Prospects of using self-fertility in breeding rye populations varieties

The advances in rye hybrid breeding are due to the use of self-fertile forms. Rye self-fertility is determined by mutations in one of the three gametophytic loci (S, Z, and T), which control the reaction of incompatibility. Attempts to construct synthetic populations by combining self-fertile forms selected by general combining ability failed because of high-rate selfing. A breeding scheme was proposed to include crosses of a line carrying a self-fertility mutation in the S locus with the population subject to improvement, selfing of the resulting hybrids, selection and intermating of the best inbred progenies, and subsequent elimination of the self-fertility mutation from the breeding material with the use of the Prx7 allozyme marker. The scheme can be employed in improvement of the existing rye varieties, their differentiation into populations differing in end use, and construction and improvement of complementary gene pools in hybrid breeding. To facilitate the implementation of the scheme, an original instrument was designed for high-throughput isozyme analysis.     

Fine mapping a self-fertility locus in perennial ryegrass

Key message

A self-fertility locus was fine mapped to a 1.6 cM region on linkage group 5 in a perennial ryegrass population. This locus was the main determinant of pollen self-compatibility.

Abstract

In grasses, self-incompatibility (SI) is characterized by a two-loci gametophytic (S and Z) mechanism acting together in the recognition and inhibition of self-pollen. Mutations affecting the expression of SI have been reported in a few grass species. In perennial ryegrass (Lolium perenne L.), a mutation independent from S and Z, and mapping on linkage group 5 (LG 5), was previously reported to produce self-fertile plants. Here, we describe fine mapping of the self-fertility (SF) gene in a perennial ryegrass population and determine whether there is any effect of other genomic regions on the pollen compatibility. The phenotypic segregation of SF showed a bimodal distribution with one mean at 49% pollen compatibility and the other at 91%. Marker-trait association analysis showed that only markers on LG 5 were significantly associated with the trait. A single gene model explained 82% of the observed variability and no effects of the other regions were detected. Using segregation and linkage analysis, the SF locus was located to a 1.6 cM region on LG 5. The flanking marker sequences were aligned to rice and Brachypodium distachyon reference genomes to estimate the physical distance. We provide markers tightly linked to SF that can be used for introgression of this trait into advanced breeding germplasm. Moreover, our results represent a further step towards the identification of the SF gene in LG 5.

     

Exploring new alleles for frost tolerance in winter rye

Key message

Rye genetic resources provide a valuable source of new alleles for the improvement of frost tolerance in rye breeding programs.

Abstract

Frost tolerance is a must-have trait for winter cereal production in northern and continental cropping areas. Genetic resources should harbor promising alleles for the improvement of frost tolerance of winter rye elite lines. For frost tolerance breeding, the identification of quantitative trait loci (QTL) and the choice of optimum genome-based selection methods are essential. We identified genomic regions involved in frost tolerance of winter rye by QTL mapping in a biparental population derived from a highly frost tolerant selection from the Canadian cultivar Puma and the European elite line Lo157. Lines per se and their testcrosses were phenotyped in a controlled freeze test and in multi-location field trials in Russia and Canada. Three QTL on chromosomes 4R, 5R, and 7R were consistently detected across environments. The QTL on 5R is congruent with the genomic region harboring the Frost resistance locus 2 (Fr–2) in Triticeae. The Puma allele at the Fr–R2 locus was found to significantly increase frost tolerance. A comparison of predictive ability obtained from the QTL-based model with different whole-genome prediction models revealed that besides a few large, also small QTL effects contribute to the genomic variance of frost tolerance in rye. Genomic prediction models assigning a high weight to the Fr–R2 locus allow increasing the selection intensity for frost tolerance by genome-based pre-selection of promising candidates.

     

Evolutionary divergence of the rye <Emphasis Type="Italic">Pm17</Emphasis> and <Emphasis Type="Italic">Pm8</Emphasis> resistance genes reveals ancient diversity

Key message

We have isolated a novel powdery mildew resistance gene in wheat that was originally introgressed from rye. Further analysis revealed evolutionary divergent history of wheat and rye orthologous resistance genes.

