CRISPR/Cas9‐mediated knockout of Ms1 enables the rapid generation of male‐sterile hexaploid wheat lines for use in hybrid seed production

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male‐sterile lines were potentially non‐transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.     

Paternal regulation of seed development in wheat hybrids

Summary Diallel crosses among Triticum boeoticum (4 lines from different geographical areas), T.urartu, Aegilops squarrosa and Ae. speltoides exhibited reciprocal differences in hybrid seed morphology, endosperm development, and embryo viability. T. urartu and Ae. squarrosa as females with T. boeotiaum and Ae. speltoides lead to shrivelled inviable seed. T.boeoticum accessions as female with Ae.speltoides also lead to shrivelled seeds. The reciprocal crosses produced plump seeds which either resembled the maternal parent or showed size differences. By altering the endospermic genome ratios, hybrid seeds with 1 (PF)/1 (PM) showed extreme shrivelling whereas those with 4 (PF)/1 (PM) were medium shrivelled to plump. Genetic experiments involving hybrids of T. boeoticum, T. urartu and T. monococcum showed that a factor is present in pollen or male gametes, which shows dosage effect and which, by interacting with the maternal genome, leads to endosperm abortion.     

Gene introgression from common wheat into Aegilops L.

Group of experiments were carried out to verify possibility of gene introgression from common wheat into Aegilops. The artificial indoor crossbreed was conducted using 7 genotypes from 4 wheat relative species as female, and common wheat as male. The experiment result shows that different species has variable cross ability. Among the 4 Aegilops species, the highest cross rate is from the combination of Aegilops tauschii × Triticum aestivum (46.49% for genotype Ae42, 22.58% for Y92), the second is from Aegilops ovata × T. aestivum (14.76% for Y100, 12.11% for Ae23), the third is from Aegilops cylindrica × T. aestivum (2.23% for Ae7, 8.50% for Y145), and the lowest is from Aegilops speltoides × T. aestivum (0.19%). Hybrid embryos from different combinations have different ability of callus initiation and germination. The hybrid embryos from A. ovata/T. aestivum and Ae. tauschii/T. aestivum have a higher level of callus initiation and germination. Ae. cylindrica/T. aestivum has a middle level, while the Ae. speltoides has a lower level. The interspecific hybrids between Aegilops and common wheat have so low fertility. In back-crosses, the seed-set rate of hybrids of Ae. ovata/T. aestivum is 3.71% and 4.36% respectively back-crossed with male and female parents, while for hybrids of Ae. cylindrica/T. aestivum, they were 0 and 0.33% respectively, and for Ae. tauschii/T. aestivum, 0.33% and 0 respectively. On selfing of the hybrids, the seed-set rate is 0 (no seed set from 9750 florets) for the combination of Ae. cylindrica/T. aestivum, 0.044% (3 selfed seeds out of 6870 florets) for Ae. ovata/T. aestivum and 0 (no seed set from 7253 florets) for Ae. tauschii/T. aestivum. The research suggests that the probability of gene introgression from T. aestivum into Aegilops species is very low in nature.     

Exploiting the <Emphasis Type="Italic">Rht</Emphasis> portfolio for hybrid wheat breeding

Key message

The portfolio of available Reduced height loci (Rht-B1, Rht-D1, and Rht24) can be exploited for hybrid wheat breeding to achieve the desired heights in the female and male parents, as well as in the hybrids, without adverse effects on other traits relevant for hybrid seed production.

