Genome-wide development and utilization of novel intron-length polymorphic (ILP) markers in <Emphasis Type="Italic">Medicago sativa</Emphasis>

Alfalfa (Medicago sativa L.) is the most widely cultivated forage legume around the world. Though development and application of microsatellite markers in large-scale was reported in this species, a systematic investigation and large-scale exploitation of intron-length polymorphic (ILP) markers has not been conducted. In the present study, the RNA-Seq sequences of alfalfa were aligned with the genomic sequences of Arabidopsis to predict the position of introns and develop ILP markers in alfalfa. A total of 693 putative ILPs were identified, and 502 ILP markers were successfully developed. Furthermore, 100 ILP markers exhibited relatively high levels of transferability to leguminous (40.0%–83.0%) and non-leguminous (21.0%–22.0%) species. Polymorphisms in 40 randomly selected MsILP markers were evaluated in 21 alfalfa accessions and collectively yielded 169 alleles with an average of 4.7 alleles per locus. The polymorphism information content (PIC) ranged from 0.15 to 0.87 with an average of 0.60, which indicated a high level of polymorphism in the MsILP markers. For the first time, we developed large-scale ILP markers in alfalfa and demonstrated their utility in transferability, which will be valuable for genetic relationship assessments, comparative genomic studies and marker-assisted breeding of leguminous and non-leguminous species.     

Relationship Among Intron Length,Gene Expression,and Nucleotide Diversity in the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Crassostrea gigas is a model mollusk, but its genetic features have not been studied comprehensively. In this study, we used whole-genome resequencing data to identify and characterize nucleotide diversity and population recombination rate in a diverse collection of 21 C. gigas samples. Our analyses revealed that C. gigas harbors both extremely high genetic diversity and recombination rates across the whole genome as compared with those of the other taxa. The noncoding regions, introns, intergenic spacers, and untranslated regions (UTRs) showed a lower level diversity than the synonymous sites. The larger introns tended to have lower diversity. Moreover, we found a negative association of the non-synonymous diversity with gene expression, which suggested that purifying selection played an important role in shaping genetic diversity. The nucleotide diversity at the 100- and 50-kb levels was positively correlated with population recombination rates, which was expected if the diversity was shaped by purifying selection or hitchhiking of advantageous mutants. Our work gives a general picture of the oyster’s polymorphism pattern and its association with recombination rates.     

Improvement of cell wall digestibility in tall fescue by <Emphasis Type="Italic">Oryza sativa</Emphasis> SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (<Emphasis Type="Italic">Helianthus annum</Emphasis>, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented.Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

Identification and Analysis of <Emphasis Type="Italic">VTC2</Emphasis> Homologs Encoding the Key Enzyme of L-Ascorbic Acid Biosynthesis in Tomato Species (<Emphasis Type="Italic">Solanum</Emphasis> Section of Lycopersicon)

The complete nucleotide sequences of 11 wild and cultivated tomato GDP-L-galactose-phosphorylase- 1 homologs (VTC2) were identified and characterized. The overall level of nucleotide variability was 9.19%. Totally 25 out of 54 cDNA SNPs were non-synonymous and resulted in amino acid substitutions. In the group of green-fruited tomato species, more SNPs were detected than in the group of red-fruited species, but the percentage of non-synonymous substitutions in red-fruited tomatoes was significantly higher (66.7% vs. 37.5%). In the translated amino acid sequences, the GDF-L-galactose-hexose-1-phosphate-guanyltransferase domain and the highly conserved HIT-motif were identified and a conserved motif specific for VTC2 of Solanoideae species was detected.     

Assessment of genetic relationships between cultivated arracacha (<Emphasis Type="Italic">Arracacia xanthorrhiza</Emphasis> Bancr.) and its wild close relatives in the area of domestication using microsatellite markers

Arracacha (Arracacia xanthorrhiza Bancr.) is an asexual propagated root crop domesticated in the Andean highlands, which exists naturally with polycarpic and monocarpic forms. Wild A. xanthorrhiza are present in the area of domestication and can occasionally be mistaken in the same field for a crop or a weed. To study genetic relationships between cultivated arracacha and the wild forms, we surveyed the diversity of 178 plant samples at 11 microsatellite (SSR) loci. As expected, wild A. xanthorrhiza forms showed a significantly higher allelic diversity for all the examined SSR markers. The cultivated pool showed an excess of heterozygosity as opposed to a deficit found in the wild compartment. High Fst values and AMOVA analysis suggest that the cultivated variety has genetically differentiated from the wild forms and is more related to the wild polycarpic than to the monocarpic. Both the wild forms were well distinguished from the cultivars. Nevertheless, among a set of F1 experimental hybrids (cultivated?×?wild polycarpic), some other genotypes were revealed, also being admixed. Our results highlight a large genetic base available in the wild populations of A. xanthorrhiza with potential implications for the utilization and breeding of this promising crop.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.     