Abstract

Wheat production is under constant threat from a number of fungal pathogens, among them is wheat powdery mildew (Blumeria graminis f. sp. tritici). Deployment of resistance genes is the most economical and sustainable method for mildew control. However, domestication and selective breeding have narrowed genetic diversity of modern wheat germplasm, and breeders have relied on wheat relatives for enriching its gene pool through introgression. Translocations where the 1RS chromosome arm was introgressed from rye to wheat have improved yield and resistance against various pathogens. Here, we isolated the Pm17 mildew resistance gene located on the 1RS introgression in wheat cultivar ‘Amigo’ and found that it is an allele or a close paralog of the Pm8 gene isolated earlier from ‘Petkus’ rye. Functional validation using transient and stable transformation confirmed the identity of Pm17. Analysis of Pm17 and Pm8 coding regions revealed an overall identity of 82.9% at the protein level, with the LRR domains being most divergent. Our analysis also showed that the two rye genes are much more diverse compared to the variants encoded by the Pm3 gene in wheat, which is orthologous to Pm17/Pm8 as concluded from highly conserved upstream sequences in all these genes. Thus, the evolutionary history of these orthologous loci differs in the cereal species rye and wheat and demonstrates that orthologous resistance genes can take different routes towards functionally active genes. These findings suggest that the isolation of Pm3/Pm8/Pm17 orthologs from other grass species, additional alleles from the rye germplasm as well as possibly synthetic variants will result in novel resistance genes useful in wheat breeding.

     

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.

     

Marker-assisted breeding of <Emphasis Type="Italic">Musa balbisiana</Emphasis> genitors devoid of infectious endogenous Banana streak virus sequences

Breeding new interspecific banana hybrid varieties relies on the use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of Musa balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus in interspecific hybrids harboring both Musa acuminata and M. balbisiana genomes. Here we provide evidence that seedy M. balbisiana diploids display diverse eBSV allelic combinations and that some eBSVs differ structurally from those previously reported. We also show that segregation of infectious and non-infectious eBSV alleles can be achieved in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. Our work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.     

Production of wheat-rye substitution lines based on winter rye cultivars with karyotype identification by means of C-banding,GISH, and SSR markers

The study presents a continuation of the research aimed at producing of wheat-rye substitution lines (2n = 42) based on the cross (Triticum aestivum L. × Secale sereale L.) × Triticum aestivum L., and using winter rye cultivars Vyatka and Vietnamskaya Mestnaya. In BC 1 F 5 two lines were identified, having karyotypes in which a pair of homologous wheat chromosomes was substituted by a homeologous pair of rye chromosomes. The chromosome composition of these lines was analyzed using C-banding, GISH, and SSR markers. It was demonstrated that karyotype of each line included a single pair of rye chromosomes and lacked wheat-rye translocations. The rye chromosomes were identified, and the chromosomes of wheat, at which the substitutions occurred, were determined. The lines generated by crosses with rye of Vyatka and Vietnamskaya Mestnaya cultivars were designated 1Rv(1A) and 5Rviet(5A), respectively. Chromosome identification and classification of the lines makes it possible to use them in breeding programs and genetic studies.     

Genetic Collection of Meiotic Mutants of Rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F₂ generations after self-fertilization of the F₁ hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome “bouquet” was impaired, and all chromosomes were univalent in meiotic metaphase I in 96.8% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase I. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with 14 univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei8-10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Delayed selfing ensures reproductive assurance in <Emphasis Type="Italic">Utricularia praeterita</Emphasis> and <Emphasis Type="Italic">Utricularia babui</Emphasis> in Western Ghats

