Crop Science Journal of Natural Resources and Life Sciences Education
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mian, M. A. R.
Right arrow Articles by Zwonitzer, J. C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Mian, M. A. R.
Right arrow Articles by Zwonitzer, J. C.
Agricola
Right arrow Articles by Mian, M. A. R.
Right arrow Articles by Zwonitzer, J. C.
Related Collections
Right arrow Other Forage Crops
Right arrow Plant Genetic Resources
Crop Science 42:944-950 (2002)
© 2002 Crop Science Society of America

PLANT GENETIC RESOURCES

Determination of Genetic Diversity in Tall Fescue with AFLP Markers

M. A. Rouf Mian*, Andrew A. Hopkins and John C. Zwonitzer

Forage Biotechnology Group, The Samuel Roberts Noble Foundation, Ardmore, OK 73401

* Corresponding author (rmmian{at}noble.org)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Little information is available regarding genetic variation in tall fescue (Festuca arundinacea Schreb). Such information is important in constructing mapping populations and targeting germplasm collection and utilization. The objective of this study was to evaluate the genetic diversity among a number of tall fescue accessions, cultivars, and selected genotypes. In one experiment, 16 persistent tall fescue plants (genotypes) were assayed by a fluorescence-labeled amplified fragment length polymorphism (AFLP) detection method using DNA samples from individual plants. In a second experiment, AFLP diversity among 18 tall fescue populations, including sources of ‘KY-31’, cultivars derived from KY-31 and accessions collected from Oklahoma and Texas, was assessed by an infrared dye-labeling method using DNA samples bulked by each population. On the basis of 461 AFLP markers from six primer combinations, the 16 individual plants were clustered in groups that largely supported the known origins of these plants. On the basis of genetic divergence, several candidate parents were identified for constructing a mapping population. Analysis of bulked DNA samples revealed that sources of KY-31 and cultivars derived from KY-31 generally clustered separately from accessions collected in the southern Great Plains. Such accessions thus provide an additional source of genetic variation for tall fescue breeding programs.

Abbreviations: AFLP, amplified fragment length polymorphism • bp, base pair • FBG, Forage Biotechnology Group • PEX, potassium ethyl xanthogenate • RAPD, random amplified polymorphic DNA • UPGMA, unweighted pair group mean average


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
TALL FESCUE, a major forage and turf grass species in the temperate regions of the world, is a hexaploid (2n = 6x = 42) with a genome size of {approx}5.27 to 5.83 x 106 kb (Seal, 1983). The genomic constitution of tall fescue is PPG1G1G2G2 with the P (2x) genome coming from F. pratensis and the G1G2 (4x) genome from F. arundinacea var glaucescens auct. [= F. arundinacea subsp. fenas (Lag.) Arcang.] (Sleper, 1985). Tall fescue is also an open pollinated species with a high level of self-incompatibility (Xu et al., 1991). There is limited information available on genetic diversity of tall fescue (Xu et al., 1994). Molecular markers should be useful in revealing the genetic diversity in this species.

Molecular markers can provide information needed to select genetically diverse parents for developing breeding and mapping populations. The AFLP markers have been successfully used to determine genetic diversity in many plant species including forage and turf grasses (Sharma et al., 1996; Mace et al., 1999; Pillay and Myers, 1999; Zhang et al., 1999; Roldan-Ruiz et al., 2000; Guthridge et al., 2001). Other molecular marker systems, including restriction fragment length polymorphisms, simple sequence repeats, random amplified polymorphic DNA (RAPD), and allozymes, have also been used for diversity assessment in various grass species (Huff et al., 1993; Xu et al., 1994; Sun et al., 1998; Kubik et al., 1999; Sun et al., 1999). Unlike RAPD markers, AFLP markers were found to be highly reproducible with an overall error rate of <2% (Tohme et al., 1996; Vos et al., 1995). The PCR-based AFLP markers are amenable to automation for high-throughput genotyping and, being anonymous, do not require any sequence information.

