Advances in Alfalfa Cytogenetics

Gary R. Bauchan* and M. Azhar Hossain

USDA-ARS, Soybean & Alfalfa Research Lab., Beltsville, MD 20705-2350

E-Mail:gbauchan@asrr.arsusda.gov

* Corresponding author.

Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other produ cts that may also be suitable.

Abstract

The knowledge of alfalfa cytogenetics has progressed significantly in the past 10 years. Most of the advancements which have been made are due to the development and utilization of image analysis and molecular cytogenetic techniques including Giemsa banding and in situ hybridization. Chromosome studies of mitotic chromosomes by C- and N-banding on the species in the Medicago sativa complex namely M. sativa ssp. coerulea, ssp. sativa, and ssp. fa lcata have been conducted and standardized karyotypes have been developed. Comparative studies between the diploid species reveal that a majority of the ssp. falcata have bands at the centromeres only and the nucleolar organizing regions, where as ssp. coerulea have a number of heterochromatic DNA bands not only at the centromeres but also on the terminal ends, interstitial bands of the short arms, and three of the largest chromosomes possess interstitial bands on their long arms. Prelimi nary studies of the tetraploid ssp. falcata have shown that in addition to the centromeric bands there are chromosomes which have additional heterochromatic DNA bands especially on the short arms of the chromosomes. Tetraploid ssp. sativa C- banding patterns display a typical autotetraploid pattern with four chromosomes per set showing similar banding patterns. Investigations into the chromosome banding of the nine germplasm sources for the alfalfa in the US have determined that the non-dorma nt types have more heterochromatic DNA than the dormant types. Polymorphisms for the number, location, and intensity of the chromosomal bands exist within and between accessions studied. B-chromosomes, isochromosomes, "mega" chromosomes, aneuploidy and de leted chromo-somes have been observed. The annual Medicago species which have been studied appear to have only bands only at their centromeres. The implications and applications of molecular cytogenetic approaches such as chromosome banding of meio tic chromosomes, in situ hybridization, and fluorescent tagging methods on the genetic manipulation and chromosome engineering for the improvement of alfalfa are discussed.

Introduction

There has been several advances in the area of alfalfa cytogenetics in the past 10 years. Prior to this there have been comprehensive chapters written on the cytogenetics of alfalfa including Lesins and Gillies (1972) Stanford et al . (1972), McCoy and Bingham (1988 and 1991) and McCoy and Echt (1992). The most recent progress can be characterized by the advancements in the use of computerized image analysis, chromosome banding, and in situ hybridization.

Bauchan and Campbell (1994) developed and utilized a computerized image analysis system to critically measure, analyze and construct the karyotype of diploid alfalfa. Since that time the development of faster computers (20 Mhz to 600 Mh z) with larger memory (512KB to 128MB RAM) has allowed for increased resolution (1048 x 960 pixels to 2096 x 1920 pixels) and direct connections with Windows based software such as spread sheets for data analysis and presentation programs for production o f pictures for analysis and publication. These advances have made it possible for even more precise karyotypic investigations and several cells per hour can be analyzed rather than one or two per day.

Johnson et al. (1984) and McCoy and Bingham (1988) stated that their attempts to band the chromosomes of Medicago were either unsuccessful or produced bands at the centromeres only and thus this technique did not aid in the advan cement of Medicago cytogenetics. Masoud et al. (1991) showed that the chromosomes of diploid M. sativa ssp. sativa (L.) L. & L. cv. CADL (cultivated alfalfa at the diploid level) had additional bands. B auchan and Hossain (1997) perfected the C-banding technique and extensively applied the technique to prove that there are additional bands and developed a standardized karyotype for diploid ssp. falcata Arcengeli and ssp. coerulea Schmalh.. Bauchan and Hossain (1998a) were the first to develop N-banding for the genus Medicago and this was the first successful utilization of N-banding to identify individual chromosomes of a dicot plant. Falistocco et al. (1995) published a karyotype of tetraploid M. sativa ssp. sativa showing that the individual chromosomes could be identified and added additional proof that alfalfa is an autotetraploid.

Schaff et al.(1990) utilized molecular cytogenetic techniques on alfalfa chromosomes. They used enzymatic techniques (strepavidin-horseradish peroxidase complex) to label a specific gene ($-tubulin) and in situ hybridize the gene to alfalfa chromosomes to identify the location of the gene on two chromosomes. Calderini et al. (1996) used fluorescent stains [4', 6-diamidion-2-phenylindole (DAPI) and chromomycin A3 CMA3)] to band the chromosomes of alfalfa showing that the nucleolar organizing regions (NOR) were high in GC content. Cluster et al. (1996) and Calderini et al. (1996) have used dual fluorescent staining (rhodamine and DAPI) for fluorescent in situ hybridization (FISH) techniques to label the 18S gene of rDNA and identify the number and location of the genes on the chromosomes.

These approaches have advanced the science of alfalfa cytogenetics and have led to a number of new discoveries about the molecular cytogenetics of the genus Medicago particularly the species in the Medicago sativa complex.

The Medicago sativa complex

The species composing the M. sativa complex includes diploid subspecies M. sativa ssp. coerulea and ssp. falcata and tetraploid ssp. sativa, ssp. falcata, and glutinosa M.B.. Diploid M. sativa ssp. coerulea and tetraploid ssp. sativa are characterized by violet or lavender flowers and coiled pods (Quiros and Bauchan, 1988). These species are adapted to a wide range of distribution including the Mediterranean, the Near and Middle East, the Caucasus, Middle, Central and South Asia. The highest concentration of variability is located in the foothills and mountain valleys of Armenia, Eastern Anatolia, Iran, Afghanistan, Central Asia, Jammu and Kashmir (Ivanov, 1977). These species readily cross with both diploid and tetraploid forms of ssp. falcata. Subspecies falcata is characterized by yellow flowers with straight to sickle-shaped pods which are distributed over a wide geographic range; from south Germ any in the west, to Siberia in the east, and from the Black Sea coast of Bulgaria in the south, to St. Petersburg in the north (Ivanov, 1977). M. sativa ssp. glutinosa is tetraploid, characterized by bright yellow or cream corolla color at t he bud stage or in recently opened flowers, changing to full yellow several hours after opening. The pods are coiled and covered with glandular hairs. It is adapted to the moist, subalpine regions of Caucasia, along river valleys. It is thought that this subspecies is the result of hybridization between M. glomerata Balb.(putative progenitor of the M. sativa complex) and ssp. falcata (Quiros and Bauchan, 1988). All of these subspecies readily intercross and have been found growing wild in nature in the same geographic locations and naturally occurring hybrids between them have been observed (Lesins and Lesins, 1979; Small and Bauchan, 1984). Quiros and Bauchan (1988) explain the confusion due to the proliferation of names of species a nd subspecies for the M. sativa - complex. caused by their morphological variability and the existence of hybrids between these species. This section will focus on the recent cytogenetic advancements for the subspecies in the M. sativa compl ex and not their natural hybrids.

Diploid M. sativa ssp. falcata

Utilizing image analysis techniques it is possible to make critical measurements of chromosomes. Diploid ssp. falcata chromosomes are slightly shorter, and average of 1.35 - 2.3um in length (Bauchan, unpublished), than ssp. coerulea chromosomes, an average of 1.50 - 2.40um (Bauchan and Campbell, 1994). The standard C- (Baucha n and Hossain, 1997) and banding (Bauchan and Hossain, 1998a) pattern can be characterized as having bands only at th