Abstract

Plant height is an important trait in wheat line breeding, but is of even greater importance in hybrid wheat breeding. Here, the height of the female and male parental lines must be controlled and adjusted relative to each other to maximize hybrid seed production. In addition, the height of the resulting hybrids must be fine-tuned to meet the specific requirements of the farmers in the target regions. Moreover, this must be achieved without adversely impacting traits relevant for hybrid seed production. In this study, we explored Reduced height (Rht) loci effective in elite wheat and exploited their utilization for hybrid wheat breeding. We performed association mapping in a panel of 1705 wheat hybrids and their 225 parental lines, which besides the Rht-B1 and Rht-D1 loci revealed Rht24 as a major QTL for plant height. Furthermore, we found that the Rht-1 loci also reduce anther extrusion and thus cross-pollination ability, whereas Rht24 appeared to have no adverse effect on this trait. Our results suggest different haplotypes of the three Rht loci to be used in the female or male pool of a hybrid breeding program, but also show that in general, plant height is a quantitative trait controlled by numerous small-effect QTL. Consequently, marker-assisted selection for the major Rht loci must be complemented by phenotypic selection to achieve the desired height in the female and male parents as well as in the wheat hybrids.

     

Phylogenetic analysis of tetraploid wheat based on nuclear DMC1 gene

Tetraploid wheat (AABB or AAGG, 2n = 4x = 28) holds an important place in Triticum. It includes two allopolyploid species, Triticum turgidum and Triticum timopheevii. Many problems concerning the phylogenetic relationships among tetraploid wheat species remain unresolved. In this study, sequences data for the nuclear DMC1 gene from 61 accessions of Triticum and Aegilops species, representing diploid and tetraploid species, were used to examine the phylogenetic relationships among tetraploid wheat. Phylogenetic trees were constructed using maximum-likelihood and neighbor-joining approaches, and gene flow and genetic differentiation values were computed. The results indicated that the A genome of tetraploid wheat originated from T. urartu rather than T. monococcum, and Aegilops speltoides was the donor of the B and G genomes. Hulled tetraploid wheat accessions formed a subclade, and naked tetraploid wheat got other subclade, indicating that at least two intermediary subspecies were involved in the evolution of T. turgidum. Triticum turgidum and T. timopheevii might have simultaneously originated from a hybridization events. These results indicated that the DMC1 gene sequences are useful for resolution of the molecular phylogenetic relationships of tetraploid wheat.     

The potential of synthetic hexaploid wheats to improve zinc efficiency in modern bread wheat

Synthetic hexaploid wheats (Triticum aestivum L) derived from crosses between durum wheat [Triticum turgidum ssp. durum (Desf.) Husn.] and diploid wheat (Aegilops tauschii Coss.) have been developed as a means of transferring desirable characteristics of Aegilops tauschii Coss. such as disease resistance and abiotic stress tolerance into modern bread wheat genotypes. In a growth room experiment using soil culture, we studied a group of 30 synthetic hexaploid wheat accessions together with modern wheat genotypes in order to identify new sources of zinc efficiency for further improvement of zinc efficiency in modern wheat genotypes. There was considerable genetic variation in expression of zinc deficiency symptoms (slight to severe), zinc efficiency (70–100%), shoot Zn concentration (5.8–10.5 and 33–53 mg/kg DW under deficient and sufficient Zn, respectively), shoot Zn content (3.8–10.6 and 34.0–64.6 μg/plant, under deficient and sufficient Zn, respectively) and Zn utilization (0.096–0.172 and 0.019-0.033 g DW/μg Zn under deficient and sufficient Zn, respectively) within synthetic accessions. The presence of synthetic accessions with greater zinc efficiency (100%) than zinc efficient modern wheat genotypes (85%) indicates that the synthetic hexaploids can be used to improve current levels of zinc efficiency in modern wheat genotypes. Synthetic hexaploids may also be a good source of high grain Zn concentration (28–66 mg Zn/kg seed DW).     

Introgression of bread wheat chromatin into tall wheatgrass via somatic hybridization

Regenerates were obtained following somatic hybridization between tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum cv. Jinan177) protoplasts. Two lines (CU and XI) were self-fertile in the first (R₀) and subsequent (R₁ and R₂) generations. The phenotype of each R₁ population was uniform. All CU progeny were phenotypically similar to the tall wheatgrass parent, while XI progeny had thinner, smoother and softer leaves. Cytological analysis showed that more wheat chromatin was present in the hybrid callus than in the R₁ and R₂ plants, and that some intercalary translocations of wheat chromosome segments were retained in the R₂ generation. AFLP profiling confirmed the presence of wheat DNA in the introgression lines. Analysis of the high molecular weight glutenin subunit content of derived seed identified three novel subunits, not present in either the wheat or the tall wheatgrass parent. Microsatellite-based profiling of the chloroplast genome of the introgression lines suggested that only chloroplast sequences from the tall wheatgrass parent were present. The specifically inherited phenomena and possible application of these hybrids are discussed. Haifeng Cui and Zhiyong Yu were contributed equally to this article.     

