Amylase Activity in Taproots of Medicago sativa L. and Lotus corniculatus L. Following Defoliation

Although the patterns of starch metabolism in taproots of alfalfa(Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatusL.) have been characterized, little is known regarding the activitiesof starch-degrading enzymes in taproots of these species. Ourobjective was to determine how defoliation influences starchdegradation and activities of amylases in taproots of alfalfaand birdsfoot trefoil. In Exp. 1, amylolytic activities andstarch concentrations in taproots of defoliated and undefoliatedplants were compared on days 0, 3, 7, 10, and 14 after defoliation.Taproot starch concentrations declined in defoliated plants,while increasing in taproots of undefoliated plants. Exoamylaseactivities in taproots of defoliated plants did not change withdefoliation, while endoamylase activities increased 2-fold indefoliated alfalfa and 50% in defoliated birdsfoot trefoil plantswhen compared to undefoliated plants. In Exp. 2, activity andisoform complement of amylases were monitored during seedlingdevelopment. High endoamylase activity was found in taprootsof both species at all samplings. In contrast, exoamylase accumulatedin taproots of alfalfa, but not birdsfoot trefoil, in a patternsimilar to starch accumulation. As in Exp. 1, defoliation increasedendoamylase, but not exoamylase activity in taproots of bothspecies. Taproots of both species contained one major and twominor endoamylase isoforms, but the electrophoretic mobilityof these isoforms differed between species. Activities of allisoforms, as indicated on starch-gel blots, increased in responseto defoliation. These results indicate that defoliation increasesactivity of taproot endoamylases, whose activity is associatedwith taproot starch degradation. Key words: Starch degradation, alfalfa, birdsfoot trefoil, enzymes     

Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth

Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.     

Starch Grain Distribution in Taproots of Defoliated Medicago sativa L

Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.     

Relationships among genetic distance,forage yield and heterozygosity in isogenic diploid and tetraploid alfalfa populations

Isogenic diploid and tetraploid alfalfa (Medicago sativa L.) was studied with molecular markers to help understand why diploid performance and breeding behavior does not always predict that of tetraploids. In a previous study of partially heterozygous alfalfa genotypes, we detected a low correlation between yields of isogenic diploid (2x) and tetraploid (4x) single-cross progenies, and genetic distances were more highly correlated with yields of tetraploids than diploids. These differences may be related to the level of RFLP heterozygosity expected among progenies derived from heterozygous parents at the two ploidy levels. The objectives of this study were to determine the relationships among genetic distance, forage yield and heterozygosity in isogenic 2 x and 4 x alfalfa populations. Four diploid genotypes were chromosome doubled to produce corresponding isogenic autotetraploids, and these genotypes were mated in 4 × 4 diallels to produce 6 single-cross families at each ploidy level for field evaluation. Allele compositions of parents were determined at 33 RFLP loci by monitoring segregation of homologous restriction fragments among individuals within progenies, and these were used to estimate RFLP heterozygosity levels for all single-cross progenies at both ploidy levels. RFLP heterozygosity rankings were identical between progenies of isogenic diploid and tetraploid parents; but significant associations (P < 0.05) between estimated heterozygosity levels and forage yield were detected only at the tetraploid level. Since tetraploid families were nearly 25% more heterozygous than the corresponding diploid families, inconsistencies in the association between molecular marker diversity and forage yields of isogenic 2 x and 4 x single crosses may be due to recessive alleles that are expressed in diploids but masked in tetraploids. The gene action involved in heterosis may be the same at both ploidy levels; however, tetraploids benefit from greater complementary gene interactions than are possible for equivalent diploids. Present address: AgResearch Grasslands, New Zealand Pastoral Agriculture Research Institute, Palmerston North, New Zealand     

Etiolated Growth as Measure of Non-structural Biomass in Lucerne Taproots

Enzymatic hydrolysis of starch in lucerne (Medicago sativa L.)taproots is the conventional method used to determine the quantityof carbohydrates allocated to regrowth. Etiolated growth froma taproot could be used to quantify total root biomass allocatedto regrowth. This study compared concentrations of non-structuralcarbohydrates, as measured by -amylase hydrolysis of starchto glucose, to concentrations of non-structural biomass, asmeasured by etiolated growth from lucerne taproots placed inan incubator and plants in situ. The concentration of starchfrom enzymatically assayed taproots was 325 g kg^-1 expressedas glucose equivalents. Etiolated growth and weight loss byrespiration from plants grown in the incubator accounted for524 g of actual biomass per kg of root. There was 46·2g kg^-1 of N, 3·1 of P, and 33·1 of K in the etiolatedgrowth. An 88% increase in etiolated growth dry weight was observedfrom plants in situ compared to taproots placed in the incubator.Accurate quantification on non-structural biomass should notbe limited to sampling just the taproot, but must included theentire root system. Compared to determining non-structural carbohydratesby enzymatic hydrolysis of starch, the procedure used in determiningnon-structural biomass by etiolate growth gave results in unitsrelative to the plant. The use of etiolate growth also providedinformation on mineral nutrient partitioning from root to shoots,was less technically demanding, and could be applied to theentire root system.Copyright 1993, 1999 Academic Press Medicago sativa, root carbohydrates, etiolated growth, taproot     

