Chromosomes
Lotus japonicus is characterized by a small genome (2n = 2x = 12; genome size per Nobuko Ohmido
Cytology and Molecular Cytogenetics 121
haploid, 472 Mb), relatively short life cycle (2–3 months) and ease of genetic manipula-tion (e.g. transformamanipula-tion, it being an autoga-mous diploid plant; Jiang and Gresshoff, 1997;
Udvardi et al., 2005). A large-scale sequencing project was initiated for the L. japonicus acces-sions Miyakojima and Gifu, and subsets of genomic sequences are now available (Sato et al., 2001; Nakamura et al., 2002; Asamizu et al., 2003; Kaneko et al., 2003; Kato et al., 2003). Sato and Tabata (2006) have constructed a high-density genetic linkage map of L. japon-icus and mapped numerous transformation-competent artificial chromosome (TAC) genomic markers (Table 8.1). These are indis-pensable for Leguminosae studies in various fields, including comparative genomics, gene identification, gene isolation and marker-assisted breeding. Consequently, many micro-satellite and simple-sequence repeat (SSR) markers, as well as derived cleaved amplified polymorphic sequences (dCAPS), have been genetically and physically mapped on the L.
japonicus genome (http://www.kazusa.or.jp/
lotus/). Co-dominant markers can be used for map-based cloning of useful protein-coding genes (i.e. transcription factor receptor-like kinase, and transporter and disease resistance genes; Sato et al., 2008).
Map-based cloning requires a dense and precise linkage map of the trait of interest, followed by establishment of the relationship between genetic and physical distances. The identification of individual mitotic promet-aphase chromosomes of L. japonicus based on condensation patterns (CPs) became feasible, and their chromosome maps were developed (Ito et al., 2000; Hayashi et al., 2001; Pedrosa et al., 2002). However, mitotic prometaphase chromosomes are much smaller than pach-ytene chromosomes, and thus the resolu-tion for genetic research is limited, probably because the mitotic chromosome length is 4.29–9.64 mm and 1.51–2.67 mm, respectively (Ito et al., 2000; Pedrosa et al., 2002).
We now discuss quantitative pachytene chromosome maps of the six L. japonicus chromosomes based on chromosome length, centromeric position, heterochromatin and euchromatin distribution pattern, as well as the position of major repetitive sequences employing FISH and an imaging method
using the chromosome image analysis sys-tem ver. 3 (CHIAS3) with high-resolution pachytene chromosomes to determine the precise integration between genetic and physical distances in the L. japonicus genome (Fig. 8.1).
The image analysis system CHIAS3 (CHIAS III, 2004) was used to analyse the L. japonicus chromosomes. A quantitative chromosome idiogram was constructed based on the digitized intensity of the fluo-rescent signals after counterstaining with DAPI. The original chromosome images for the construction of the idiogram were RGB images, each with 8-bit grey levels. The procedure used to construct the idiogram was as follows: first, the 24-bit RGB images were converted into 8-bit grey images of R, G and B stack images. The chromosome area was delimited based on the DAPI (B) image for each chromomere, and the chromomere indices were established. Midrib lines were drawn along the axis of the chromosome, and the fluorescence intensity of Cy3 (R), FITC (G) and DAPI (B) measured. Next, the average fluorescence profile was computed by meas-uring the fluorescent intensities of more than three chromosomes from signal-detected images. Finally, the idiogram was constructed based on the average fluorescence profile. The numerical values of the fluorescent intensities of chromomeres were converted into mono-chrome binary band patterns.
The genomic library of L. japonicus was also constructed via TAC, selected on the basis of the sequences of SSR and dCAPS from L. japonicus (Sato and Tabata, 2006). TAC clones were selected from the 3-D DNA pools of the TAC libraries by PCR to amplify SSRs.
The TAC clones used for FISH mapping are listed in Table 8.1. The 45S ribosomal RNA (rDNA) gene derived from rice and 5S rDNA isolated from L. japonicus were employed. The high copy numbers of tandem repeat DNA, LjTR1, LjTR2, LjTR3 and LjTR4, and the ret-roelements, LjRE1 and LjRE2 with the highest copy numbers, were isolated and cloned from the L. japonicus genome (Sato et al., 2008).