Gene flow in genetically modified wheat

Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow distances can be efficiently estimated with population-level PCR analyses.     

Map-based analysis of the tenacious glume gene Tg-B1 of wild emmer and its role in wheat domestication

The domestication of wheat was instrumental in spawning the civilization of humankind, and it occurred through genetic mutations that gave rise to types with non-fragile rachises, soft glumes, and free-threshing seed. Wild emmer (Triticum turgidum ssp. dicoccoides), the tetraploid AB-genome progenitor of domesticated wheat has genes that confer tenacious glumes (Tg) that underwent genetic mutations to give rise to free-threshing wheat. Here, we evaluated disomic substitution lines involving chromosomes 2A and 2B of wild emmer accessions substituted for homologous chromosomes in tetraploid and hexaploid backgrounds. The results suggested that both chromosomes 2A and 2B of wild emmer possess genes that inhibit threshability. A population of recombinant inbred lines derived from the tetraploid durum wheat variety Langdon crossed with a Langdon — T. turgidum ssp. dicoccoides accession PI 481521 chromosome 2B disomic substitution line was used to develop a genetic linkage map of 2B, evaluate the genetics of threshability, and map the gene derived from PI 481521 that inhibited threshability. A 2BS linkage map comprised of 58 markers was developed, and markers delineated the gene to a 2.3 cM interval. Comparative analysis with maps containing the tenacious glume gene Tg-D1 on chromosome arm 2DS from Aegilops tauschii, the D genome progenitor of hexaploid wheat, revealed that the gene inhibiting threshability in wild emmer was homoeologous to Tg-D1 and therefore designated Tg-B1. Comparative analysis with rice and Brachypodium distachyon indicated a high level of divergence and poorly conserved colinearity, particularly near the Tg-B1 locus. These results provide a foundation for further studies involving Tg-B1, which, together with Tg-D1, had profound influences on wheat domestication.     

Quality of synthetic hexaploid wheat containing null alleles at Glu-A1 and Glu-B1 loci

Triticum turgidum ssp. dicoccon PI94668 and PI349045 were identified as containing null alleles at Glu-A1 and Glu-B1 loci in previous investigation. Sequencing of the respective HMW-GS genes Ax, Bx, Ay and By in both accessions indicated equal DNA lengths with gene silencing caused by 1 to 4 in-frame stop codon(s) in the open reading frames. Six synthetic hexaploid wheat lines were produced by crossing PI94668 or PI349045 with six Aegilops tauschii by spontaneous chromosome doubling of unreduced gametes. As expected, these amphiploids had three different HMW-GS: Dx 3.1^t?+?Dy11*^t, Dx2.1^t?+?10^t and Dx2^t?+?Dy12^t in Glu-D1 but double nulls in Glu-A1 and Glu-B1. Quality tests showed that most quality parameters in two T. turgidum ssp. dicoccon parents were very low due to the lack of HMW-GSs. However, incorporation of HMW-GS from Ae. tauschii in six synthetic hexaploid wheat lines significantly increased most quality related parameters. The potential values of these wheat lines in improving the quality of wheat are discussed.     

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.     

Reproductive and genetic roles of the maternal progenitor in the origin of common wheat (Triticum aestivum L.)

