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 the centromeric regions and a large band at the nucleolar organizer regions (NOR) of the satellited chromosome 8 (Fig. 1a). Occasionally a n interstitial band occurs in the long arm of the satellited (SAT) chromosome .
A comparison of the mitotic banding pattern to meiotic pachytene chromosomes (Gillies, 1970a) shows a relatively good correlation between mitotic heterochromatic bands and the chromatic knobs (chromomeres) found at pachytene. Gillies (1 970a) and Ho and Kasha (1972) found that a majority of the chromomeres at pachytene in M. sativa ssp. falcata were located on either side of the centromeres and there were no prominent telomeric knobs except when an entire arm was chromatic. In a majority of the accessions studied using C-banding of somatic chromosomes there were no bands found on the short arms of chromosomes. However, in a survey of 17 accessions of diploid ssp. falcata representing the wide geographic distribution of the subspecies, Bauchan and Hossain (1999a) discovered that 10 accessions possessed 1 to 14 additional C-bands when compared to the standard karyotype for ssp. falcata. Polymorphisms for the number, locations and intensities of C-bands were dete cted among and between accessions. Only 9% of the plants studied contained displayed polymorphisms. C-bands may exist at the terminal ends of all the chromosomes and interstitial bands were detected on both the short arms and the long arms of chromosomes 1, 2, 3 and 6. The origin of these additional bands could have preexisted or could be the result of crossing over product from hybridization with other subspecies in the Medicago sativa complex. The absence of diagnostic bands on the chromosomes ma kes it difficult to critically analyze the karyotype of this subspecies, however, with the aid of an computerized image analysis system (Bauchan and Campbell, 1994) it is possible to develop karyotypes based on chromosome measurements.
Chromosome modifications such as isochromsomes for the short arms of chromosome 2 and 6 were also observed (Bauchan and Hossain, 1999a). Isochromosomes are monocentric chromosomes with homologous arms that are mirror images of one anoth er (Rieger et al.,1991). Isochromosomes have the potential of determining the dosage effect of genes. No isochromosomes or telosomes have been observed for the long arm of any of the species studied thus far in the Medicago sativa complex. Three in dividual seedlings from two different accessions were found to exhibit B-chromosomes (Hossain and Bauchan, 1999). B-chromosomes are usually smaller than the normal A chromosomes and are generally heterochromatic; they normally do not influence the viabili ty and phenotype of the organism; they do not pair with the A chromosomes; they can affect mitotic behavior by lagging and elimination, polymitosis, or preferential distribution (Reiger, et al., 1991). Since Medicago sativa spp. are allogamous, it is presumed that polymorphisms might exist within its genome, which could be detected by banding and molecular techniques.
M. sativa ssp. coerulea
Bolton and Greenshileds (1950) were the first to publish a photomicrograph of the somatic chromosomes of diploid M. sativa ssp. coerulea. At that time they were unable to distinguish the chromosomes, however they d id recognize two chromosomes possessing satellites. The first karyotype was published by Buss and Cleveland (1968) and additional karyotypes have been developed by Agarwal and Gupta, (1983) and Bauchan and Campbell (1994). A majority of the karyotypes of diploid M. sativa ssp. coerulea has been based on meiotic pachytene chromosomes (Buss and Cleveland, 1968; Clement and Stanford, 1963; Gillies, 1968; Gillies and Bingham, 1971; Ho and Kasha, 1972). Kasha, et al. (1970) developed a standardiz ed pachytene karyotype of alfalfa which reconciled the differences which were found in the previous studies.
The somatic chromosome karyotype of this subspecies consists of one pair of satellited chromosomes (chromosome 8), four pairs of submetacentric chromosomes (chromosomes 1-4), and three pairs of short metacentric chromosomes (chromosomes 5-7). Chromosome 8 possesses the NOR of the alfalfa genome, the SAT chromosomes are a diagnostic feature of the karyotype. The large submetacentric chromosome pair 1 is easy to distinguish if the chromosomes have not spiralized too much during pretreatme nt. Occasionally a tertiary constriction can be found on chromosome 4. Using a computerized image analysis system it is possible to determine the homologous chromosomes and develop a karyotype based on chromosome arm lengths, arm ratios and total chromoso me length (Bauchan and Campbell, 1994).
