analysis of segregation in cross between wild soybean (glycine soja) and cultivated soybean (glycine max) based on morphology, agronomic traits and ssr marker

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY ANALYSIS O

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MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

ANALYSIS OF SEGREGATION IN CROSS

BETWEEN WILD SOYBEAN (Glycine soja) AND CULTIVATED SOYBEAN (Glycine max) BASED

ON MORPHOLOGY, AGRONOMIC TRAITS

AND SSR MARKER

SUPERVISORS STUDENT

MSc NGUYEN TRI YEN CHI Student code: 3082585

Session: 34 (2008-2013)

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MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

ANALYSIS OF SEGREGATION IN CROSS

BETWEEN WILD SOYBEAN (Glycine soja) AND

CULTIVATED SOYBEAN (Glycine max) BASED

ON MORPHOLOGY, AGRONOMIC TRAITS

AND SSR MARKER

SUPERVISORS STUDENT

MSc NGUYEN TRI YEN CHI Student code: 3082585

Session: 34 (2008-2013)

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APPROVAL

SUPERVISOR STUDENT

Dr TRUONG TRONG NGON NGUYEN QUANG DAT

Can Tho, June, , 2013

PRESIDENT OF EXAMINATION COMMITTEE

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Abstract

F 2 population made by cross between wild soybean and cultivated soybean was analyzed for the segregation of phenotype and genotype based on morphological characters, agronomic characters and SSR marker The cross was generated before between the female parent Xanh Ha Bac (Glycine max) and the male parent Soja 182 (Glycine soja) The results showed that the segregation of pod color fitted to (1:2:1) ratio The hilum color segregated in (3:1) ratio Four characters showed high heritability including days to flowering (R1), plant height at (R1), plant height at (R8), and number of nodes in main stem The population was divided into three groups, they were determinate, indeterminate and semi-indeterminate There was diversity among these groups for plant height and number of nodes in main stem All of six SSR loci showed the segregation ratio of (1:2:1) in

F 2 population No segregation distortion was recorded among these SSR loci

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Content

Abstract i

Content ii

1 Introduction 1

2 Materials and methods 2

2.1 Materials 2

2.2 Methods 2

2.2.1 Experiment design 2

2.2.2 Phenotypic characters score 2

2.2.3 Genotypic analysis 3

2.2.3.1 DNA extraction 3

2.2.3.2 PCR amplification 3

2.2.3.3 Analysis of PCR products 5

2.2.4 Statistics analysis 5

2.2.4.1 Analysis of morphological characters 5

2.2.4.2 Analysis of agronomic characters 5

2.2.4.3 Heritability of characters 5

3 Results and discussion 6

3.1 Inheritance of morphological traits 6

3.1.1 Hypocotyl color, flower color, number of leaflets, and leaflet shape 6

3.1.2 Pod color 8

3.1.3 Seed coat color 9

3.1.4 Hilum color 10

3.2 Stem types 10

3.3 Heritability of agronomic characters 10

3.4 Inheritance of growing characters 11

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4 Conclusions and suggestions 21

4.1 Conclusions 21

4.2 Suggestions 21

References .22

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1 Introduction

The soybean (Glycine max) is one of important crops

around the world This species of legume provides many products for many uses such as foods for human and breeding, materials in industries, improving soil

In Vietnam, soybean has been cultivating for thousands years Because of the importance of soybean in country agricultural economy, the improvement of soybean varieties is a necessary requirement

Wild soybean, Glycine soja, has several useful traits such

as pest resistance and drought tolerant Conversely, cultivated soybean rarely contain these abilities Therefore, transferring these traits of wild soybeans into cultivated soybeans will open possibility to make new varieties that meet the cultivation need The introduction of wild soybean germplasm into cultivated soybean seems to be a promising method to create a new subject of soybean breeding

To serve breeding better, scientists need to know more clearly the segregation mode of F2 population However, there are

few complete researches on this area up to now

Objective: the aim of this study is to analyze the inheritance

mode of the several agronomic characters and six loci SSR in the early segregating generation from the cross between wild soybean and cultivated soybean

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2 Materials and methods

2.1 Materials

+ Xanh Ha Bac cultivar (Glycine max, landrace)

+ Accession Soja 182 (Glycine soja, Korea origin)

+ F2 population of the cross between Xanh Ha Bac (as female parent) and Soja 182 (as male parent)

+ Chemicals and equipments for planting, DNA extraction, PCR, and electrophoresis