Determination of genetic diversity in tall fescue and other outcrossing species is complicated by the fact that genetic variation exists among populations (cultivars or accessions), as well as among individuals within populations. For this reason, genetic diversity studies on outcrossing species have been traditionally conducted by separately profiling DNA from a number of individuals within each population. Diversity within and among populations is then determined by analyzing the DNA profiles of these individuals. Xu et al. (1994) concluded that 16 plants were sufficient to adequately sample the genetic diversity within a cultivar.

Often, the initial objective of DNA profiling of populations is to determine diversity among populations in order to develop genetically distinct subsets of populations in a breeding program or to check for duplicates in a gene bank. In these cases, it may be possible to determine diversity among populations by profiling bulked DNA of the individuals. Sweeny and Danneberger (1997) distinguished synthetic cultivars of perennial ryegrass (Lolium perenne L.) by screening seed-derived bulked DNA with RAPD markers. Yu and Pauls (1993) estimated the genetic distances among heterogeneous populations of alfalfa (Medicago sativa L.) using RAPD data generated from bulked DNA of 10 individual plants from each population. These estimates of genetic distances corresponded to the known relatedness of the alfalfa populations.

It is believed that KY-31 is the predominant tall fescue cultivar in the eastern USA (Ball et al., 1993). Several cultivars are known derivatives of KY-31, including ‘Penngrazer’, ‘Stargrazer’ (Alderson and Sharp, 1994), and ‘Jesup’ (Bouton et al., 1997). Other cultivars, such as ‘GA-5’ (Bouton et al., 1993), were selected from germplasm from the eastern USA that may trace largely to KY-31. It is unknown to what extent tall fescue germplasm found in the southern Great Plains differs from KY-31.

Information regarding genetic diversity in tall fescue is needed both as an initial step in identifying parents for constructing a mapping population, and to determine if germplasm collected from the southern Great Plains represents an additional source of genetic variation relative to KY-31. Thus, the objective of this study was to evaluate the genetic diversity among a number of tall fescue accessions, cultivars, and selected genotypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Experiment 1
Genetic diversity was assessed among 16 tall fescue plants (genotypes) selected as possible parents for construction of tall fescue mapping populations with potential for persistence in the southern Great Plains (Table 1). Fifteen plants, from among >2000 plants representing more than 170 accessions, were selected for persistence following multiple seasons of heavy grazing pressure near Ardmore, OK (1998–2000, unpublished data). The clone designated HD28-56, selected for high digestibility, was supplied by Dr. David Sleper, University of Missouri. These 16 tall fescue plants were selected as possible parents for construction of tall fescue mapping populations with potential persistence in the southern Great Plains.


View this table:
[in this window]
[in a new window]
  		Table 1. Geographical origin and pedigree of 16 persistent tall fescue plants (genotypes) surveyed for amplified fragment length polymorphism.

 
Approximately 200 mg of tissue from young leaves of each plant was collected in a 2.0-mL eppendorf tube, immediately frozen in liquid N, and ground to fine powder. The DNA was extracted with the potassium ethyl xanthogenate (PEX) DNA isolation protocol described by Williams and Ronald (1994) with modifications. The ground tissue was mixed well with 1 mL of the PEX buffer and incubated at 60°C for 1 h.

The DNA was extracted from duplicate leaf samples of each of the 16 plants, and the AFLP procedure was performed on the duplicate DNA samples in order to confirm the repeatability of the AFLP markers. The AFLP procedure was conducted according to the protocol supplied with the AFLP plant mapping kit of PE Applied Biosystems (Foster City, CA) with minor modifications. The restriction-ligation reactions were carried out with EcoRI and MseI restriction enzymes according to the protocol. The preselective amplification was done with the EcoRI + A and MseI + C primers in a thermocycler for 24 cycles set at 94°C denaturation (20 s), 56°C annealing (30 s), and 72°C extension (2 min). The initial hold was at 72°C for 2 min, and the final extension was at 60°C for 30 min. The amplification product was diluted 20-fold in 0.1x TE buffer and stored at -20°C until used for selective amplification reactions. Selective amplification was performed using the EcoRI + AGG primer 5'–end labeled with blue (6-Fam) fluorescent tag and six MseI + 4 primers. Selective amplifications for this experiment were done according to the ABI protocol.