The C- (Bauchan and Hossain, 1997) and N-banding (Bauchan and Hossain, 1998a) pattern of this diploid species displayed several more bands than diploid ssp. falcata with a majority of the bands located on their short arms (Fig. 1 b). The location and intensity of the bands in each chromosome is unique which allows for the precise identification of individual chromosomes for karyotypic studies. Generally the standardized karyotype of this subspecies exhibits in addition to centrome ric bands, which are the most prominent of the bands, telomeric bands in all the chromosomes short arms; all of the chromosomes except chromosome 7 have interstitial bands in their short arms; and chromosomes 1, 2, and 3 each have bands on their long arms (Fig. 2). N-banding reveals an additional interstitial band on the long arm of chromosome 5. The SAT chromosome is characterized as having a centromeric band, a prominent NOR band, and a telomeric and interstitial band on the long arm (Bauchan and Hossai n, 1997 and 1998a). Masoud et al. (1991) reported the first C-banded karyotype of diploid M. sativa ssp. sativa cv. CADL (cultivated alfalfa at the diploid level). However, they observed mostly centromeric and tel omeric bands and only a few interstitial bands.
Comparison of the C- and N– banding patterns reveals somewhat corresponding banding patterns. All of the short arms have a terminal bands in both C- and N-banding, however, the intensity of the bands varies from very prominent to faint. The interstitial bands on the short arms also vary in their intensity but they also vary in their locations, i.e. the C-band on chromosome 1 is located near the middle of the short arm whereas the N-band is located closer to the terminal end rather than the centromere. A characteristic feature of the N-banding pattern is a very intense, darkly stained interstitial band on the long arm of chromosome 3 (Bauchan and Hossain, 1996). N-banded chromosomes exhibit very poor contrast between the darkly stained h eterochromatin and the lighter stained euchromatin, therefore C-banding has been used much more extensively used than N-banding.
Studies on the polymorphisms in banding patterns in 14 accessions, which represent the wide geographic distribution of the accessions in the U.S. germplasm collection, showed a majority of the plants containing cells which were identica l to the standardized karyotype. However, in half of the accessions which exhibited polymorphisms the heterochromatic DNA bands differed in their intensity, location and total number of bands among and between accessions. In some accessions telomeric band s were missing and in others, bands were missing on only one of the chromosomes in a pair. Some plants contain chromosome with two bands on their long arms. A very large "mega" SAT chromosome was detected (possibly due to a translocation), isochromosomes, and trisomic and tetrasomic plants have also been observed (Hossain and Bauchan, 1999b).
Gillies (1970a) and Ho and Kasha (1972) concluded based on the pachytene karyotype of diploid ssp. falcata that it did not differ significantly from the pachytene karyotype of diploid ssp. coerulea (Gillies, 1968). However , upon close observation of the pachytene karyotypes, all of the M. sativa ssp. coerulea chromosomes have chromomers on the telomere of their short arms, whereas there were no telomeres on the short arms of diploid ssp. falcata except when the entire short arm of the chromosome was heterochromatinized. Thus, it appears that diploid ssp. falcata contains a lower amount of heterochromatic DNA than ssp. coerulea. However, this will depend on the accession which was studied as there is variation in amount of heterochromatic DNA from accession to accession (Bauchan and Hossain, 1999). C- and N-banding studies of mitotic chromosomes by Bauchan and Hossain (1997 and 1998a) have shown conclusively that diploid ssp. coerulea possesses more heterochromatic DNA than diploid ssp. falcata. Upon a reexamination of the heterochromatic distribution in both mitotic and meiotic chromosomes these studies agree that diploid ssp. falcata contains a lower amount of hetero chromatin than ssp. coerulea.
