Development of molecular markers based on LTR retrotransposon in the Cleistogenes songorica genome.

Long terminal repeat retrotransposons (LTR-RTs) contribute a large fraction of many sequenced plant genomes and play important roles in genomic diversity and phenotypic variations. LTR-RTs are abundantly distributed in plant genomes, facilitating the development of markers based on LTR-RTs for a variety of genotyping purposes. Whole-genome analysis of LTR-RTs was performed in Cleistogenes songorica. A total of 299,079 LTR-RTs were identified and classified as Gypsy type, Copia type, or other type. LTR-RTs were widely distributed in the genome, enriched in the heterochromatic region of the chromosome, and negatively correlated with gene distribution. However, approximately one-fifth of genes were still interrupted by LTR-RTs, and these genes are annotated. Furthermore, four types of primer pairs (PPs) were designed, namely, retrotransposon-based insertion polymorphisms, inter-retrotransposon amplified polymorphisms, insertion site-based polymorphisms, and retrotransposon-microsatellite amplified polymorphisms. A total of 350 PPs were screened in 23 accessions of the genus Cleistogenes, of which 80 PPs showed polymorphism, and 72 PPs showed transferability among Gramineae and non-Gramineae species. In addition, a comparative analysis of homologous LTR-RTs was performed with other related grasses. Taken together, the study will serve as a valuable resource for genotyping applications for C. songorica and related grasses.

Genome-wide development and application of miRNA-SSR markers in Melilotus genus.

Genetic diversity of plants is the brace of biodiversity and diversity within species, between species, and of ecosystems. SSR markers are the most preferable molecular marker tool that has been successfully used to study the genetic diversity of plant species. Development of miRNA-SSR markers has been deed in animals but is still limited in plants. In this study, 365 precursors miRNA were extracted from Melilotus albus (Ma) genome and used to design Ma miRNA-SSR primers. 137 Ma primer pairs (79 from known and 58 from novel pre-miRNAs) were obtained. 66 pairs of Ma miRNA-SSR primers were selected with polymorphisms and expected fragment size. The polymorphisms of primers were evaluated in 60 individuals of 15 Ma accessions. A total of 66 primer pairs showed high polymorphism, with average polymorphic information content of 0.49 among 15 Ma accessions and 0.63 among 18 Melilotus species, indicating that these primers have high polymorphisms. The number of alleles produced per primer ranged from 2 to 6 with an average of 3.6 alleles per locus in Ma accessions, and 2 to 10 numbers of alleles with a mean of 5.24 alleles per locus in Melilotus spp. For further studies, the genetic relationship was examined and the cluster analysis showed that 15 Ma accessions were grouped in three groups, on the other hand, 18 Melilotus species clustered into two groups. The analysis of molecular variance (AMOVA) revealed that 64.82% of the variation was found within the species and 35.18% between the species. The population structure analysis showed similar results with PCA analysis in that 18 species were grouped in two groups. In addition, 16,450 miRNA target genes were identified and used for GO and KEGG analysis. This is the first study to develop miRNA-SSR molecular markers in Melilotus spp., which has a great potential for marker-assisted, genetic improvement, genotyping applications, QTL analysis, and molecular-assisted selection studies for plant breeders and other researchers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01086-z.

Genome-Wide Identification and Development of LTR Retrotransposon-Based Molecular Markers for the Melilotus Genus.

Melilotus is an important genus of legumes with industrial and medicinal value, partly due to the production of coumarin. To explore the genetic diversity and population structure of Melilotus, 40 accessions were analyzed using long terminal repeat (LTR) retrotransposon-based markers. A total of 585,894,349 bp of LTR retrotransposon sequences, accounting for 55.28% of the Melilotus genome, were identified using bioinformatics tools. A total of 181,040 LTR retrotransposons were identified and classified as Gypsy, Copia, or another type. A total of 350 pairs of primers were designed for assessing polymorphisms in 15 Melilotus albus accessions. Overall, 47 polymorphic primer pairs were screened for their availability and transferability in 18 Melilotus species. All the primer pairs were transferable, and 292 alleles were detected at 47 LTR retrotransposon loci. The average polymorphism information content (PIC) value was 0.66, which indicated that these markers were highly informative. Based on unweighted pair group method with arithmetic mean (UPGMA) dendrogram cluster analysis, the 18 Melilotus species were classified into three clusters. This study provides important data for future breeding programs and for implementing genetic improvements in the Melilotus genus.

The genome of Cleistogenes songorica provides a blueprint for functional dissection of dimorphic flower differentiation and drought adaptability.

