A genetic linkage map is a valuable tool for quantitative trait locus mapping, map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa, one of the most important forage crops in the world, is autotetraploid, allogamous, and highly heterozygous, characteristics that have impeded the construction of a high-density linkage map using traditional genetic marker systems. Using genotyping-by-sequencing (GBS), we constructed low-cost, reasonably high-density linkage maps for both maternal and paternal parental genomes of an autotetraploid alfalfa F1 population. The resulting maps contain 3591 single-nucleotide polymorphism markers on 64 linkage groups across both parents, with an average density of one marker per 1.5 and 1.0 cM for the maternal and paternal haplotype maps, respectively. Chromosome assignments were made based on homology of markers to the M. truncatula genome. Four linkage groups representing the four haplotypes of each alfalfa chromosome were assigned to each of the eight Medicago chromosomes in both the maternal and paternal parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and clearly identified the known translocation between Chromosomes 4 and 8. In addition, a small inversion on Chromosome 1 was identified between M. truncatula and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that greatly improved genome coverage relative to previous maps and that will facilitate investigation of genome structure. GBS could be used in breeding populations to accelerate molecular breeding in alfalfa.
Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.
A lfalfa, a perennial forage crop, experiences seasonal changes in growth patterns and morphology in the temperate regions of the world. Fall dormancy, referring to the characteristic growth reduction and decumbent shoot orientation of certain genotypes in autumn, typically occurs in late summer and early autumn as temperature declines and photoperiod shortens (Castonguay et al., 2006;McKenzie et al., 1988). For practical purposes, the dormancy level of alfalfa cultivars is ABSTRACT Alfalfa (Medicago sativa L.) is a widely planted perennial forage crop. Fall dormancy is generally negatively correlated with winter injury in alfalfa. To understand the genetic basis of the two traits, we identified quantitative trait loci (QTL) controlling autumn growth and winter injury using a tetraploid alfalfa F 1 population. In total, 601 marker alleles were scored from 78 restriction fragment length polymorphism (rFLp), 123 simple-sequence repeat (SSr), and 48 single nucleotide polymorphism (SNp) markers. Linkage maps were constructed for each parent separately. Both maps contained eight linkage groups (LGs), with a length of 898 cM for WISFAL-6 and 845 cM for ABI408. Using interval mapping, we identified 15 QTL from an across-environment analysis and 71 QTL within individual environments for autumn plant height; winter injury; and autumn shoot, crown, and root biomass across four Iowa environments. of the 71 QTL, 42 were identified at 18 chromosomal locations that were identified in multiple environments for the same trait. possible pleiotropic QTL that contributed to dry weight of shoot, crown, and taproot were found, which partially explained the observed genetic correlations between those traits. However, few QTL were related to both autumn plant height and winter injury, supporting the observation of no genetic correlation between the two traits in this study. These results indicated that the two traits could be manipulated independently and, possibly, efficiently improved using marker-assisted selection. Because most QTL identified in this study were mapped to intervals of at least 10 cM, validation and localization in additional populations is needed to facilitate application of marker-assisted selection.
SummaryBiomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon‐tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions such as light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 down‐regulated or up‐regulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 down‐regulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, down‐regulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.
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