Chromosome analysis using specific fluorochrome chromomycin A3 (CMA) banding Pattern in Citrus

Presented by: NOORANI, M1

Advisor: Dr. Chitose HONSHO

Date: 30-10-2014

Part A• Introduction

• Key Point of Presentation

Part B

• Research paper 1

• Knowledge gape

• Plant materials and Methods

• Results and discussion

Part C

• Research paper 2

• Knowledge gape

• Plant materials and Methods

• Results and discussion

• Achieved knowledge

• Discussion section

Citrus Taxonomy Reasons why citrus Taxonomy is

complicated

Citrus is a major crop with a complex taxonomy

for breeders

Family Rutacea consists of six genera

Citrus, Fortunella, Poncirus, Clymenia,

Eremocitrus and Microcitrus

Citrus genus has three true species

Citrus medica (Citron), C mexima (pummelo) and

C reticulata (sweet orange)

Citrus paradise (grape fruit) C sinensis and Citrus

limon (lemon) are hybrids.

1. Large no. of cultivars

2. Wide hybridization (intergeneric and interspecific)

3. Polyploidy

4. Mutations

5. Polyembryony ( Nucellar embryony)

6. Unexpected relationship of the manifold varieties

to one another.

7. Wide cross compatibility

Breeding tropical and subtropical fruits (P.K. RAY)Yamamoto. M et al., (2007)

• Scientists applied many techniques to study Phylogenic relationships of citrus• Karyotypic study is useful technique to understand phylogenic and evolutionary trends (SHAN et al., 2003)

Traditionally staining method

Aceto-carmine

Aceto-orcein

Or Feulgen’s sln

Modern staining with fluorescent

DAPI (4',6-diamidino-2-phenylindole)

Chromomycin A3 ( CMA)

Karyotype : length and position of centromere, constriction of chromosome Stain AT, Useful compare to

traditional, but can not show specific banding pattern in citrus (

Khan 2007)

Less informative b/c citrus mitotic chromosome small (Khan 2007)

Promising, GC with A3

Let us study about chromosome analysis while using guanine-cytosine (GC) with specific fluorochrome chromomycin A3 (CMA).

•Verification of chromosomes •Cytogenetic characterization •Identification of hybrids• Phylogenic relationship of different species and genera

• Chromomycin A3 (CMA)•Chromosome karyotyping •Phylogenic relationshipe

CMA Banding Patterns of Chromosomes in Major Citrus Species

(Yamamoto et al,. 2007)

B : Research paper 1:

Let us study together

Objective of the research paper:

• The CMA banding patterns ofimportant species have been revealed.• CMA banding analysis of subgenus ofCitrus papedas has not been reported.•This study clarified chromosome bandingpatterns of papedas and their phylogenicrelationship.

Subgenus : Papedas

Source of image: www.google.com

Knowledge gape

Materials and Methods

Results and discussion

Materials and Methods

Results and discussion

Swingle systematics 1943

For monoembryonic young leaves (3-5mm), adult trees

1mm root tip from germinated seeds (dark 25 c)

Low no. of embryoand low no. of seeds

Fixation, enzymatic maceration (Fukui 1996), 2% Giemsa staining and 0.1g/L CMA (Hizume 1991)

• All accessions had 2n = 18 chromosomes.

• No variation in CMA banding patterns within seedlings of any species

• 7 types CMA- positive banding

Results and Discussion

Results and discussion …

CMA Banding Pattern

Same CMA band, b/c of C medica

homologous ch while in citrus did not.

(A, B and C )But in this study did not possed B

Simplest k ( D and E)Represent C. reticulata

Subgenus papeda b/c Dst was not in citrus

Chromosome configuration different from Citrus

Similarity in both, and it seems C. latipes was ancester of C. maxima

.

Very different C.C from Citrus and

papeda and similar with Yuzu

C. limon, C. aurantium, C. sinensis, aurantifolia, paradisi were proved hybride in previous studies.

2: CMA Staining Analysis of Chromosomes in Citrus Relatives, Clymenia, Eremocitrus and Microcitrus.

(Yamamoto et al,. 2008)

c : Research paper 2

Objective of the research paper:

• (Rutaceae) family has six genera, i.e., Citrus, Fortunella, Poncirus, Clymenia,Eremocitrus, and Microcitrus.•The CMA banding pattern of first three genera has been analyzed by many scientists.• However, CMA banding analysis of the other three genera has not progressed.•In this study, they clarified the variability of CMA chromosome banding patterns in severalspecies belonging to Clymenia, Eremocitrus, and Microcitrus and discussed theirPhylogenic relationships.

Table: shows plant materials

young leaves (3-5mm), adult trees . Fixation, enzymatic maceration and air drying (Fukui 1996), 2% Giemsa staining and 0.1g/L CMA (Hizume 1991)

Chromosome staining 2% Giemsa in phosphat buffer 15 min. after confirmation, de-stain, 0.1 g/L CMA

• All accessions had 2n = 18 chromosomes.

• No variation in CMA banding patterns within seedlings of any species

• 6 types CMA- positive banding

• High chromosomal variability

2 and 4 Identical CMA banding

pattern

1, 2, 3, and 4 were similar with C, D, E

while australasica B, C, D, E

Commonly observed in citrus and Poncirus, pre-dominant D, E

Did not fall in the same category of citrus, Poncirus, Fortunella

M. warburgiana, fruits and leaves are distinct from other Microcitrus.Indora has oil in the mature leaves

Resemblance , B , not similar morphological traits and DNA analysis. Maybe ancestral type

C. Medica ( 2B+8D+8E) primitive type of citrus, P. trifoliata ( 2B+10D+6E). however ( 1, 2,3) show resemblance but instead of B, they showed C. This results may indicate the existence of ancestral type.

• The CMA banding patterns of Cl. polyandra, M. australis, M. inodora, M. warburgiana, and Sydney hybrid were reported for the first time in this study.

• CMA banding pattern of two species were not identical but similar to previous study.

• Variability in Microcitrus chromosome

CMA is useful technique for

• Verification of chromosome numbers and karyotyping

• AT region and GC region

• Phylogenic relationship among citrus

• Determination of heterozygosity and variability in the DNA base composition.

• Yamamoto. M et al., (2007) CMA Banding Patterns of Chromosomes in Major Citrus Species. J. Japan. Soc. Hort. Sci. 76 (1): 36-40. 2007.

• Yamamoto.M et al., (2008) CMA Staining Analysis of Chromosome in Citrus Relatives, Clymenia, Eremocitrus and Microcitrus. J. Japan. Soc. Hort. Sci. 77 (1): 24-27. 2008.

Thanks for your kind attention