1 | INTRODUCTION
The tribe Triticeae (Poaceae) represented an important gene pool for
genetic improvement of cereal crops and forage grasses. It included
approximately 450 species that distributed in a wide range of ecological
habitats over the temperate and subtropical and tropic alpine regions
(Dewey, 1984). The majority of species in Triticeae were allopolyploids,
with ploidy levels ranging from diploid (2n=2x) to dodecaploid (2n=12x).
Natural hybridization between different genera or species oftenoccurred
in the Triticeae. As early as 1926, the natural hybrid ofTriticum - Aegilops - Secale was reported (Von
Tschermak & Bleier, 1926). Stebbins et al. reported a series of natural
hybrids in the Triticeae: natural hybrids of E. condensatus andE. triticoides (Stebbins & Walters, 1949); triploid hybrids ofAgropyron and Elymus (Stebbins & Singh, 1950); natural
hybrids of Elymus and Sitanion (Stebbins & Vaarama,
1954). At the same time, they performed artificial hybridization,
artificial and natural hybrids for morphology, chromosome pairing, seed
set and other aspects of comparison, speculated that the possible origin
of natural hybrids (Stebbins & Walters, 1949; Stebbins & Singh, 1950;
Stebbins & Vaarama, 1954).
Roegneria C. Koch was a relatively large perennial genus in
Triticeae, and includes approximately 130 species, most of which were
tetraploid with StY -genome, nearly 70 of which were found in
China (Yang et al., 2008). Roegneria species not only provided
genetic material for the improvement of forage crops but could also be
used as potential contributors of genes for cereal crops (Keng, 1959).
Predecessors
have reported some studies on the hybrids of Roegneria , such as a
hybrid of Roegneria and Hordeum (Zhou et al., 1995), a
hybrid of R. ciliaris and Leymus multicaulis (Zhang et
al., 2008). These hybrids were created by the artificial hybridization
and could not replace the value of natural hybrids. In recent years,
researchers had discovered some natural hybrids of Roegneria . For
example, Zeng et al. (2012) had discovered the pentaploid natural
hybrids (StStYYP ) between Roegneria (StY ) andKengyilia (StYP ).
In the process of plant system evolution, hybridization was the direct
cause of the formation of diploid and polyploid species and the
production of reticulate evolution (Rieseberg, 1995; Soltis & Soltis,
1993). However, it was not easy to accurately identify whether a species
was a hybrid and to explore origin of hybrids (Rauscher et al., 2002).
Early identification of hybridization was mainly based on morphological
characteristics, and it was often based on the morphology intermediate
of the parents to infer whether a plant came from a hybridization.
However, the reliability of morphological markers was low, and
morphology intermediate was not always related to hybridization. It may
also be caused by convergent evolution or environment. Therefore,
morphological markers could not be used alone to identify hybrids
(Rieseberg, 1995). Cytological markers have been used as important
criteria for hybridization, including karyotype analysis, meiotic
pairing analysis, Genomic in situ hybridization (GISH) and
Fluorescence in situ hybridization (FISH), which could be used to
identify and analyze natural hybrids (Han et al., 2004; Mao et al.,
2017). For example, Using FISH and GISH techniques, the two parents ofElytrigia ×mucronata could be studied through signal sites
on chromosomes (Paštová et al., 2019). However, due to the high parental
chromosome homology of interspecific hybrids, it was difficult to
explore origin of hybrids by FISH and GISH. Therefore, even if a species
has been determined from morphology or cytogenetics to be a hybrid or
hybrid origin, it still needs to be verified with some other evidence
(Soltis et al., 1992). Phylogenetic analysis could not only reflect the
genetic relationship between hybrids and parents, but also overcome the
shortcomings of non-dominance and
insufficient
repetitiveness of other molecular markers. The method was also the first
choice for identifying natural hybrids (Quijada et al., 1997; Sang et
al., 1995). Since genes at different sites in the genome of diploid or
polyploid hybrids derived from different parent species, these genes
have different evolutionary processes. This was the basis for detecting
hybridization by phylogenetic analysis (Yu et al., 2011). Single- or
low-copy nuclear genes, which were less susceptible to concerted
evolution, could serve as useful markers for studies of phylogenetic
relationships (Lei et al., 2018; Sha et al., 2010). Among the available
nuclear sequences,
DNA
meiotic recombinase 1 (DMC 1) gene sequences have been used to
examine hybridization events (Tang et al., 2017). The
chloroplast DNA (cp DNA) is
maternally inherited in grasses (Smith et al., 2006). Among the
available chloroplast sequences,
ribosomal protein S16 (rps 16) were used to identify the maternal
donor of genera in Triticeae (Yan et al., 2014).
The experimental field of Sichuan
Academy of Grassland Science (SAGS) located on Northwest Sichuan
Plateau, China (Hongyuan county, Sichuan Province, 31°51′ to 33°33′ N,
101°51′ to 103°22′ W) at altitude 3500 m. Two species ofRoegneria [Roegneria stricta Keng and Roegneriaturczaninovii (Drob.) Nevski] were planted very close in SAGS.
We harvested the seeds of the two species and then individual planting.
After three years growing, we found that 23 putative hybrids (5.75%,
23/400) randomly distributed in R. stricta field and 54 putative
hybrids (16.36%, 54/330) randomly distributed in R.
turczaninovii field (Figures 2 a-c). These putative hybrids grew
stronger than around plants and their seed set was very low
(0.23%-5.59%, Figure 3). They showed intermediate morphological
characters of R. stricta and R. turczaninovii , such
as
pubescence of leaf, basal leaf
sheath
and stem node (Figures 1 d-o).
Both of R. stricta and R. turczaninovii were tetraploid
perennial species (2n=4x=28) with the StY genome. R.
stricta come from Luhuo County, Sichuan, China while R.
turczaninovii origins from Linxi County, Inner Mongolia, China. Because
these two species had the same ploidy and genome constitutions, natural
hybridizations might occur between them if they were grown together for
a long time. In the current study, we hypothesized that the sterile
plants were hybrids of R. stricta and R. turczaninovii . To
determine if this is indeed the case, we conducted different methods
including morphological analysis, fertility analysis, karyotype, meiotic
pairing analysis, in situ hybridization and DNA sequence analysis
in these putative hybrids and their accompanying plants. The results
provided useful resources for origin and formation mechanism of natural
hybrids, species evolution in the Triticeae, and laid material
foundation for breeding new varieties.