Comparative Analysis of Chloroplast Pan-Genomes and Transcriptomics Reveals Cold Adaptation in Medicago sativa.

Alfalfa (Medicago sativa) is a perennial forage legume that is widely distributed all over the world; therefore, it has an extremely complex genetic background. Though population structure and phylogenetic studies have been conducted on a large group of alfalfa nuclear genomes, information about the chloroplast genomes is still lacking. Chloroplast genomes are generally considered to be conservative and play an important role in population diversity analysis and species adaptation in plants. Here, 231 complete alfalfa chloroplast genomes were successfully assembled from 359 alfalfa resequencing data, on the basis of which the alfalfa chloroplast pan-genome was constructed. We investigated the genetic variations of the alfalfa chloroplast genome through comparative genomic, genetic diversity, phylogenetic, population genetic structure, and haplotype analysis. Meanwhile, the expression of alfalfa chloroplast genes under cold stress was explored through transcriptome analysis. As a result, chloroplast genomes of 231 alfalfa lack an IR region, and the size of the chloroplast genome ranges from 125,192 bp to 126,105 bp. Using population structure, haplotypes, and construction of a phylogenetic tree, it was found that alfalfa populations could be divided into four groups, and multiple highly variable regions were found in the alfalfa chloroplast genome. Transcriptome analysis showed that tRNA genes were significantly up-regulated in the cold-sensitive varieties, while rps7, rpl32, and ndhB were down-regulated, and the editing efficiency of ycf1, ycf2, and ndhF was decreased in the cold-tolerant varieties, which may be due to the fact that chloroplasts store nutrients through photosynthesis to resist cold. The huge number of genetic variants in this study provide powerful resources for molecular markers.

A genomic perspective on the important genetic mechanisms of upland adaptation of rice.

BACKGROUND: Cultivated rice consists of two important ecotypes, upland and irrigated, that have respectively adapted to either dry land or irrigated cultivation. Upland rice, widely adopted in rainfed upland areas in virtue of its little water requirement, contains abundant untapped genetic resources, such as genes for drought adaptation. With water shortage exacerbated and population expanding, the need for breeding crop varieties with drought adaptation becomes more and more urgent. However, a previous oversight in upland rice research reveals little information regarding its genetic mechanisms for upland adaption, greatly hindering progress in harnessing its genetic resources for breeding and cultivation. RESULTS: In this study, we selected 84 upland and 82 irrigated accessions from all over the world, phenotyped them under both irrigated and dry land environments, and investigated the phylogenetic relations and population structure of the upland ecotype using whole genome variation data. Further comparative analysis yields a list of differentiated genes that may account for the phenotypic and physiological differences between upland and irrigated rice. CONCLUSIONS: This study represents the first genomic investigation in a large sample of upland rice, providing valuable gene list for understanding upland rice adaptation, especially drought-related adaptation, and its subsequent utilization in modern agriculture.

Comprehensive Identification of the ß-Amylase (BAM) Gene Family in Response to Cold Stress in White Clover.

White clover (Trifolium repens L.) is an allopolyploid plant and an excellent perennial legume forage. However, white clover is subjected to various stresses during its growth, with cold stress being one of the major limiting factors affecting its growth and development. Beta-amylase (BAM) is an important starch-hydrolyzing enzyme that plays a significant role in starch degradation and responses to environmental stress. In this study, 21 members of the BAM gene family were identified in the white clover genome. A phylogenetic analysis using BAMs from Arabidopsis divided TrBAMs into four groups based on sequence similarity. Through analysis of conserved motifs, gene duplication, synteny analysis, and cis-acting elements, a deeper understanding of the structure and evolution of TrBAMs in white clover was gained. Additionally, a gene regulatory network (GRN) containing TrBAMs was constructed; gene ontology (GO) annotation analysis revealed close interactions between TrBAMs and AMY (α-amylase) and DPE (4-alpha-glucanotransferase). To determine the function of TrBAMs under various tissues and stresses, RNA-seq datasets were analyzed, showing that most TrBAMs were significantly upregulated in response to biotic and abiotic stresses and the highest expression in leaves. These results were validated through qRT-PCR experiments, indicating their involvement in multiple gene regulatory pathways responding to cold stress. This study provides new insights into the structure, evolution, and function of the white clover BAM gene family, laying the foundation for further exploration of the functional mechanisms through which TrBAMs respond to cold stress.