Two microliters of the selective amplification product was mixed with 12.0 µL of deionized formamide and 0.5 µL of GeneScan-500 ROX (Perkin-Elmer Applied Biosystems, Foster City, CA) internal size standard. The samples were electrophoresed on an ABI 310 Capillary Genetic Analyzer (Perkin-Elmer Applied Biosystems) with an injection time of 8 s and run time of 28 min. Raw data were analyzed with GeneScan analysis software (Version 2.1, Perkin-Elmer Applied Biosystems) and the resulting GeneScan trace files were imported into Genographer Version 1.2 (http://hordeum.oscs.montana.edu/genographer). The AFLP fragments between 60 to 500 base pair (bp) were scored in Genographer as present (A) or absent (B). Scores were recoded (by changing an A to 1 and a B to 0) and formatted for analyses.

Genetic distances among genotypes were calculated according to the Nei and Li (1979) similarity definition: Sij = 2a/(2a + b + c), where Sij is the similarity between two individuals, i and j; a is the number of bands present in both samples i and j; b is the number of bands present in i and absent in j; and c is the number of bands present in j and absent in i. The distance matrix was used for construction of a dendrogram by the unweighted pair group mean average (UPGMA) method (SAS Institute, 1989).

Experiment 2
Eighteen tall fescue populations [seven cultivars, two plant introductions, and nine Forage Biotechnology Group (FBG) collections] were included in this experiment (Table 2). Origins of the cultivars are described by Alderson and Sharp (1994) and Bouton et al. (1997). Seeds of FBG collections were collected in bulk from the site of origin, except 98TF5 and 98TF6. For these accessions, 35 and 23 genotypes, respectively, were selected from stressful microsites at the site of origin and subsequently placed in separate polycross isolations near Ardmore, OK, to produce seeds. The accessions PI 269850 and PI 204447 were among the most and least persistent accessions, respectively, in the grazing tolerance evaluation trial mentioned above.


View this table:
[in this window]
[in a new window]
  		Table 2. Geographical origin and pedigree of 18 fescue populations surveyed for amplified fragment length polymorphism diversity.

 
Approximately equal amounts of young expanding leaf tissues were collected from 16 4-wk-old seedlings of each of the 18 tall fescue populations. Equal amounts of leaf tissues of the 16 seedlings from each population were bulked together, freeze dried, and ground for DNA extraction according to the protocol described by Xu et al. (1991).

The AFLP procedure was performed according to the protocol of Vos et al. (1995) that is supplied by Life Technologies (Gaithersberg, MD) with the AFLP Analysis System I, with modifications. Approximately 500 ng of DNA of each bulked sample was simultaneously and completely digested with EcoRI and MseI at 37°C for 3 h. The restricted DNA fragments were ligated to EcoRI and MseI adapters overnight at 37°C, and the product was diluted to 500 µL with TE buffer. Preamplification reactions were done with EcoRI + A and MseI + C AFLP primers.

The amplification product was diluted 10-fold in TE buffer and stored at -20°C until used for selective amplifications. Selective amplification was done with eight combinations of EcoRI + 3 and MseI + 4 primers: E-AAG/M-CCAG, E-AAG/M-CCGC, E-AAG/M-CCTA, E-AAG/M-CGCG, E-AAG/M-CTCG, E-AAG/M-CGCT, E-ACG/M-CCGC, and E-ACG/M-CCTA. The infra red dye labeled EcoRI primers were purchased from LI-COR, Inc. (Lincoln, NE). Selective amplifications were performed in a final volume of 10 µL containing 2 µL of the dilute preamplification product, 15 ng of the MseI selective primers, 0.5 µL of 1 µM IRD-800 labeled EcoRI primer, 0.4 units of Taq polymerase, 1x PCR buffer, 2.0 mM MgCl2, and 200 µM deoxynucleotide triphosphates.