2.2 Methods

The experiments were carried out in the Molecular Biology Laboratory and the greenhouse of Biotechnology Research & Development Institute from September of 2012 to March of 2013

2.2.1 Experiment design

The experiments were designed following a completely randomized design with three replications Each replication consisted of one plot of Xanh Ha Bac, one plot of Soja 182 and four plot of F2 generation

2.2.2 Phenotypic characters score

Morphological characters including hypocotyls color, leaflet number, leaflet shape, stem type, flower color, pod color, seed color, and hilum color were scored

Agronomic characters such as days to first flowering (R1), plant height at (R1), plant height at (R8), node number in main stem, pod number, branch number, etc were also scored

Standard for scoring was based on Characterization Record Sheet of Asian Vegetable Research and Development Center

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2.2.3 Genotypic analysis

2.2.3.1 DNA extraction

DNA was extracted from young leaves of parents, F1, and

13 F2 plants according to CTAB protocol (Rogers and Bendich, 1988)

DNA quality were tested by electrophoresis on agarose 0,8% gel Good DNA samples were used for PCR analysis

2.2.3.2 PCR amplification

PCR was conducted with six SSR primers (Table 1)

Table 1 List of SSR primers

TGCGCCATTTATTCTTCA Satt

GCGCATTGCACATTAGGTTTTCTGTT Satt

GCGTGTGCAAAATGTTCATCATCT Satt

CACTGCTTTTTCCCCTCTCT Satt

TGCCGCGAGATTAATATAATTTGT Satt

TCCGCGAGATAAATTCGTAAAAT Satt

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The components of each reaction and the PCR cycle was shown in Table 2 and Figure 1

Table 2 The reaction components of PCR

Figure 1 PCR program

(Soybase; Wang et al., 2010)

Annealing temperature (Ta) was specifically set for each primer (from 47oC to 60oC)

∞

35 cycles

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2.2.3.3 Analysis of PCR products

10µl of each PCR product was mixed with 4µl of loading buffer and subjected to electrophoresis on 3% agarose Electrophoresis was conducted at 40V in the 23cm tank that contents TBE 1X

SSR bands were detected by Bio-Rad XRS

2.2.4 Statistics analysis

2.2.4.1 Analysis of morphological characters

Chi-square test was used to compute the fitness of segregation of morphological characters to the Mendel ratio (3:1

or 1:2:1) The characters including pod color, seed coat color, and hilum color

2.2.4.2 Analysis of agronomic characters

Frequency distribution of three characters (days to flowering (R1), plant height at (R1), and plan height at (R8)) was made

ANOVA were used to analyze parameters for almost characters

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3 Results and discussion

3.1 Inheritance of morphological traits

3.1.1 Hypocotyl color, flower color, number of leaflets, and leaflet shape

There was no variation in hypocotyl color, flower color, number of leaflets and leaflet shape of F2 population These results were due to no difference between male and female parents in above traits

The hypocotyl color and flower color of all populations were purple (Figure 2 and 3)

Number of leaflets in all populations were three The leaflet shape of all populations was ovate (Figure 4)

Figure 2 Hypocotyl color of parents, F 1 , and F 2 plants

(January, 12th, 2013)

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Figure 3 Flower color of parents, F 1 , and F 2 plants

(February, 10th, 2013)

Figure 4 Leaflet shape of parents, F 1 , and F 2 plants

th

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3.1.2 Pod color

The character of pod color segregated in Mendel ratio (1:2:1) There were three types of pod color in F2 population, the tan color contributes 25.9%, brown contributes 51.3%, and black contributes 22.8% (Figure 6)

Figure 5 Pod colors of parents, F 1 , and F 2 plants

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61.3% 18.9%

19.8%

black brow pale

3.1.3 Seed coat color

The character of seed coat color did not segregate in Mendel ratio F2 population generated three types of seed coat color, 61.3% of pale, 19.8% of black, and 18.9% of brown (Figure 8)

Figure 7 Seed coat colors of parents, F 1 , and F 2 plants

(April, 5th, 2013)

Figure 8 Distribution of seed coat colors in F population

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25.3%

black brown

3.1.4 Hilum color

There were two colors of hilum in F2 population, black and brown (Figure 9) The segregation of hilum color was fitted to 3:1 ratio