Common wheat (Triticum aestivum L., AABBDD genome) is thought to have emerged through natural hybridization between Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). Hybridization barriers and doubling of the trihaploid F₁ hybrids’ genome (ABD) via unreduced gamete fusion had key roles in the process. However, how T. turgidum, the maternal progenitor, was involved in these mechanisms remains unknown. An artificial cross‐experiment using 46 cultivated and 31 wild T. turgidum accessions and a single Ae. tauschii tester with a very short genetic distance to the common wheat D genome was conducted. Cytological and quantitative trait locus analyses of F₁ hybrid genome doubling were performed. The crossability and ability to cause hybrid inviability did not greatly differ between the cultivars and wild accessions. The ability to cause hybrid genome doubling was higher in the cultivars. Three novel T. turgidum loci for hybrid genome doubling, which influenced unreduced gamete production in F₁ hybrids, were identified. Cultivated T. turgidum might have increased the probability of the emergence of common wheat through its enhanced ability to cause genome doubling in F₁ hybrids with Ae. tauschii. The ability enhancement might have involved alterations at a relatively small number of loci.     

Seed bank dynamics govern persistence of Brassica hybrids in crop and natural habitats

Background and Aims Gene flow from crops to their wild relatives has the potential to alter population growth rates and demography of hybrid populations, especially when a new crop has been genetically modified (GM). This study introduces a comprehensive approach to assess this potential for altered population fitness, and uses a combination of demographic data in two habitat types and mathematical (matrix) models that include crop rotations and outcrossing between parental species.Methods Full life-cycle demographic rates, including seed bank survival, of non-GM Brassica rapa × B. napus F₁ hybrids and their parent species were estimated from experiments in both agricultural and semi-natural habitats. Altered fitness potential was modelled using periodic matrices including crop rotations and outcrossing between parent species.Key Results The demographic vital rates (i.e. for major stage transitions) of the hybrid population were intermediate between or lower than both parental species. The population growth rate (λ) of hybrids indicated decreases in both habitat types, and in a semi-natural habitat hybrids became extinct at two sites. Elasticity analyses indicated that seed bank survival was the greatest contributor to λ. In agricultural habitats, hybrid populations were projected to decline, but with persistence times up to 20 years. The seed bank survival rate was the main driver determining persistence. It was found that λ of the hybrids was largely determined by parental seed bank survival and subsequent replenishment of the hybrid population through outcrossing of B. rapa with B. napus.Conclusions Hybrid persistence was found to be highly dependent on the seed bank, suggesting that targeting hybrid seed survival could be an important management option in controlling hybrid persistence. For local risk mitigation, an increased focus on the wild parent is suggested. Management actions, such as control of B. rapa, could indirectly reduce hybrid populations by blocking hybrid replenishment.     

Variation Associated with an AEGILOPS UMBELLULATA Chromosome Segment Incorporated in Wheat. II. Peroxidase and Leucine Aminopeptidase Isozymes

Zymograms were analyzed of a number of Triticum aestivum derivatives which incorporated a segment of the Aegilops umbellulata chromosome bearing resistance to leaf rust. Evidence has been presented which suggests that genes involved in the production of two peroxidases and a single peptidase are located on the short arm of wheat chromosome 6B. One peroxidase isozyme, attributed to the presence of the Aegilops segment, was seen in only one of the resistant lines (Transfer) and it was postulated that this peroxidase band was present in a suppressed state in a number of lines. Possible differences in the A and B genomes of T. aestivum and T. dicoccum were discussed.     

温敏不育系A3314在中国不同生态地点的育性表现

利用作者参与发明的ZL00105488．0专利方法选育的小麦温度敏感不育系A3314在中国元谋、杨陵、石家庄、互助、依安、贵阳、武威7个不同纬度地点种植的自交结实率,结合各点光温条件的分析表明：A3314在黄淮冬麦区、云贵冬麦区、西北春麦区、东北春麦区各点,按当地小麦生产正季播种均表现稳定雄性不育;而在黄淮和云贵冬麦区春播(夏播)则自交结实,适宜条件下自交结实率可达60％以上。说明该温敏不育系的雄性育性受温度的制约,而与日长无明显相关。根据A3314的育性表现,推测它在中国大部分小麦产区均可安全用于杂交小麦制种。     

Genetic Diversity of the Cytoplasm in Triticum and Aegilops. VIII. Fraction I Protein of 39 Cytoplasms