Composition and Structure of Starch from Taproots of Contrasting Genotypes of Medicago sativa L

The objective of this study was to examine the composition and branch chain lengths of alfalfa (Medicago sativa L.) taproot starch during starch utilization and reaccumulation in response to defoliation. Genotypes were propagated vegetatively and well-established plants were sampled at defoliation and at weekly intervals thereafter. Starch granules from root tissues were dispersed in dimethyl sulfoxide and starch components separated using gel permeation chromatography. Root starches also were debranched enzymically, and branch chain lengths were examined. Results indicate that, irrespective of starch concentration, starch from taproots of the high starch genotype was composed of approximately 80% high molecular weight starch with I₂-Kl absorbance characteristics similar to amylopectin. The remaining 20% of the starch was low molecular weight with I₂-Kl absorbance characteristics similar to amylose. Starches of the low starch genotype contained approximately 85% high molecular weight polysaccharide at high root starch concentrations (>50 grams per kilogram). At low root starch concentrations (<10 grams per kilogram), starch from the low starch genotype had nearly equal proportions of low and high molecular weight polysaccharide. The I₂-Kl absorbance properties of the low molecular weight starches from roots of the low starch genotype indicated that some branching may be present. The distribution of chain lengths from amylopectin did not change during starch degradation and reaccumulation for the high starch genotype. In the low starch genotype, the proportion of low molecular weight branches having a degree of polymerization between 1 and 30 was decreased at the very low starch concentrations observed on the 14th day of regrowth. Higher concentrations and/or quantities of starch in roots of the high starch genotype were not associated with greater rate of herbage regrowth, when compared to the low starch genotype.     

Development of an RFLP map in diploid alfalfa

Summary We have developed a restriction fragment length polymorphism (RFLP) linkage map in diploid alfalfa (Medicago sativa L.) to be used as a tool in alfalfa improvement programs. An F₂ mapping population of 86 individuals was produced from a cross between a plant of the W2xiso population (M. sativa ssp. sativa) and a plant from USDA PI440501 (M. sativa ssp. coerulea). The current map contains 108 cDNA markers covering 467.5 centimorgans. The short length of the map is probably due to low recombination in this cross. Marker order may be maintained in other populations even though the distance between clones may change. About 50% of the mapped loci showed segregation distortion, mostly toward excess heterozygotes. This is circumstantial evidence supporting the maximum heterozygote theory which states that relative vigor is dependent on maximizing the number of loci with multiple alleles. The application of the map to tetraploid populations is discussed.     

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn ₁ and nn ₂ )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F₁ plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F₂ population segregating the nn ₁ and nn ₂ genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F₁ plants.     

Partitioning of Nitrogen Derived from N2 Fixation and Reserves in Nodulated Medicago sativa L. During Regrowth

Nodul{macron}ted alfalfa plants were grown hydroponically. Inorder to quantify N₂ fixation and remobilization of N reservesduring regrowth the plants were pulse-chase-labelled with ¹⁵N.Starch and ethanol-soluble sugar contents were analysed to examinechanges associated with those of N compounds. Shoot removalcaused a severe decline in N₂ fixation and starch reserves within6 d after cutting. The tap root was the major storage site formetabolizable carbohydrate compounds used for regrowth; initiallyits starch content decreased and after 14 d started to recoverreaching 50% of the initial value on day 24. Recovery of N₂fixation followed the same pattern as shoot regrowth. Afteran initial decline during the first 10 d following shoot removal,the N₂ fixation, leaf area and shoot dry weight increased sorapidly that their levels on day 24 exceeded initial values.Distribution of ¹⁵N within the plant clearly showed that a significantamount of endogenous nitrogen in the roots was used by regrowingshoots. The greatest use of N reserves (about 80% of N incrementin the regrowing shoot) occurred during the first 10 d and thencompensated for the low N₂ fixation. The distribution of N derivedeither from fixation or from reserves of source organs (taproots and lateral roots) clearly showed that shoots are thestronger sink for nitrogen during regrowth. In non-defoliatedplants, the tap roots and stems were weak sinks for N from reserves.By contrast, relative distribution within the plant of N assimilatedin nodules was unaffected by defoliation treatment. Key words: Medicago sativa L., N₂ fixation, N remobilization, N₂ partitioning, regrowth     

Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)?

Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei.     