Numerous bladderwort (Utricularia) species are distributed worldwide, but their reproductive biology is rarely investigated. Bladderworts are known to depend on tiny organisms to meet a significant proportion of their energy requirement by trapping them in bladders. However, information on the extent of their reliance on insects for pollination success is limited. We examined the reproductive strategy of two Utricularia species viz. Utricularia praeterita and U. babui, endemic to Western Ghats, India. The main aspects of the investigation involved floral biology, breeding system, pollination mechanism, and reproductive success. Flowers of both the species are structured for outbreeding through entomophilous floral suites, herkogamy, protandrous dichogamy and sensitive lobes of the stigma. With nearly 65% natural fruit-set, both the species appeared to be sufficiently open-pollinated. However, pollinators failed to show in plants of U. praeterita while in U. babui there was an apparent mismatch between the extent of fruit-set and pollinator visits. The study demonstrated that in the absence/insufficient visits of pollinators, the two species resort to autonomous selfing. In U. babui, denser patches of plants appeared to be crucial for attracting the pollinators. Both species are self-compatible, and reproductive success is predominantly achieved by delayed autonomous selfing. The sensitive stigma in the species fails to prevent selfing due to diminished herkogamy during the late anthetic stages. It is inferred that in the pollinator-limited environment, delayed selfing contributes to absolute natural fecundity in U. praeterita, while it produces a mixed progeny in U. babui.     

Dissecting the genetic basis of heavy panicle hybrid rice uncovered <Emphasis Type="Italic">Gn1a</Emphasis> and <Emphasis Type="Italic">GS3</Emphasis> as key genes

Key message

Shuhui498 (R498) is an elite parent of heavy panicle hybrid rice by pyramiding the rare gn1a and null gs3 alleles. This finding reveals the genetic basis and great potential application in future breeding of R498.

Abstract

The heavy panicle trait, defined as 5 g or more of grain weight per panicle, is one of the target traits in super-high-yield rice breeding programs. The use of heavy panicle-type hybrid rice has been shown to be a successful strategy for super-high-yield breeding programs, particularly under the environmental conditions of high humidity and deficient solar radiation in southwestern China. However, the genetic components of the heavy panicle trait in hybrid rice remain elusive. Here, we report that the combination of loss-of-function mutations in Grain number 1a (Gn1a) and Grain Size 3 (GS3) is responsible for the heavy panicle phenotype of the elite hybrid rice restorer line Shuhui498 (R498). The null gn1a allele is the determinant factor for heavy panicles through increased grain number, while gs3 is associated with grain size and weight. R498 pyramided the two major null alleles, resulting in heavy panicles with a high grain number and large grains. Clustering analysis revealed that the null gn1a^R498 allele is a rare haplotype which has been innovatively utilized in R498, underscoring the great potential of R498 for breeding purposes. Our research thus sheds light on the distinct genetic compositions of heavy panicle-type rice and may potentially facilitate super-high-yield rice breeding.

     

Development of molecular markers for breeding of double flowers in Japanese gentian

Double flowers are valuable floral traits in most floricultural plants. We recently revealed that a double-flowered mutant of Gentiana scabra was caused by an insertion of a retrotransposable element (Tgs1) into GsAG1, one of the C-class MADS-box genes in gentian. In this study, we developed a PCR-based molecular DNA marker to distinguish double- and single-flower phenotypes at the young seedling stage in Japanese gentian plants. To test the validity of the markers, 17 F₂ populations were produced by selfing F₁ plants crossed between the double-flower mutant and seven breeding lines. Multiplex PCR demonstrated that the Tgs1 insertion in GsAG1 cosegregated with the double-flower phenotype in two F₂ populations, indicating that the PCR-based DNA marker was useful to discriminate between double- and single-flower phenotypes in advance of flowering in Japanese gentian. Given that Japanese gentians lack variation in flower shape and require a long breeding period, the DNA marker developed here will be helpful for efficient breeding of double-flowered cultivars in the future.     

Evolutionary relationships in the genus <Emphasis Type="Italic">Secale</Emphasis> revealed by DArTseq DNA polymorphism