Repeated sequences are mapped on the L. japonicus genome (Ohmido et al., 2010). LjRE1, a highly repeated retroele-ment, has long terminal repeats (LTRs) and
Table 8.1. Repetitive sequences, 45S rDNA, 5S rDNA and transformation-competent artificial chromosome (TAC) clones used as probes for fluorescence in situ hybridization (FISH). Linkage and physical position data are cited from the Lotus genome database (Lotus japonicus News, 2011).
Marker C bne name
Size (bp)
Position (cM)a Mb
Physical location
Chromosome
Chromosomal position (%)b LjRE1 Ty-1 Retroelement
(copia type)
12,069 Dispersed
LjRE2 Ty-3 Retroelement (gypsy type)
6840 Centromeres
LjTR1 Tandem repeat 190 Constitutive
hetrochromatin
LjTR2 Tandem repeat 237 Euchromatin
LjTR3 Tandem repeat 172 Hetrochromatin
LjTR4 Tandem repeat 172 Chromosome
terminal region
45S rDNA Ribosomal RNA gene 2,5 and 6
5S rDNA Ribosomal RNA gene
2
TM0088 LjT15K21 0.0 0.1 1 0.1
TM0063 LjT09L22 4.8 14.8 1 8.7
TM0910 LjT42H23 71.0 87.2 1 98.1
TM0904 LjT33P02 4.0 6.8 2 7.4
TM0153 LjT28L17 10.8 15.5 2 11.4
TM0081 LjT01G01 24.6 24.3 2 –
TM0225 LjT27K02 25.8 25.7 2 –
TM0124 LjT26I01 33.8 34.6 2 57.0
TM0008 LjT10B11 44.2 42.1 2 58.3
TM0021 LjT04I02 60.9 57.9 2 –
TM0031 LjT16N13 68.5 72.3 2 59.9
TM0380 LjT18K09 72.9 80.6 2 94.9
TM0793 LjT23013 0.0 0.005 3 4.2
TM0059 Lj13M14 6.9 7.2 3 5.8
TM0436 LjT13N17 10.5 11.2 3 6.5
TM0111 LjT40002 26.8 27.8 3 17.8
TM0246 LjT34I09 42.0 50.9 3 53.0
TM0217 LjT09C16 74.8 81.9 3 71.7
TM0261 LjT34I09 83.2 88.2 3 90.5
TM0288 LjT36E18 2.0 1.7 4 3.1
TM0131 LjT21G09 21.3 19.4 4 9.1
TM0087 LjT14P20 28.6 31.8 4 34.6
TM0042 LjT10L16 69.2 68.2 4 87.8
TM0089 LjT14E05 0.4 0.7 5 0
TM0048 LjT05P01 27.6 25.7 5 –
TM04148 LjT30P03 44.1 52.2 5 85.5
TM0180 LjT03D07 54.1 61.8 5 95.0
TM0260 LjT47K21 54.9 62.5 5 95.7
TM1383 LjT26K12 1.7 1.2 6 5.7
TM0057 LjT03B03 27.6 32.8 6 48.4
TM1240 LjT33P12 66.6 68.1 6 92.4
aLinkage position;
bphysical position from the end of the short arm of the corresponding chromosome; – the location of the signal shows much variation, with successful detection uncommon.
Cytology and Molecular Cytogenetics 123
Fig. 8.1. Procedure for development of the quantitative idiogram by CHIAS on L. japonicus chromosome 5. A, original RGB image of chromosome 5; B–F, layers of midrib line of the chromosome, Cy3, FITC, DAPI and chromomere-index image; G, fluorescence profiles (FPs) of DAPI, Cy3 and FITC were measured along the midrib line. Each FISH signal was localized into precise chromosomal position; H–J, straightened images of DAPI, Cy3 and FITC, respectively; K–M, idiograms constructed from FP value;
K, index idiogram segmented by each chromomere; L, FP image idiogram; M, quantitative pachytene chromosome idiogram with localization of TAC clones.