Tetraploid M. sativa ssp. falcata
Preliminary studies of six accessions of tetraploid ssp. falcata offer a surprising result. Most of the plants possess chromosomes which have C-bands in addition to the normal centromeric bands (Fig. 1c). There is a range from an accession which contains the fewest number of additional bands, 4 pairs of chromosomes have a extra telomeric band on their short arms, whereas the rest of the chromosomes only have a centromeric bands; two accessions which have multiple bands on each chromosome and a banding pattern similar to doubled diploid ssp. coerulea. In general a majority of the heterochromatic bands appear in the short arms of the chromosomes with only two chromosomes possessing interstitial bands on their long ar ms (Bauchan and Hossain, unpublished). All of the accessions studied had yellow flowers with sickle shaped pods. However, additional studies need to be conducted to prove that these accessions are not the product of hybridization with M. sativa ssp . sativa.
Tetraploid M. sativa ssp. sativa
Analysis of somatic chromosomes of tetraploid alfalfa has been previously reported (Agarwal and Gupta, 1983; Falistocco, 1987; Sclarbaum et al., 1988, Falistocco, et al., 1995). Agarwal and Gupta (1983) karyotyped several Med icago species with chromosome measurements made using an ocular micrometer, thus the accuracy of the measurements is questionable. Falistocco (1987) measured chromosomes from photomicrographs, resulting in much larger chromosome measurements, e.g., th e total length ranged between 9 and 12 u, than what has been reported for the genus Medicago. Schlarbaum et al., (1988) karyotyped tetraploid alfalfa from plants that had been r egenerated from tissue culture. Generally it is known that plants regenerated from a tissue culture system potentially can have chromosomes which have been altered (Nagarajan and Walton, 1987).
In a study of the nine germplasm sources of alfalfa in the US (Bauchan and Hossain, 1998b), C-banding polymorphisms were detected in the number, position and intensity of terminal and interstitial bands within a germplasm source. Howeve r, morphometric measurements and C-banding studies reveal that alfalfa has four nearly identical sets of chromosomes which allows for the development of an accurate karyotype (Fig.1d), (Falistocco et al., 1995 and Bauchan and Hossain, 1998b). The similari ty of the chromosome morphology and the C-banding pattern among homologous is not always perfect, however, there is enough resemblance between the chromosomes to made it possible to group alfalfa chromosomes into eight sets of four chromosomes.
The mitotic banding patterns of tetraploid M. sativa ssp. sativa resembles the distribution of the heterochromatic knobs found in meiotic pachytene chromosomes (Gillies, 1970b). However, the mitotic chromosomes in the tetr aploid did not appear to be shorter and did not appear to possess a larger amount of heterochromatin than the diploid subspecies M. sativa ssp. coerulea which was observed in pachytene chromosomes by Gillies (1970b)
A wide range of differences were observed between the nine germplasm sources. The ‘Falcata’ germplasm source is strikingly different from the other germplasm sources due to a fewer number of terminal and interstitial bands. ‘Falcata’ ch romosomes have primarily C-bands at their centromeres. Non-dormant alfalfa germplasm sources which include African, Chilean, Peruvian and Indian germplasm exhibit the largest number of heterochromatic bands. The African chromosomes possess the highest amo unt of heterochromatic DNA and resemble the banding pattern of a presumably doubled diploid M. sativa ssp. coerulea. The banding pattern of Chilean and Peruvian chromosomes are similar. Indian chromosomes have the fewest number of bands when compared to the other non-dormant sources especially on the long arms of their chromosomes. The ranking of germplasm sources from highest amount of heterochromatic DNA C-bands to lowest number of heterochromatic DNA C-bands studied thus far is as follows :‘African’>’Peruvian’>‘Chilean’> ‘Flemish>’Indian’> ’Varia’>’Ladak’>’Turkistan’>’Falcata’. It is interesting to note that the more winter hardy germplasm sources have fewer C-bands indicating the incorporation of ‘Falcata’ germplas m in alfalfa (Bauchan and Hossain, 1998b).