The electrophoretic characteristics of the cytoplasmically controlled large subunit of the Fraction I protein of 36 alloplasmic and three euplasmic control lines are reported. These lines, representing the cytoplasms of 32 Triticum and Aegilops species, had either H- or L-type large subunits in their Fraction I protein; the diploid Triticum and most Aegilops species, including Ae. bicornis and Ae. sharonensis, had the L-type subunits; whereas, all the polyploid Triticum species (emmer, timopheevi, common wheats), Ae. speltoides, Ae. aucheri, and Ae. longissima had H-type subunits. Therefore, section Sitopsis of Aegilops exhibits interspecific heterogeneity. The H-type is believed to have originated in the Sitopsis section from an L-type subunit because of the prevalence of the latter among the diploid species.     

Genetic architecture of male fertility restoration of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> cytoplasm and fine-mapping of the major restorer locus <Emphasis Type="Italic">Rf3</Emphasis> on chromosome 1B

Key message

Restoration of fertility in the cytoplasmic male sterility-inducing Triticum timopheevii cytoplasm can be achieved with the major restorer locus Rf3 located on chromosome 1B, but is also dependent on modifier loci.

Abstract

Hybrid breeding relies on a hybrid mechanism enabling a cost-efficient hybrid seed production. In wheat and triticale, cytoplasmic male sterility based on the T. timopheevii cytoplasm is commonly used, and the aim of this study was to dissect the genetic architecture underlying fertility restoration. Our study was based on two segregating F₂ triticale populations with 313 and 188 individuals that share a common female parent and have two different lines with high fertility restoration ability as male parents. The plants were cloned to enable replicated assessments of their phenotype and fertility restoration was evaluated based on seed set or staining for pollen fertility. The traits showed high heritabilities but their distributions differed between the two populations. In one population, a quarter of the lines were sterile, conforming to a 3:1 segregation ratio. QTL mapping identified two and three QTL in these populations, with the major QTL being detected on chromosome 1B. This QTL was collinear in both populations and likely corresponds to Rf3. We found that Rf3 explained approximately 30 and 50% of the genotypic variance, has a dominant mode of inheritance, and that the female parent lacks this locus, probably due to a 1B.1R translocation. Taken together, Rf3 is a major restorer locus that enables fertility restoration of the T. timopheevii cytoplasm, but additional modifier loci are needed for full restoration of male fertility. Consequently, Rf3 holds great potential for hybrid wheat and triticale breeding, but other loci must also be considered, either through marker-assisted or phenotypic selection.

     

Distribution of the fertility-restoring gene <Emphasis Type="Italic">Rf3</Emphasis> in common and spelt wheat determined by an informative SNP marker

Male sterility induced by the cytoplasm of Triticum timopheevii Zhuk. has shown potential for hybrid seed production in common wheat (Triticum aestivum L.). As hybrids produced by this method are often partially sterile, fertility restoration is crucial for implementing this technology in breeding practice. Several restorer genes were identified, of which Rf3 is one of the most effective genes for achieving restoration. Previous studies located Rf3 on chromosome 1B in common and spelt wheat. However, the distribution of Rf3 in these taxa remained unclear. In the present study, we genetically mapped Rf3 using a BC₁ population derived from CMS-Sperber and the restorer line Primepi (N = 193). After marker validation in four independent BC₁ populations and a diversity panel, we evaluated the distribution of Rf3 in 524 common wheat and 30 European spelt genotypes. In the mapping population, the SNP marker IWB72107 cosegregated with Rf3, whereas IWB14060 was mapped 2.0 cM distal on chromosome 1BS. Surveying the linkage between IWB72107 and Rf3 in the four validation populations revealed map distances that ranged from 0.4 to 2.3 cM. Validation of IWB72107 in the diversity panel showed that it is suitable for marker-assisted selection and related applications. Using this marker, we estimated that 8.8% of the common wheat lines and 66.7% of the spelt cultivars carried the restoring Rf3 allele. We propose that Rf3 explains the restoration capacity of a large proportion of European common wheat lines.