1st step
2nd step
3rd step
Fluorescence profile (g)
(h)
(k) (l) (m)
TM0048 TM0260
DAPI (i) Cy3 (j) FITC 250 200 150
Grey value
100 50
0 –150 –100 –50 0
Centromere DAPI
Cy3 FITC
50 100
Pixels (a)
(b)
Object Layer Cy3 image
FITC image
DAPI image
Index image (c)
(d)
(e) (f)
gag-polymerase genes, and is characterized as a Ty-1 copia-type retroelement (Sato et al., 2008). LjRE1 is dispersed throughout
euchro-matin and heterochroeuchro-matin regions. The second largest retroelement, LjRE2, charac-terized by a Ty-3 gypsy-type retroelement,
was localized on the centromeric regions of L. japonicus chromosomes. The fluorescent intensity of the LjRE2 retroelement differed among the six chromosomes; the intensity on chromosome 1 was strong but was weak on chromosome 5. This variation was due to differences in the copy numbers at the peri-centromeric regions of each chromosome.
The tandem repeat sequence LjTR1 (190 bp unit size) comprised 4.6% of TAC clones in the L. japonicus genomic library, which was revealed using end sequences from anchored TACs (Sato et al., 2008). FISH data have shown that LjTR1 was localized at the highly con-densed constitutive heterochromatic regions in the L. japonicus nucleus and chromosomes (Fig. 8.2). LjTR2 (237 bp unit size) comprised 4.1% of TAC clones and was localized at the decondensed euchromatic regions of all chromosomes (Fig. 8.2). Furthermore, LjTR3 (172 bp unit size) comprised 1.4% of the TAC clones and was localized at specific hetero-chromatin regions; LjTR4 (172 bp unit size) was localized at the terminal region of all
chromosomes, except for the short arms of chromosomes 1 and 2 (data not shown).
An integrated map based on the mitotic chromosome, the pachytene map and link-age map was developed for six individual L. japonicus chromosomes using data on the positions of TAC clones and somatic chromo-some maps (Ito et al., 2000). The comparison of these maps shows the centromeric posi-tion and some interstitial regions, albeit with some recombination distortion (Fig. 8.2).
Based on the recombinant frequency, the dis-tance at the terminal regions is apparently evaluated as larger than the physical distance of the chromosomes. The distance between TM0111 and TM0246, including the centro-mere and heterochromatin, is 2.80 cM/mm, while the terminal region between TM0793 and TM0059 on the short arm is 27.6 cM/
mm. These findings suggest that in L. japoni-cus, the recombination frequency at the centromeric region is suppressed by approxi-mately tenfold compared with the terminal region. However, the recombination ratio
Fig. 8.2. Relationships among cytological features, recombination frequency and the chromosome structure of chromosome 3 by FISH mapping of seven TAC clones in L. japonicus. Interphase image represents the FISH mapping of LjTR1 and 45SrDNA.
LjTR1
LjTR1 LjTR1
Mitosis prometaphase
Meiosis pachytene
Linkage map
TM0217 TM0246 TM0111 TM0436 TM0059 TM0793
6.9 cM Chromosome map < Genetic map
→ recombination hot spot
Chromosome map > Genetic map
→ recombination cold spot
Chromosome map > Genetic map
→ recombination cold spot 3.6 cM
17.1 cM
16.0 cM
33.2 cM
8.8 cM TM0261
Cytology and Molecular Cytogenetics 125
of the terminal region between TM0217 and TM0261 is similar (2.90 cM/mm) to that of the centromeric region. The large constitu-tive heterochromatic block comprising LjTE1 found between TM0217 and TM0261 should influence suppression of the recombination frequency on chromosome 3.
The quantification of chromosome density by CHIAS3, in situ localization of repetitive sequences and high-resolution mapping of genes and/or markers by FISH are expected to facilitate the analysis of gene density, segment duplication and other chro-mosome rearrangements and to yield inte-grated maps for legumes (Ohmido et al., 2010). In particular, probes applicable for Lotus, red clover, soybean and other legumes will help in developing a framework for a common genomics of legumes (Ohmido et al., 2007). Molecular cytogenetics may con-tribute to this goal, for example in the case of rice and tomato (de Jong et al., 1999; Cheng et al., 2001). From the integration of link-age data, chromosome density and physical localization of DNA markers and/or genes, basic research as well as legume breeding will benefit.
8.3 Integrated Chromosome