The tetraploid C-banded karyotype by Falistocco, et al. (1995) differs from the karyotype of ‘African’ primarily in the reduced number of bands which they reported. A number of reasons could explain the differences. First they used diff erent pre-treatments of the chromosomes, our experience has shown that an ice water pretreatment for 18h gives the maximum number of bands and thus the ability to distinguish the individual chromosomes from each other more critically, based on the larger number of landmark bands. Two bands which are located next to each other may appear as a single band if the chromosomes are too contracted thus reducing the number of detectable bands. Second, they used the Italian variety ‘Turrena’ which may have some M. sativa ssp. falcata germplasm in it’s background. Thus, this variety may have a reduced amount of constitutive heterochromatic DNA than the ‘African’ germplasm source.
Annual Medicago species
The annual Medicago species sometime referred to as medics are close relatives to alfalfa and they contain a number of agronomic traits such as insect resistance due to glandular hairs on the stems, leaves and pods which coul d be beneficial to alfalfa. The medics have not been well studied cytogeneticially. The first published photomicrographs of annual Medicago chromosomes was presented by Lesins and Lesins in 1961. Both Heyn (1963) and Lesins and Lesins (1979) provid e an overview of the cytogenetics of the annual Medicago species. Since that time Schlarbaum et al. (1984), Mariani and Falistocco (1991), and Mariani et al. (1996) have studied the 2n=16 species M. arabica, M. blancheana, M. cilia ris, M. doliata, M. granadensis, M. intertexta, M. muricoleptis, and M. noëana, M. rotata by developing karyotypes based on physical measurements of the chromosomes from photomicrographs. Bauchan and Elgi n (1984) discovered that the two tetraploid species, M. scutellata and M. rugosa have 2n=30 chromosomes rather than the recorded 32. Because M. scutellata possesses two pairs of SAT chromosomes it was suggested that this species was a n allotetraploid derived from the hybridization of a 2n=16 and a 2n=14 species and then spontaneously chromosome doubled. This began the search to the putative progenitors to these species. Falistocco and Falcinelli (1991) and Mariani et al. (1996) develo ped karyotypes of the 2n=14 species, M. murex, M. polymorpha, M. preacox and M. rigidula. Based on karyotypic similarity and some RFLP molecular data Mariani et al. (1996) suggested that there were four species, M. intertext a, M. murocoleptis, M. polymorpha and M. murex which were the most likely candidates for progenitors to M. scutellata and M. rugosa. It is interesting to note that all four species which are 2n=14 have one pair of ch romosomes which are larger than all the rest suggesting that these species evolved by translocation and loss of some DNA (Mariani et al. 1996). Studies on the banding of the annual species have yielded only bands at the centromeres (Mariani and Falistocco , 1990 and 1991; Falistocco and Falcinelli, 1993), thus this technique appears not to add valuable cytogenetic markers for karyotypic analysis or identification of individual chromosomes. In a recent study of the chromosomes of M. truncatula, using fluorescent in situ hybridization (FISH) techniques, Cerah, et al. (1999) we able to distinguish the chromosomes of two different types of M. truncatula (R-108-1 and Jemalong J5).
Cytogenetics and Breeding
Utilization of chromosome number counting and chromosome morphology differences has been used as evidence for the production of interspecific hybrids between perennial and annual Medicago species (Sangduen et al. 1982; Piccir illi and Arcioni, 1992), hybrids between perennial wild Medicago species and alfalfa (summarized by McCoy and Smith, 1986) and somatic hybrids produced via cell fusion (Pupilli et al. 1995 and Nenz et al 1996). The cytogenetic evidence used to dete rmine the production of the hybrids was primarily somatic chromosome numbers, with the number and morphology of SAT chromosomes as key features, and chromosome pairing at MI in microsporocytes. C- (Bauchan and Hossain, 1997) and N-banding (Bauchan and Hos sain, 1998a) techniques have been used to determine the hybridity of crosses between diploid M. sativa ssp. falcata and M. sativa ssp. coerulea . Because of the contrasting differences between banding patterns, ssp. falcata only having bands at their centromeres and multiple bands in ssp. coerulea, it was easy to identify hybrids between the two species. It was also easy to identify individual chromosomes of ssp. coerulea (Bauchan and Hossain, 1997 and 1998 a). Utilizing banding techniques it should be possible to discern the exchange of bands in the F2 and subsequent generations as well as backcross progenies. This same technique could be used to identify interspecific hybrids between ssp. coe rulea/ssp. sativa and annual species because so far all the annual species which have been studied have only centromeric bands. Identification of aneuploids for specific chromosomes can be accomplished by using banding techniques. Critical stud ies of the aneuploids can locate genes of agronomic importance on individual chromosomes.