Cleistogenes songorica (2n = 4x = 40) is a desert grass with a unique dimorphic flowering mechanism and an ability to survive extreme drought. Little is known about the genetics underlying drought tolerance and its reproductive adaptability. Here, we sequenced and assembled a high-quality chromosome-level C. songorica genome (contig N50 = 21.28 Mb). Complete assemblies of all telomeres, and of ten chromosomes were derived. C. songorica underwent a recent tetraploidization (~19 million years ago) and four major chromosomal rearrangements. Expanded genes were significantly enriched in fatty acid elongation, phenylpropanoid biosynthesis, starch and sucrose metabolism, and circadian rhythm pathways. By comparative transcriptomic analysis we found that conserved drought tolerance related genes were expanded. Transcription of CsMYB genes was associated with differential development of chasmogamous and cleistogamous flowers, as well as drought tolerance. Furthermore, we found that regulation modules encompassing miRNA, transcription factors and target genes are involved in dimorphic flower development, validated by overexpression of CsAP2_9 and its targeted miR172 in rice. Our findings enable further understanding of the mechanisms of drought tolerance and flowering in C. songorica, and provide new insights into the adaptability of native grass species in evolution, along with potential resources for trait improvement in agronomically important species.

Genome-wide development of miRNA-based SSR markers in Cleistogenes songorica and analysis of their transferability to Gramineae/non-Gramineae species.

Simple sequence repeat (SSR) markers are commonly used for many genetic applications, such as map construction, fingerprinting, and genetic diversity analyses, due to their high reproducibility, polymorphism, and abundance. Endogenous miRNAs play essential roles in plant development and gene expression under diverse biotic and abiotic stress conditions. In the present study, we predicted 110 miRNA-SSR primer pairs from 287 precursor miRNAs. Among 110 primer pairs, 85 were successfully amplified and examined for transferability to other Gramineae and non-Gramineae species. The results showed that all 82 primer pairs yielded unambiguous and strong amplification, and across the 23 studied Cleistogenes accessions, a total of 385 alleles were polymorphic. The number of alleles produced per primer varied from 3 to 11, with an average of 4.69 per locus. The expected heterozygosity (He) ranged from 0.44 to 0.88, with an average of 0.74 per locus, and the PIC (Polymorphism Information Content) values ranged from 0.34 to 0.87, with an average of 0.69 per locus. Furthermore, 1422 miRNA target genes were predicted and analyzed using the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. In conclusion, the results showed that an miRNA-based microsatellite marker system can be applicable for genetic diversity and marker-assisted breeding studies.

Analysis of Six Transcription Factor Families Explores Transcript Divergence of Cleistogamous and Chasmogamous Flowers in Cleistogenes songorica.

Cleistogenes songorica is a cultivated turfgrass species that employs a mixed breeding system. To determine the morphological differences and molecular mechanisms of the chasmogamous (CH) and cleistogamous (CL) flowers of this species, we evaluated seed traits and analyzed six transcription factor (TF) families related to floral development. The seed traits from the CH and CL flowers were significantly different. In total, 12 CsAP2, 13 CsSPL, 9 CsGRF, 21 CsMYB, 15 CsMADS, and 1 CsLFY differentially expressed genes were identified from the transcriptome of the C. songorica flowers, which was further supported by evolutionary relationships and conserved motifs. All collinear gene pairs had a Ka/Ks ratio <1. Analysis of the promoters and miRNAs of the TFs revealed that the members of the six TF families may coregulate the divergence of CH and CL flowers during evolution. Two CsAP2, 8 CsSPL, 6 CsGRF, 3 CsMYB (targeted by miR172s, miR156s, miR396a/b and miR159a/b, respectively), and 15 CsMADS genes as well as 1 CsLFY gene may be involved in the development of CH and CL flowers. This study is the first to analyze the differences between CH and CL flowers at the TF-family level, which will help in the understanding of dimorphic turfgrasses.

Mining Late Embryogenesis Abundant (LEA) Family Genes in Cleistogenes songorica, a Xerophyte Perennial Desert Plant.

Plant growth and development depends on its ability to maintain optimal cellular homeostasis during abiotic and biotic stresses. Cleistogenes songorica, a xerophyte desert plant, is known to have novel drought stress adaptation strategies and contains rich pools of stress tolerance genes. Proteins encoded by Late Embryogenesis Abundant (LEA) family genes promote cellular activities by functioning as disordered molecules, or by limiting collisions between enzymes during stresses. To date, functions of the LEA family genes have been heavily investigated in many plant species except perennial monocotyledonous species. In this study, 44 putative LEA genes were identified in the C. songorica genome and were grouped into eight subfamilies, based on their conserved protein domains and domain organizations. Phylogenetic analyses indicated that C. songorica Dehydrin and LEA_2 subfamily proteins shared high sequence homology with stress responsive Dehydrin proteins from Arabidopsis. Additionally, promoter regions of CsLEA_2 or CsDehydrin subfamily genes were rich in G-box, drought responsive (MBS), and/or Abscisic acid responsive (ABRE) cis-regulatory elements. In addition, gene expression analyses indicated that genes from these two subfamilies were highly responsive to heat stress and ABA treatment, in both leaves and roots. In summary, the results from this study provided a comprehensive view of C. songorica LEA genes and the potential applications of these genes for the improvement of crop tolerance to abiotic stresses.