The selective amplification product was mixed with 4 µL of the loading dye, and the mixture was denatured at 95°C for 3 min and immediately placed on ice. One µL of the denatured samples was loaded on a 6.5% polyacrylamide gel containing 7 M urea, and electrophoresis was conducted with a constant power of 40 W at a constant temperature of 47°C for 4 h in a LI-COR 4000L DNA sequencer (LI-COR, Inc.). The sequencer automatically saved the raw AFLP data in a TIFF format. The raw data from the TIFF file were scored after importing the data into the AFLP Quantar software Version 1.0 (KeyGene Products B.V., The Netherlands). Fragments between 50 and 700 bp were scored with the majority falling in the 100- to 400-bp range. Only clearly visible fragments were scored with the AFLP Quantar software. Low intensity fragments were not scored. The software assigned a score of +, present; -, absent; or ?, not sure to each sample for each of the fragments selected for scoring. Each score was then checked manually by visual observation of TIFF images to confirm or to make necessary changes to the computer assigned scores. Statistical analysis of the data was performed as for Exp. 1.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Experiment 1
Initial testing of AFLP techniques on tall fescue demonstrated that selective amplification with the EcoRI + 3 and MseI + 3 primer combinations produced too many fragments ({approx}200) to be resolved on polyacrylamide gels, whereas EcoRI + 3 and MseI + 4 primer combinations generally produced the desired number of fragments (80–100). A total of 461 AFLP (261 polymorphic and 200 monomorphic) fragments were scored from six primer combinations. The E-AGG and M-CTGC primer combination produced the greatest number of polymorphic fragments (61), while the E-AGG and M-CTCG primer combination yielded the fewest polymorphic fragments (33). The number of monomorphic fragments for each primer pair varied from 30 to 50. The majority of scored fragments were in the 80- to 350-bp range; however, fragments were found across the entire size range of 60 to 500 bp (Fig. 1) . Duplicate DNA samples of each plant were placed next to each other (e.g., HD28-56a and HD28-56b in Fig. 1 were the two duplicate samples from the HD28-56 plant) for comparing the repeatability of the AFLP fragments. Most AFLP fragments were either present or absent in both DNA samples of each plant, indicating a very good repeatability. However, nearly 2.5% of the AFLP fragments indicated lack of repeatability, that is, these fragments were present in only one of the two DNA samples taken from a plant. Fragments that lacked repeatability in this experiment were considered as missing for data analyses.



View larger version (51K):
[in this window]
[in a new window]
  		Fig. 1. The AFLP profiles of duplicate samples of 16 tall fescue genotypes generated with the E-AAG/M-CAGC selective primer combination. Duplicate samples of each genotype were placed next to each other for comparison of the reproducibility of the AFLP bands.

 
Genetic distances among genotypes varied from 0.22 to 0.53 (Table 3). Genotypes TFA2-4 and TFA2-7, both selected from the accession 97TF1, had the minimum genetic distance (0.22). Six of the seven plants selected from 97TF1 were grouped into one cluster along with HD28-56 (Fig. 2 and Table 1). The HD28-56 clone is a selection out of a composite population created by intercrossing several PIs and US collections (Dr. D.A. Sleper, 2001, personal communication). Thus, HD28-56 may share some common ancestry with 97TF1, which is a collection from Oklahoma. Plant TFA2-15, selected out of PI 508604 from Argentina, clustered with plant TFA2-10, which was selected out of PI 512313 from Spain. It is possible that these two plant introductions have a common origin. Plant TFA2-6, derived from PI 269850 from Tunisia, did not cluster with any other entry in this study. The AFLP results were in general agreement with available information regarding origins of these plants.