Till this day, there is not much known about the phylogeny of the Secale genus; therefore, in our research, we tried to shed some lights on the issue of rye (Secale genus) taxonomy. The genetic diversity and phylogenetic relationships were evaluated using 13,842 DArTseq? polymorphic markers. The model-based clustering (STRUCTURE software) separated our 84 samples into three main clusters: perennial cluster, annual cluster, and S. sylvestre cluster. The same result was obtained using Neighbour Joining tree and self-organizing maps. Secale sylvestre, S. strictum, and S. cereale are the three main species of the Secale genus. Three samples of rye are in basal positions in phylogenetic trees. These accessions share ancient morphological characters and are probably the ancestors of different lineages within Secale. Annual Secale taxa, with the exception of S. sylvestre, create one mutual taxon. We have found out that the semi-perennial taxa of S. cereale var. multicaule and S. strictum subsp. ciliatoglume are genetically closest to the annual species of S. cereale. Phylogenetic signals for semi-perennial and annual taxa are also present in S. strictum subsp. africanum. SNP-based analysis revealed that evolution of annual S. cereale has already begun in S. strictum subsp. africanum. The results showed that S. vavilovii cannot be considered as a separate species but a subspecies of S. cereale Secale cereale subsp. dighoricum is a hybrid. It is still not clear whether we can consider S. strictum subsp. strictum and S. strictum subsp. kuprijanovii as two separate species.     

Pollination biology and breeding system in five nocturnal species of <Emphasis Type="Italic">Oenothera</Emphasis> (Onagraceae): reproductive assurance and opportunities for outcrossing

The capacity to produce seed, both by selfing and outcrossing, or mixed mating strategies, is considered a mechanism for overcoming unpredictable pollinator availability. In the present study, we investigate breeding system, insect visitations and the role of insect visitors in the pollination of five species of Oenothera subsect. Oenothera. Field experiments showed that autonomous selfing occurs at bud stage, prior to the opening of the flower. Control flowers showed similar seed set to hand-pollinated flowers, whereas emasculated flowers and those subject to open pollination set fewer seed. These results are consistent with the hypothesis that the investigated Oenothera exhibit a great capacity for autonomous selfing and that selfing is selected in order to provide reproductive assurance. Although flowers were visited mostly by nocturnal lepidopterans, these insects did not precipitate pollination and are thus considered nectar thieves. Conversely, analysis of pollen loads and behavior during foraging by diurnal insect visitors revealed that honeybees and bumblebees are the probable pollinators. We conclude that production of flowers capable of autonomous selfing at bud stage, followed by anthesis and opportunities for outcrossing, probably improves the invasive potential of these Oenothera in Europe, together with a rapid increase in their populations, even when pollinators are scarce.     

Using SSR markers for hybrid identification and resource management in Vietnamese <Emphasis Type="Italic">Acacia</Emphasis> breeding programs

We used a set of 16 SSR markers to check the identity of pure-species and hybrid clones in Vietnam’s Acacia auriculiformis, Acacia mangium, and acacia hybrid (A. mangium × A. auriculiformis) breeding programs. The statistics package HIest, applied to a large synthesized population, enabled accurate allocation of genotypes to the two pure species, F₁ and F₂ inter-specific hybrids and backcrosses, based on estimates of hybridity and heterozygosity. The hybridity status of putatively pure A. mangium and A. auriculiformis clones in adjacent clonal seed orchards was checked. Four out of 100 clones selected as A. mangium were found to be backcrosses (A. mangium × F₁ inter-specific hybrid) while out of 96 clones selected as A. auriculiformis, two were F₁ hybrids and two were backcrosses (A. auriculiformis × F₁ hybrid). The markers were then applied to check the hybridity status of 160 putative acacia F₁ hybrid genotypes that had been selected on morphological criteria from open-pollinated progenies collected from A. auriculiformis and A. mangium parents. Many selections based on morphology were found to be mistaken. Only thirteen of 63 clones originating from A. auriculiformis mothers were F₁ hybrids, four were backcrosses, and the remaining 46 were pure A. auriculiformis. Fewer mistakes were evident for clones selected from A. mangium mothers, with 82 out of 89 clones confirmed as F₁ hybrids, three as backcrosses, and four as pure A. mangium. The occurrence of F₁ hybrids and backcrosses in pure-species seed orchards and their progeny shows that inter-species contamination is an issue requiring management in both pure-species and in hybrid breeding of these species in Vietnam. Examination of genetic distances among verified clones showed patterns of relatedness that were consistent with pedigree records. Implications for resource management as well as for breeding and clonal selection strategies are considered.     