Alfalfa breeders have been attempting to incorporate the genes of wild species into cultivated alfalfa through the use of interspecific hybridization via sexual crosses (see Quiros and Bauchan 1988; McCoy and Bingham, 1988 for summary), embryo rescue (Bauchan 1987), ovule embryo rescue (McCoy and Smith, 1986) and somatic hybridization (Pupilli et al.1995; Nenz et al.1996) with some success. A majority of the crosses which have been made, used either ssp. coerulea or ssp. sativ a as one of the parents for obvious reasons due their economic importance. Some of the attempted crosses could have been unsuccessful in incorporating wild genes due to the lack of pairing and thus lack of crossing over caused by large amounts of hete rochromatin in the short arms of the chromosomes. Heterochromatin is characterized by the presence of middle and highly repetitive sequences, often in tandem arrays of satellites, tightly bound to protein (Bickmore and Craig, 1997). Heterochromatin interf erers with crossing over and thus a reduction in the formation of chiasmata (Dyer, 1964; John and Lewis, 1965; Sybenga, 1975). A better candidate for crossing with wild species is diploid ssp. falcata or tetraploid ssp. falcata with much red uced heterochromatin. Diploid ssp. falcata which does not have heterochromatin on its chromosome arms will provide many more opportunities for chiasmata formation and thus exchange of genetic material can occur by crossing over. Because ssp. fal cata readily crosses with ssp. sativa it may be used as a bridge species between ssp. sativa and wild species of Medicago. Chromosome banding can also be used to identify the introgression of individual chromosomes, arms and/or se gments from the wild species into alfalfa as well as the exchange of genetic materials through crossing over which has been done extensively in wheat for it’s improvement (Friebe et al.1991).
Banding of meiotic chromosomes can be accomplished (Hossain and Bauchan, unpublished), thus it is possible to identify which chromosomes are involved in pairing at metaphase. An interesting study would be to observe the meiotic pairing between ssp. falcata and ssp. coerulea. At the diploid level meiotic pairing occurs between ssp. falcata and sp. coerulea chromosomes. The multiple bands which exist in ssp. coerulea and the lack of bands on diploid ssp. falcata can be used to show that they pair. Do the chromosomes of these two species pair in this same manner at the tetraploid level? Do the chromosomes pair completely at random as evidenced from genetic studies (Stanford, 1951) or is there some preferential pairing between falcata-falcata and coerulea-coerulea or falcata-coerulea as suggested by Cleveland and Sanford (1959)? These questions should be answered in the next few years.
Clement and Stanford (1963) pointed out, based on pachytene analysis, that the short arms of diploid alfalfa are heterochromatic thus chiasmata and crossing over is limited or are restricted on the short arms of the chromosomes. The exi stence of highly heterochromatinized short arms has been shown to be true based on C- and N-banding of ssp. coerulea, ssp. sativa and tetraploid ssp. falcata (Bauchan and Hossain, 1997 and 1998a). These finding supports the con clusion which Stanford et al. (1972) made regarding the fact that autotetraploid alfalfa forms very few multivalents probably due to the reduction of chiasmata because of heterochromatin on the short arms of the chromosomes. There could also be un describ ed gene(s) responsible for chromosome pairing. Banding studies of the meiotic chromosomes could answers this question through the identification of individual chromosomes and there pairing behavior.
The existence of large blocks of heterochromatic DNA on the short arms of the chromosomes of ssp. coerulea, ssp. sativa and tetraploid ssp. falcata could explain the difficulty molecular geneticists are having when mapping the genome of alfalfa. Heterochromatic DNA interferes with chiasmata formation and thus crossing over near these regions is reduced (Kaltsikes and Gustfason, 1984). This could explain the phenomenon of segregation distortion which has been observe d (Osborn et al. 1998).
