids.
Table 1. Chromosome constitutions of F2 plants derived from D2138 and three wheat hybrids.
HybridsF2
Plants		Disomic
6VbS.2VbL		Monosomic
6VbS.2VbL		Monosomic
2VbL		Monosomic
6VbS		Monosomic
iso-2VbL		Monosomic
iso-6VbS		Wheat
Chromosome
Variation
D2138 × CM6227015624486645
D2138 × MY1126245224233-79
D2138 × JM2228614612117296
Total81861546081178220
Table
Table 2. Agronomic and grain quality traits of D2138 derived populations and the wheat parents.
Table 2. Agronomic and grain quality traits of D2138 derived populations and the wheat parents.
LinesPH
(cm)		TPPSLTKW
(g)		GPC
(%)		WGC
(%)		ZEL
(mL)		GHABS
MY1184.23.59.233.614.434.545.559.456.9
CM6295.34.011.859.812.129.126.654.252.4
JM2268.88.89.727.614.340.568.941.159.6
D213891.2 *10.0 *10.3 *33.617.5 *44.2 *67.3 *52.861.4 *
D2138/MY11
−6VbS85.59.010.631.115.339.557.1 *50.461.9
+6VbS91.4 *11.39.732.8 *16.1 *43.5 *50.758.1 *62.4
D2138/CM62
−6VbS93.19.411.349.7 *14.938.144.853.161.8
+6VbS94.911.311.648.216.4 *39.2 *46.7 *59.9 *60.9
D2138/JM22
−6VbS72.27.410.0 *31.7 *14.939.654.851.662.3
+6VbS84.7 *7.89.230.915.58 *39.853.457.2 *63.7 *
PH Plant height, TPP Tillers per plant, SL spike length, TKW thousand-kernel weight, GPC grain protein content, WGC wet gluten content, ZEL Zeleny sedimentation value, GH grain hardness values, ABS water absorption, respectively. * Significant difference at p < 0.05 to the the progenies without homozygous Dasypyrum chromosomes.
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MDPI and ACS Style

Jiang, C.; Jiang, W.; Liu, M.; Wang, H.; Yang, E.; Yang, Z.; Li, G. Characterization of a Wheat-Dasypyrum breviaristatum Chromosome Addition and Its Derived Progenies Carrying Novel Dasypyrum-Specific Gliadin Genes. Agronomy 2022, 12, 1673. https://doi.org/10.3390/agronomy12071673

AMA Style

Jiang C, Jiang W, Liu M, Wang H, Yang E, Yang Z, Li G. Characterization of a Wheat-Dasypyrum breviaristatum Chromosome Addition and Its Derived Progenies Carrying Novel Dasypyrum-Specific Gliadin Genes. Agronomy. 2022; 12(7):1673. https://doi.org/10.3390/agronomy12071673

Chicago/Turabian Style

Jiang, Chengzhi, Wenxi Jiang, Min Liu, Hongjin Wang, Ennian Yang, Zujun Yang, and Guangrong Li. 2022. "Characterization of a Wheat-Dasypyrum breviaristatum Chromosome Addition and Its Derived Progenies Carrying Novel Dasypyrum-Specific Gliadin Genes" Agronomy 12, no. 7: 1673. https://doi.org/10.3390/agronomy12071673

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