Marker-assisted pyramiding of bacterial blight and gall midge resistance genes into RPHR-1005, the restorer line of the popular rice hybrid DRRH-3

Bacterial blight (BB) of rice caused by the pathogen Xanthomonas oryzae pv. oryzae and the insect gall midge (GM) (Orseolia oryzae) are two major constraints of rice production. The present study was carried out to improve RPHR-1005, a stable restorer line of the fine-grain-type rice hybrid DRRH-3, for BB and GM resistance through marker-assisted backcross breeding (MABB). Two major GM resistance genes, Gm4 and Gm8, and a major BB resistance gene, Xa21, were selected as target genes for transfer to RPHR-1005. Two sets of backcrosses were carried out to combine either Xa21 + Gm4 or Xa21+ Gm8 into RPHR-1005 using breeding lines in the genetic background of ISM possessing either Gm4 or Gm8 along with Xa21. Foreground selection was performed for Xa21, Gm4, Gm8, and the major fertility restorer genes Rf3 and Rf4 using gene-specific markers, while 61 polymorphic simple sequence repeat (SSR) markers were used for background selection and marker-assisted backcrossing was continued until BC₂ generation. A promising homozygous backcross-derived plant at the BC₂F₂ generation possessing Xa21 + Gm4, and another possessing Xa21 + Gm8, were intercrossed to stack the target resistance genes. At ICF ₄ (inter-crossed F₄) , three promising lines possessing the three target resistance genes in a homozygous condition along with fine-grain type, complete fertility restoration, and better panicle exsertion than RPHR-1005 have been identified. Among these, a single line, # RPIC-16-65-125, showed better yield, was highly resistant to BB and GM, was of medium–slender grain type, and had complete fertility restoration along with better panicle exsertion and taller plant type than RPHR-1005. This is the first report of combining resistance against BB and GM in the genetic background of a hybrid rice parental line.     

Genetic mapping and salt tolerance of a novel <Emphasis Type="Italic">D1</Emphasis>-allelic mutant of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height and grain shape are important traits that may affect yield in rice, and they therefore have enormous importance in breeding. A dwarf small-grain mutant (S525) was identified among progeny of the Indica rice restorer line ‘Xida 1B’ (wild type) raised from seeds treated with ethyl methanesulfonate. The dwarf and small-grain phenotypes were stably inherited after multi-generation selfing. Field-grown mutant plants showed the phenotypes of dwarfism, broad leaves, and small round grains. Genetic mapping and sequencing confirmed that S525 was a novel d1-allelic mutant. A single-base transition (G to A) in the functional dwarfism gene D1 at the conjunction site of the 11th intron caused excision or duplication of the 11th exon in the mRNA and resulted in translation of a defective G_α protein. The S525 showed enhanced salt tolerance compared with the wild type (WT), and the expression of genes associated with salt tolerance quantitatively increased in response to treatment with 200 mM NaCl. The S525 may be useful for future investigation of G_α functions and in the breeding of new dwarf rice cultivars.     

Identification of a specific molecular marker for the rice blast-resistant gene <Emphasis Type="Italic">Pigm</Emphasis> and molecular breeding of thermo-sensitive genic male sterile leaf-color marker lines

Rice blast is a damaging disease caused by Magnaportheoryzae. Marker-assisted selection of blast resistance genes could help develop cultivars with blast resistance. Pigm is a broad-spectrum blast-resistant gene. However, few rice resources contain Pigm. In this study, the Pigm gene donor Gumei4 (GM4) was investigated. By analyzing different regions of Pigm sequences, we found that marker G8900 was a specific molecular marker of Pigm gene in GM4. Correlation analysis between molecular marker detection and identification of rice blast disease nursery revealed that G8900 could be used in marker-assisted selection (MAS) of Pigm. Furthermore, we introduced Pigm gene into the KT27S line (a blast-susceptible yellow-green-leaf-color mutant) in G8900-assisted breeding and identified three new yellow-green-leaf-color marker lines that are resistant to blast. The agronomic and economic traits of the three new lines are similar to those of their parental lines. The identification and application of Pigm-specific molecular marker in breeding of yellow-green-leaf-color marker line could play an important role in the production of disease-resistant hybrid rice.