Genome-wide analysis of the CDPK gene family and their important roles response to cold stress in white clover.

Calcium-dependent protein kinases (CDPKs) are an important class of calcium-sensitive response proteins that play an important regulatory role in response to abiotic stresses. To date, little is known about the CDPK genes in white clover. White clover is a high-quality forage grass with high protein content, but it is susceptible to cold stress. Therefore, we performed a genome-wide analysis of the CDPK gene family in white clover and identified 50 members of the CDPK genes. Phylogenetic analysis using CDPKs from the model plant Arabidopsis divided the TrCDPK genes into four groups based on their sequence similarities. Motif analysis showed that TrCDPKs within the same group had similar motif compositions. Gene duplication analysis revealed the evolution and expansion of TrCDPK genes in white clover. Meanwhile, a genetic regulatory network (GRN) containing TrCDPK genes was reconstructed, and gene ontology (GO) annotation analysis of these functional genes showed that they contribute to signal transduction, cellular response to stimuli, and biological regulation, all of which are important processes in response to abiotic stresses. To determine the function of TrCDPK genes, we analyzed the RNA-seq dataset and found that most TrCDPK genes were highly up-regulated under cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiments, implying that TrCDPK genes are involved in various gene regulatory pathways in response to cold stress. Our study may help to further investigate the function of TrCDPK genes and their role in response to cold stress, which is important for understanding the molecular mechanisms of cold tolerance in white clover and improving its cold tolerance.

QTL mapping of yield components and kernel traits in wheat cultivars TAM 112 and Duster.

In the Southern Great Plains, wheat cultivars have been selected for a combination of outstanding yield and drought tolerance as a long-term breeding goal. To understand the underlying genetic mechanisms, this study aimed to dissect the quantitative trait loci (QTL) associated with yield components and kernel traits in two wheat cultivars `TAM 112' and `Duster' under both irrigated and dryland environments. A set of 182 recombined inbred lines (RIL) derived from the cross of TAM 112/Duster were planted in 13 diverse environments for evaluation of 18 yield and kernel related traits. High-density genetic linkage map was constructed using 5,081 single nucleotide polymorphisms (SNPs) from genotyping-by-sequencing (GBS). QTL mapping analysis detected 134 QTL regions on all 21 wheat chromosomes, including 30 pleiotropic QTL regions and 21 consistent QTL regions, with 10 QTL regions in common. Three major pleiotropic QTL on the short arms of chromosomes 2B (57.5 - 61.6 Mbps), 2D (37.1 - 38.7 Mbps), and 7D (66.0 - 69.2 Mbps) colocalized with genes Ppd-B1, Ppd-D1, and FT-D1, respectively. And four consistent QTL associated with kernel length (KLEN), thousand kernel weight (TKW), plot grain yield (YLD), and kernel spike-1 (KPS) (Qklen.tamu.1A.325, Qtkw.tamu.2B.137, Qyld.tamu.2D.3, and Qkps.tamu.6A.113) explained more than 5% of the phenotypic variation. QTL Qklen.tamu.1A.325 is a novel QTL with consistent effects under all tested environments. Marker haplotype analysis indicated the QTL combinations significantly increased yield and kernel traits. QTL and the linked markers identified in this study will facilitate future marker-assisted selection (MAS) for pyramiding the favorable alleles and QTL map-based cloning.

Genome-Wide Association Mapping of Seedling Drought Tolerance in Winter Wheat.