Molecular Cytogenetics - Perspective
The advent of in situ hybridization techniques has open up a wide area yet to be explored in alfalfa. Schaff et al. (1990) and Calderini et al. (1996) showed that it is possible to tag a known gene sequence and identify the l ocation of the gene(s) on the chromosome. This should lead to the development of a gene map with the genes located on specific alfalfa chromosomes. Researchers in other crops have begun to integrate physical maps and molecular genetic maps. Initial comparative studies of physical and genetic maps have shown that differences in mapping distances occur between these maps. Ganal et al. (1989) and Segal et al. (1992) found that the distances between a marker on a physica l map can have a 100-fold difference from the genetic map. Gustafson (1990) showed that there is very little recombination between the centromere and the NOR region in rye chromosomes, thus explaining the differences in the mapping distances. This may als o be true for the short arms of alfalfa due to the presence of heterochromatic DNA. Therefore, if the technique of chromosome walking or chromosome landing is to be used to isolate a gene of interest without knowing its specific location, the physical and genetic distance which lies between the gene of interest and the molecular markers should to be determined.
Sequenced plant genes from alfalfa or other plant species, RFLPs, SSRs, etc. can be used as probes, labeled with flourescent dyes and placed on the chromosomes. Recent advances in the use of fluorescent labeling has made it possible to label two different probes with contrasting fluorescent stains in order to identify if the probes are linked or unlinked [Cluster et al. (1996); Calderini et al. (1996)]Using multiple probes and several fluorechromes, Mukai et al. (1993) has been able to identify all three genomes of wheat by chromosome painting. Possibly this technology could be used to identify the genomes of ssp. s ativa and other wild Medicago species in interspecific hybrids.
The development of an aneuploid series of diploid alfalfa would be especially helpful in the development of a chromosomal gene map. Kasha and McLennan (1967) were able to isolate several primary trisomics (2n=2x+1=17). However, only tri somics for chromosomes 1, 4, 6, 7 and 8 were identified using pachytene analysis (Gillies, 1977). McCoy and Echt (1992) presented an example of how chromosome addition lines (trisomics) can be used to locate genes on specific chromosomes of alfalfa. A tri ploid (3X) hybrid would be produced from a cross between diploid M. sativa ssp. sativa (2X) and a chromosome doubled diploid M. papillosa (4X). This triploid hybrid would be backcrossed to diploid M. papillosa and the progeny s creened for trisomics which would contain 2 genomes of M. papillosa and the one extra chromosome would be from M. sativa. These trisomic addition lines could then be irradiated to induce chromosome breakage of the M. sativa chromosome to produce smaller pieces of the chromosome which could be analyzed to determine what genes are located on these chromosome fragments. The use of addition lines in this manner is being used in oat-corn addition lines to map the genome of corn ( Ananiev, et al. 1998). The use of C-banding techniques to identify individual chromosomes can also be used to identify additional primary trisomics and possibly other aneuploids such as monosomics and nullisomics.
Another technique which has been used in other plant species is genomic in situ hybridization (GISH). Total DNA is extracted from a particular species, fluorescently labeled and applied to the chromosome spreads of another plant species (Heslop-Harrison and Schwaracher, 1996). This technique could be used to answer questions on the evolution of the M. sativa complex, the relationship between the tetraploid annual medic species and the diploid species, the incorporation of alien gene sequences, and chromosomal rearrangements.
The cytogenetics of alfalfa has lapsed behind many of the other major crops. However, with a the application of new molecular cytogenetic techniques and a concerted effort by the research community of alfalfa cytogen etics is poised at the dawn of a new millennium for greatness.
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Figure 1. Karyotypes of species within the M. sativa complex. a. Diploid M. sativa ssp. falcata, b. M. sativa ssp. coerulea. c. Tetraploid M. sativa ssp. falcata, and d. M. sativa ssp. sativa.
Figure 2. Ideogram of the C-banded M. sativa ssp. coerulea.