View this table:
[in this window]
[in a new window]
  		Table 3. The among genotype distance matrix for 16 tall fescue plants (genotypes) surveyed with amplified fragment length polymor-phism (AFLP) markers in Exp. 1.

 


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. Dendrogram of 16 tall fescue genotypes based on 461 AFLP markers. A name in the parenthesis indicates the tall fescue accession from which the plant immediately above it was selected.

 
Genetic distance scores for TFA2-6 (0.42–0.53) and TFA2-12 (0.35–0.42) indicate that these genotypes could be used in a cross with any one of a number of plants examined in order to construct a mapping population. However, it may be difficult to obtain fertile progeny from crosses between North African genotypes, such as TFA2-6, and other tall fescue germplasm (Evans et al., 1973; Hunt and Sleper, 1981). Molecular marker data will also need to be coupled with phenotypic data to identify parents for constructing a mapping population. Based on genetic similarity, TFA2-11 would clearly not be a good genotype to use in crosses with 97TF1 plants in order to construct a mapping population.

Experiment 2
A total of 343 AFLP (193 polymorphic and 150 monomorphic) fragments were scored from the eight primer combinations. The E-AAG/M-CCTA primer combination produced the greatest number of polymorphic fragments (49), whereas the primer combination E-AAG/M-CGCG produced the fewest number of polymorphic fragments (20).

Genetic distances among populations ranged from 0.05 for KY-31K and KY-31M, to 0.54 for 98TF2 and PI 204447 as well as 98TF6 and PI 269850 (Table 4). The UPGMA of AFLP data resulted in meaningful groupings of the 18 tall fescue populations. Three distinct clusters were found in the dendrogram generated with the UPGMA clustering method (Fig. 3) . Cluster 1 includes four FBG collections (97TF1, 98TF4, 98TF5, and 98TF3) from Oklahoma.


View this table:
[in this window]
[in a new window]
  		Table 4. Among population genetic dissimilarity coefficients for 18 tall fescue populations based on amplified fragment length polymorphism markers generated from bulked DNA samples.

 


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3. The UPGMA tree of 18 tall fescue populations based on 343 AFLP markers generated from bulked DNA samples.

 
Cluster 2 includes several collections from the southern Great Plains (98TF1, 98TF2, and 98TF6) and one cultivar (GA5). GA5 is a cultivar derived from eastern USA ecotypes (Bouton et al., 1993). Our data indicate that GA5 is different from KY-31, given that GA5 did not cluster with any entries labeled as KY-31 that were included in this study.

Cluster 3 includes three separate sources of KY-31 (KY-31G from Univ. of Georgia, KY-31K produced in Kansas, and KY-31M produced in Missouri), several cultivars derived from KY-31 (Stargrazer, Jesup, and Penngrazer), and one collection from Texas (99GCJ1). We speculate that 99GCJ1, collected from a roadside, is a recent planting of KY-31.

Two plant introductions, one from Turkey (PI 204447) and one from Tunisia (PI 269850), did not group with any other entry in the study, implying that each PI was genetically distinct from other populations in the study.

Four of the FBG collections were grouped in Cluster 1, three in Cluster 2, and one in Cluster 3, indicating that these accessions are somewhat genetically diverse. The exact pedigrees of these FBG accessions are not known except for their collections sites. KY-31 is thought to be the seed source for 98TF2 (Tommy Pickard, 1998, personal communication) and 98TF4 (Brian Freking, 1998, personal communication), whereas seeds from Missouri, quite possibly KY-31, were used to establish 98TF5 (John Spears, 1998, personal communication). All of these sites were planted circa 1980. Similarly, KY-31 is thought to have been used to plant 98TF6 in the 1950s (G.T. Easly, 1998, personal communication).