In the southern Great Plains of the United States, winter wheat grown for dual-purpose is often planted early, which puts it at risk for drought stress at the seedling stage in the autumn. To map quantitative trait loci (QTL) associated with seedling drought tolerance, a genome-wide association study (GWAS) was performed on a hard winter wheat association mapping panel. Two sets of plants were planted in the greenhouse initially under well-watered conditions. At the five-leaf stage, one set continued to receive the optimum amount of water, whereas watering was withdrawn from the other set (drought stress treatment) for 14 days to mimic drought stress. Large phenotypic variation was observed in leaf chlorophyll content, leaf chlorophyll fluorescence, shoot length, number of leaves per seedling, and seedling recovery. A mixed linear model analysis detected multiple significant QTL associated with seedling drought tolerance-related traits on chromosomes 1B, 2A, 2B, 2D, 3A, 3B, 3D, 4B, 5A, 5B, 6B, and 7B. Among those, 12 stable QTL responding to drought stress for various traits were identified. Shoot length and leaf chlorophyll fluorescence were good indicators in responding to drought stress because most of the drought responding QTL detected using means of these two traits were also detected in at least two experimental repeats. These stable QTL are more valuable for use in marker-assisted selection during wheat breeding. Moreover, different traits were mapped on several common chromosomes, such as 1B, 2B, 3B, and 6B, and two QTL clusters associated with three or more traits were located at 107-130 and 80-83 cM on chromosomes 2B and 6B, respectively. Furthermore, some QTL detected in this study co-localized with previously reported QTL for root and shoot traits at the seedling stage and canopy temperature at the grain-filling stage of wheat. In addition, several of the mapped chromosomes were also associated with drought tolerance during the flowering or grain-filling stage in wheat. Some significant single-nucleotide polymorphisms (SNPs) were aligned to candidate genes playing roles in plant abiotic stress responses. The SNP markers identified in this study will be further validated and used for marker-assisted breeding of seedling drought tolerance during dual-purpose wheat breeding.

Genome-Wide Association Mapping of Seedling Heat Tolerance in Winter Wheat.

Heat stress during the seedling stage of early-planted winter wheat (Triticum aestivum L.) is one of the most abiotic stresses of the crop restricting forage and grain production in the Southern Plains of the United States. To map quantitative trait loci (QTLs) and identify single-nucleotide polymorphism (SNP) markers associated with seedling heat tolerance, a genome-wide association mapping study (GWAS) was conducted using 200 diverse representative lines of the hard red winter wheat association mapping panel, which was established by the Triticeae Coordinated Agricultural Project (TCAP) and genotyped with the wheat iSelect 90K SNP array. The plants were initially planted under optimal temperature conditions in two growth chambers. At the three-leaf stage, one chamber was set to 40/35°C day/night as heat stress treatment, while the other chamber was kept at optimal temperature (25/20°C day/night) as control for 14 days. Data were collected on leaf chlorophyll content, shoot length, number of leaves per seedling, and seedling recovery after removal of heat stress treatment. Phenotypic variability for seedling heat tolerance among wheat lines was observed in this study. Using the mixed linear model (MLM), we detected multiple significant QTLs for seedling heat tolerance on different chromosomes. Some of the QTLs were detected on chromosomes that were previously reported to harbor QTLs for heat tolerance during the flowering stage of wheat. These results suggest that some heat tolerance QTLs are effective from the seedling to reproductive stages in wheat. However, new QTLs that have never been reported at the reproductive stage were found responding to seedling heat stress in the present study. Candidate gene analysis revealed high sequence similarities of some significant loci with candidate genes involved in plant stress responses including heat, drought, and salt stress. This study provides valuable information about the genetic basis of seedling heat tolerance in wheat. To the best of our knowledge, this is the first GWAS to map QTLs associated with seedling heat tolerance targeting early planting of dual-purpose winter wheat. The SNP markers identified in this study will be used for marker-assisted selection (MAS) of seedling heat tolerance during dual-purpose wheat breeding.

Analysis of elite variety tag SNPs reveals an important allele in upland rice.

Elite crop varieties usually fix alleles that occur at low frequencies within non-elite gene pools. Dissecting these alleles for desirable agronomic traits can be accomplished by comparing the genomes of elite varieties with those from non-elite populations. Here we deep-sequence six elite rice varieties and use two large control panels to identify elite variety tag single-nucleotide polymorphism alleles (ETASs). Guided by this preliminary analysis, we comprehensively characterize one protein-altering ETAS in the 9-cis-epoxycarotenoid dioxygenase gene of the IRAT104 upland rice variety. This allele displays a drastic frequency difference between upland and irrigated rice, and a selective sweep is observed around this allele. Functional analysis indicates that in upland rice, this allele is associated with significantly higher abscisic acid levels and denser lateral roots, suggesting its association with upland rice suitability. This report provides a potential strategy to mine rare, agronomically important alleles.