Nucleotide Diversity and Molecular Evolution of the <Emphasis Type="Italic">ALK</Emphasis> Gene in Cultivated Rice and its Wild Relatives

Gelatinization temperature (GT), an important parameter for rice cooking quality, is mainly regulated by the ALK gene encoding starch synthase IIa. Here, we reported the nucleotide diversity of the ALK gene in 122 cultivated accessions and 199 wild rice accessions that were collected around the Pearl River Basin in China. A total of 93 single nucleotide ploymorphisms (SNPs) were identified, with an average of one SNP per 40 bp. Tajima D statistics revealed that the DNA sequences covering the last exon have probably evolved under balancing selection. Based on two functional SNPs (an A to G substitution at 4198 bp and a GC to TT dinucleotide substitution at 4330/4331 bp), three haplotypes, G/GC, G/TT, and A/GC, were identified in both wild and cultivated accessions, with the G/GC haplotype being predominant. Interestingly, the A/GC haplotype was exclusively found in the wild accessions from Guangdong province, while the G/TT haplotype was only present in the wild accessions from Jiangxi province and Hainan Island. This suggests that the G/TT and A/GC variants may have arisen independently and undergone balancing selection on separate haplotypes in multiple populations. Our result supports earlier hypothesis that cultivated rice was independently domesticated from multiple domestication events in China. Our study aids in the understanding of the domestication process that led to the improvement of rice grain quality.     

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.     

Anthropogenic dispersion of selected germplasm creates a geographic mosaic of contrasting maternal lineages in <Emphasis Type="Italic">Crescentia cujete</Emphasis> from Mesoamerica

The modification of the genetic/phenotypic composition of plant populations through artificial selection occurs both through time and space. We analyzed the role of human dispersal on the geographic distribution of maternal lineages of Crescentia cujete in Mesoamerica. We sampled 28 homegarden (224 individuals) and 12 wild populations (159 individuals). Semi-structured interviews provided information on the origin of cultivated trees. Six chloroplast microsatellites allowed for the identification of 21 haplotypes, four of them exclusively in 83% of homegarden trees. Wild haplotypes from local C. cujete and Crescentia alata were found at low frequencies (17%) under cultivation. Cultivated and wild haplotypes constituted two different haplogroups. Accordingly, barriers to seed dispersal were detected among neighboring cultivated and wild populations. Recorded events of human dispersal of cuttings and seeds attaining up to >?200 km agreed with homegardens’ lower diversity (Nei’s h?=?0.55, dropping to 0.32 when excluding wild haplotypes). Wild populations displayed high diversity (h?=?0.71) and isolation by distance, in agreement with physiographic provinces. Our results support the native status of wild C. cujete and a Pre-Columbian introduction of cultivated lineages that generated a novel genetic mosaic superimposed on native maternal lineages. The results reveal the active role of farmers in maintaining the identity of cultivated lineages through time, while chloroplast capture from local congeners has increased the diversity of maternal lineages under cultivation. Additional data are needed on the origins of cultivated lineages, but our results contribute new insights into tree domestication in this center of crop diversity.     

Physiological and biochemical changes in different drought-tolerant alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) varieties under PEG-induced drought stress

Medicago sativa L. cv. Longzhong is a nutritious forage plant in dryland regions of the Loess Plateau with strong drought tolerance and broad adaptability. To understand the adaptation mechanism of alfalfa (M. sativa L. cv. Longzhong) to drought stress, growth, and physiological parameters including levels of chlorophyll content, osmotic adjustment, reactive oxygen species (ROS), and antioxidant enzymes and antioxidants were measured under simulated levels of drought (? 0.40, ? 0.80, ? 1.20, ? 1.60, and ? 2.00 MPa). The changes in M. sativa L. cv. Longzhong were compared with those of plants of M. sativa L. cv. Longdong control (Variety I) suited to moderate rainfall areas and M. sativa L. cv. Gannong No. 3 (Variety II) suited to irrigated areas. The results showed that root–shoot ratio, the chlorophyll (a + b) and osmolytes contents, the degree of lipid peroxidation and ROS production, and the levels of antioxidative enzymes and antioxidants increased significantly with increasing drought stress, whereas plant height, aboveground biomass, chlorophyll a/b ratio, leaf water potential (Ψ₁), and relative water content (RWC) decreased in response to drought. The Longzhong variety responded early to beginning drought stress (between 0 and ? 0.4 MPa) compared with the controls. Under drought stress (between ? 0.4 and ? 2.0 MPa), the Longzhong variety had significantly higher belowground biomass, root–shoot ratio, Ψ₁, RWC, catalase (CAT) activity and reduced glutathione content than those of Varieties I and II, but hydrogen peroxide and hydroxyl free radical (OH·) contents were significantly lower. Step regression analysis showed that OH·, CAT, malondialdehyde, superoxide anion-free radical (O ₂ ^·? ), and superoxide dismutase of Longzhong had the most marked response to drought stress. In conclusion, the stronger drought tolerance of the Longzhong variety might be due to its higher water-holding capacity, root–shoot ratio, and ability to coordinate enzymatic and non-enzymatic antioxidant systems, which coordinate the peroxidation and oxidative systems.     