Figure 9 Distribution of hilum color in F 2 population

There was no linkage between seed coat color and pod color

3.2 Stem types

Xanh Ha Bac is a determinate variety, while Soja 182 is a indeterminate variety There were three stem types in F2 population They were determinate, indeterminate and semi-determinate (account for 16.67%)

3.3 Heritability of agronomic characters

Days to first flowering, plant height at (R8), and number of nodes in main stem were three characters that gave high heritability (h2>70%) (Table 3)

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The heritability of plant height at flowering (R1) and number of pods per plant were moderate (70%>h2>30%)

Number of branches had low heritability (16%)

Table 3 Total F 2 variance, error variance and

heritability in F 2 population

Characters

Plant height at flowering

σ 2 F2 : phenotypic variance; σ 2 e : error variance, h 2 : heritability

3.4 Inheritance of growing characters

Days to first flowering showed a normal distribution (Figure 10)

Days to fisrt flowering had high heritability (h2 = 78.1%, Table 3) However this trait also affected by environmental factors

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Figure 10 Distribution of days to flowering in F 2 population

Plant height at flowering (R1) gave a positive skewed distribution with one peak at 26.5 cm (Figure 11)

Figure 11 Distribution of plant height at flowering (R1) in F 2

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Plant height at (R8) also gave a positive skewed distribution but with two peak at 38 cm and 76 cm (Figure 12) Analysis of variance showed that there was a significant difference at 1% between three groups of stem types for plant height at flowering (R1)

Figure 12 Distribution of plant height at (R8) in F 2

population

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3.5 Genotypic inheritance

All of six loci had ability to separate two species of soybean (Xanh Ha Bac and Soja 182) on 3% agarose gel Almost markers could successfully amplify DNA and generated clear bands, except Satt173 and Satt243 Bands sizes had slight difference to sizes that were known in William cultivar

Figure 13 Band type at locus Satt173

M: ladder

P1: female parent Xanh Ha Bac

P2: male parent Soja 182

F1: F1 of cross Xanh Ha Bac x Soja 182

1 – 13: F2 individuals of cross Xanh Ha Bac x Soja 182

Satt173 gave two band sizes, about 197bp for Soja 182 and about 210bp for Xanh Ha Bac Bands sizes for Soja 182 was similar to the size that was known in William cultivar Distance between two bands was 13bp

210bp

197bp

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M: ladder

Satt197 gave two band sizes, about 210bp for Soja 182 and about 190bp for Xanh Ha Bac Both sizes was slightly longer than the sizes that was known in William cultivar Distance between two bands was 30bp

210bp

190bp

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M: ladder

Satt231 gave two band sizes, about 237bp for Soja 182 and about 210bp for Xanh Ha Bac Bands sizes for Soja 182 was similar to the size that was known in William cultivar Distance between two bands was 27bp

210bp

237bp

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M: ladder

Satt243 gave two band sizes, about 210bp for Soja 182 and about 220bp for Xanh Ha Bac Both sizes was closely similar to the sizes that was known in William cultivar Distance between two bands was 10bp

210bp

220bp

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M: ladder

Satt495 gave two band sizes, about 245bp for Soja 182 and about 255bp for Xanh Ha Bac Distance between two bands was 10bp

255bp

245bp

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M: ladder

Satt373 gave two band sizes, about 254bp for Soja 182 and about 290bp for Xanh Ha Bac Distance between two bands was 36bp This marker could clearly separated bands

290bp

254bp

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All of six loci SSR segregated in Mendel ratio (1:2:1) There was no segregation of loci deviated from Mendel ratio (Table 4) The segregation of these SSR markers suggested that the segregation of QTLs associated with these marker would also fitted to Mendel ratio (Table 5)

Table 4 Chi square test for segregation of six makers

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4 Conclusions and suggestions

All of six SSR markers were able to distinguish Xanh Ha Bac variety from Soja 182 accession

All of six loci SSR showed the segregation ratio of 1 : 2 : 1

in F2 population

4.2 Suggestion

Studying further on the F2 population as well as other

crosses to make exact conclusion

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REFERENCES

Rogers, S.O and A Bendich 1988 Extraction of DNA from

plant tissues In Plant molecular biology manual Section

A6 Kluwer Academic Publisher, Dordreht, A6:1-11 Wang, M., L Run-zhi, Y Wan-ming, and D Wei-jun 2010 Assessing the genetic diversity of cultivars and wild

soybeans using SSR markers African Journal of

Biotechnology, 9(31):4857-4866

Website

http://soybase.org/ (October,5 th , 2012)

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