Molecular Diversity and Population Structure of a Worldwide Collection of Cultivated Tetraploid Alfalfa (Medicago sativa subsp. sativa L.) Germplasm as Revealed by Microsatellite Markers

Information on genetic diversity and population structure of a tetraploid alfalfa collection might be valuable in effective use of the genetic resources. A set of 336 worldwide genotypes of tetraploid alfalfa (Medicago sativa subsp. sativa L.) was genotyped using 85 genome-wide distributed SSR markers to reveal the genetic diversity and population structure in the alfalfa. Genetic diversity analysis identified a total of 1056 alleles across 85 marker loci. The average expected heterozygosity and polymorphism information content values were 0.677 and 0.638, respectively, showing high levels of genetic diversity in the cultivated tetraploid alfalfa germplasm. Comparison of genetic characteristics across chromosomes indicated regions of chromosomes 2 and 3 had the highest genetic diversity. A higher genetic diversity was detected in alfalfa landraces than that of wild materials and cultivars. Two populations were identified by the model-based population structure, principal coordinate and neighbor-joining analyses, corresponding to China and other parts of the world. However, lack of strictly correlation between clustering and geographic origins suggested extensive germplasm exchanges of alfalfa germplasm across diverse geographic regions. The quantitative analysis of the genetic diversity and population structure in this study could be useful for genetic and genomic analysis and utilization of the genetic variation in alfalfa breeding.     

Molecular,cytological and morpho-agronomical characterization of hexaploid somatic hybrids in Medicago

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.This paper was supported by the National Research Council of Italy, Special Project RAISA, Sub-project No.2 paper No. 1911     

Characterization of a Small Sulphur-Rich Storage Albumin in Seeds of Alfalfa (Medicago sativa L.)

A 2S albumin fraction was characterized in seeds of alfalfa{^{Medicago sativa} L.). This low molecular weight (LMW) familyof disulphide-bonded proteins represents a major nitrogen andsulphur storage reserve for the alfalfa seed Characteristicof seed storage proteins, the 2S albumins are abundant in nitrogen-richglutarrune/glutamate/asparagine/aspartate (32%) In addition,this LMW fraction is high in cysteine (9%) and methionine (4%),amino acids which are under-represented in legume seed globulins.These 2S proteins start to accumulate during the early cotyledonstage of development, and are mobilized following germinationPulse-chase labelling experiments show that the 2S proteinsare synthesized as 'preproproteins', similar to 2S proteinsin other seeds. However, alfalfa 2S albumins are immunologicallyunrelated to these proteins. Key words: Seed development, sulphur-containing 2S storage protein, alfalfa (Medicago sativa)     

High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis

Summary A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F₁ progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment.Contribution No. 89-524-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Cold Acclimation, Freezing Resistance and Protein Synthesis in Alfalfa (Medicago sativaL. cv. Saranac)

Mohapatra, S. S., Poole, R. J. and Dhindsa, R. S. 1987. Coldacclimation, freezing resistance and protein synthesis in alfalfa(Medicago sativa L. cv. Saranac).—J. exp. Bot. 38: 1697–1703. Changes in freezing resistance (percent survival at —10°C), pattern of protein synthesis and translatable mRNApopulation during cold acclimation of alfalfa (Medicago sativaL. cv. Saranac) have been examined. Two days of cold acclimationat 4 °C increased freezing resistance from about 6% to 40%,protein content by 200% and total RNA content by 100%. Acclimationfor longer periods did not cause further increases in freezingresistance, protein content or RNA content. Examination of proteinchanges by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) coupled with protein staining, and by fluorographyof in vivo labelled proteins separated by SDS-PAGE, showed thatseveral proteins are increasingly or newly synthesized duringcold acclimation. Analysis of in vitro translation productsby SDS-PAGE and fluorography shows changes in the populationof translatable mRNAs. It is concluded that in this varietyof alfalfa cold acclimation for only 2 d is sufficient to confermaximum freezing resistance, and that changes in proteins duringcold acclimation are regulated most probably at the transcnptionallevel. Key words: Freezing resistance, protein synthesis, cold acclimation, SDS-PAGE, Medicago sativa L.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

Use of the Pressure Bomb for Hydraulic Conductance Studies

Stem sections of alfalfa (Medicago sativa L.) were subjectedto forced flows of both water and high molecular weight dextransolutions. In either case, stems responded in accordance withthe Ohm's law analogy for flow, i.e., flow was a linear functionof pressure, at pressures ranging from 7.0–500 kPa andvacuum from 25.69 kPa. Since conductance values were the samefor both pressure and vacuum powered flows, it is possible toextrapolate conductance values determined with a pressure bombto negative pressure gradients experienced by plants. Key words: Pressure bomb, Conductance, Medicago sativa     

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.     

Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa

Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid alfalfa. Ten SSR markers were incorporated into an existing F₂ diploid alfalfa RFLP map and also mapped in an F₂ tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa. Received: 10 September 1999 / Accepted: 11 November 1999