Estimation of loci involved in non-shattering of seeds in early rice domestication

Rice (Oryza sativa L.) is widely cultivated around the world and is known to be domesticated from its wild form, O. rufipogon. A loss of seed shattering is one of the most obvious phenotypic changes selected for during rice domestication. Previously, three seed-shattering loci, qSH1, sh4, and qSH3 were reported to be involved in non-shattering of seeds of Japonica-type cultivated rice, O. sativa cv. Nipponbare. In this study, we focused on non-shattering characteristics of O. sativa Indica cv. IR36 having functional allele at qSH1. We produced backcross recombinant inbred lines having chromosomal segments from IR36 in the genetic background of wild rice, O. rufipogon W630. Histological and quantitative trait loci analyses of abscission layer formation were conducted. In the analysis of quantitative trait loci, a strong peak was observed close to sh4. We, nevertheless, found that some lines showed complete abscission layer formation despite carrying the IR36 allele at sh4, implying that non-shattering of seeds of IR36 could be regulated by the combination of mutations at sh4 and other seed-shattering loci. We also genotyped qSH3, a recently identified seed-shattering locus. Lines that have the IR36 alleles at sh4 and qSH3 showed inhibition of abscission layer formation but the degree of seed shattering was different from that of IR36. On the basis of these results, we estimated that non-shattering of seeds in early rice domestication involved mutations in at least three loci, and these genetic materials produced in this study may help to identify novel seed-shattering loci.     

Morphological variation,management and domestication of ‘maguey alto’ (<Emphasis Type="Italic">Agave inaequidens</Emphasis>) and ‘maguey manso’ (<Emphasis Type="Italic">A. hookeri</Emphasis>) in Michoacán,México

Background

Agave inaequidens and A. hookeri are anciently used species for producing the fermented beverage ‘pulque’, food and fiber in central Mexico. A. inaequidens is wild and cultivated and A. hookeri only cultivated, A. inaequidens being its putative wild relative. We analysed purposes and mechanisms of artificial selection and phenotypic divergences between wild and managed populations of A. inaequidens and between them and A. hookeri, hypothesizing phenotypic divergence between wild and domesticated populations of A. inaequidens in characters associated to domestication, and that A. hookeri would be phenotypically similar to cultivated A. inaequidens.

Methods

We studied five wild and five cultivated populations of A. inaequidens, and three cultivated populations of A. hookeri. We interviewed agave managers documenting mechanisms of artificial selection, and measured 25 morphological characters. Morphological similarity and differentiation among plants and populations were analysed through multivariate methods and ANOVAs.

Results

People recognized 2–8 variants of A. inaequidens; for cultivation they select young plants collected in wild areas recognized as producing the best quality mescal agaves. Also, they collect seeds of the largest and most vigorous plants, sowing seeds in plant beds and then transplanting the most vigorous plantlets into plantations. Multivariate methods classified separately the wild and cultivated populations of A. inaequidens and these from A. hookeri, mainly because of characters related with plant and teeth size. The cultivated plants of A. inaequidens are significantly bigger with larger teeth than wild plants. A. hookeri are also significatly bigger plants with larger leaves but lower teeth density and size than A. inaequidens. Some cultivated plants of A. inaequidens were classified as A. hookeri, and nearly 10% of A. hookeri as cultivated A. inaequidens. Wild and cultivated populations of A. inaequidens differed in 13 characters, whereas A. hookeri differed in 23 characters with wild populations and only in 6 characters with cultivated populations of A. inaequidens.

Conclusions

Divergence between wild and cultivated populations of A. inaequidens reflect artificial selection. A. hookeri is similar to the cultivated A. inaequidens, which supports the hypothesis that A. hookeri could be the extreme of a domestication gradient of a species complex.

     

Comparison of bioethanol production from cultivated versus wild <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> and <Emphasis Type="Italic">Gracilaria gigas</Emphasis>

The seaweed genus Gracilaria is a potential candidate for the production of bioethanol due to its high carbohydrate content. Gracilaria is abundant throughout the world and can be found in both wild and cultivated forms. Differences in the ecological factors such as temperature, salinity, and light intensity affecting wild and cultivated specimens may influence the biochemical content of seaweeds, including the carbohydrate content. This study aimed to investigate the proximate composition and potential bioethanol production of wild and cultivated G. gigas and G. verrucosa. Bioethanol was produced using separate hydrolysis fermentation (SHF), employing a combination of enzymatic and acid hydrolysis, followed by fermentation with Saccharomyces cerevisiae ATCC 200062. The highest carbohydrate content was found in wild G. gigas. The highest galactose and glucose contents (20.21 ± 0.32 and 9.70 ± 0.49 g L^?1, respectively), as well as the highest production of bioethanol (3.56 ± 0.02 g L^?1), were also found in wild G. gigas. Thus, we conclude that wild G. gigas is the most promising candidate for bioethanol production. Further research is needed to optimize bioethanol production from wild G. gigas. Domestication of wild G. gigas is a promising challenge for aquaculture to avoid overexploitation of this wild seaweed resource.