Assuming our speculation regarding origins of these FBG accessions is correct, natural selection has likely occurred within these populations, allowing them to be distinguished from KY-31. Genetic shifts have been documented for plant populations exposed to grazing (Brummer and Bouton, 1991; Vaylay and van Santen, 1999). Such shifts could have occurred in accessions such as 98TF2, 98TF4, 98TF5, 98TF6, and PDF that were all collected from pastures. Natural selection for adaptation to edaphic and climatic conditions of the southern Great Plains has likely occurred as well, since genetic shifts appear to have taken place in populations that have evidently not been exposed to grazing, such as 97TF1 and 98TF3, which were collected from roadsides, and 98TF1, which was collected from training grounds at Fort Sill, OK. For 98TF5 and 98TF6, additional genetic shifts may have been caused by selection of the genotypes used to construct these accessions.

Alternatively, the FBG accessions may have originated from germplasm other than KY-31. This might have occurred if seed marketed as KY-31 was improperly identified. In the present research, KY-31 sources clustered closely, indicating proper labeling of seeds. A large number of seed sources would need to be examined to determine the prevalence of mislabeled KY-31 seed. In light of results from Exp. 1 and 2, as well as favorable persistence information, many of the accessions evaluated in this research are being incorporated into the FBG breeding program. Among these are 98TF1, 98TF2, and 98TF4, which were subsequently found to be largely free of endophyte (Neotyphodium ceonophialum (Morgan-Jones & Gams.) Glenn, Bacon & Hanlin comb. nov.). Genetic differences between Mediterranean accessions, such as PI 269850, and tall fescue collected from the southern Great Plains can be large. Barring prohibitive genetic barriers, these two germplasm pools may be a reasonable starting point in an effort to identify heterotic groups of tall fescue for use in a semihybrid breeding strategy, as outlined by Brummer (1999).

Using a DNA bulking strategy, we were able to distinguish among 18 tall fescue populations. Yu and Pauls (1993), Sweeny and Danneberger (1997), and Warburton et al. (2000) also assessed molecular marker diversity among heterogeneous populations of alfalfa, perennial ryegrass, and maize (Zea mays L.), respectively based on bulked DNA samples. Instead of working with separate DNA samples from a number of individuals from each population, one can determine diversity among populations based on bulked DNA samples. It should then be possible to study diversity within chosen populations as needed. This strategy could be applied to assess diversity of accessions from outcrossing species in a breeding program or germplasm collection.

Tall fescue collected from sites in the southern Great Plains generally represents a germplasm resource that is different from KY-31. In addition, such germplasm contains variation beyond that found in cultivars derived from KY-31. As such, further collections of tall fescue from the southern Great Plains are warranted.

Received for publication August 14, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




This article has been cited by other articles:


Home page
 Crop Sci.Home page
M. A. R. Mian, J. C. Zwonitzer, Y. Chen, M. C. Saha, and A. A. Hopkins
AFLP Diversity within and among Hardinggrass Populations
Crop Sci., October 27, 2005; 45(6): 2591 - 2597.
[Abstract] [Full Text] [PDF]

Home page
 Crop Sci.Home page
Y. Q. Wu, C. M. Taliaferro, G. H. Bai, and M. P. Anderson
Genetic Diversity of Cynodon transvaalensis Burtt-Davy and Its Relatedness to Hexaploid C. dactylon (L.) Pers. as Indicated by AFLP Markers
Crop Sci., March 28, 2005; 45(3): 848 - 853.
[Abstract] [Full Text] [PDF]

Home page
 Crop Sci.Home page
L. Pallottini, E. Garcia, J. Kami, G. Barcaccia, and P. Gepts
The Genetic Anatomy of a Patented Yellow Bean
Crop Sci., May 1, 2004; 44(3): 968 - 977.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mian, M. A. R.
Right arrow Articles by Zwonitzer, J. C.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Mian, M. A. R.
Right arrow Articles by Zwonitzer, J. C.
Agricola
Right arrow Articles by Mian, M. A. R.
Right arrow Articles by Zwonitzer, J. C.
Related Collections
Right arrow Other Forage Crops
Right arrow Plant Genetic Resources

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome