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Molecular Mapping of Resistance to Sugarcane Mosaic Virus in Maize

Xinhai Li and Shihuang Zhang

Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Email Xinhaili2002@yahoo.com.cn

Abstract

Sugarcane mosaic virus (SCMV) is the most important viral disease of maize (Zea mays L.) in China. The SSR linkage maps were constructed based on a population consisting of 184 F2 individuals from the cross Huangzao4Ye107, covering 1543cM on ten chromosomes of maize with an average interval length of 17.3 cM. The 184 corresponding F3 families were evaluated for SCMV resistance under artificial inoculation at Tangshan in 2000 and at Beijing in 2001. With the method of composite interval mapping, three QTL conferring resistance to SCMV were identified in the region of chromosomes 3.04, 6.01 and 10.04 in both environments, adjacent to markers phi053, phi423796 and phi062, respectively. While the other two QTL on chromosomes 1.01 and 5.05 were found only in 2001, adjacent to markers umc1160 and umc1822, respectively. The QTL on chromosomes 1.01 and 5.05 expressed significant dominant or over-dominant gene effects, and the QTL on chromosomes 3.04, 6.01 and 10.04 displayed additive or partial dominant effects. All QTL identified could contribute to 33.9% and 69.4% of phenotypic variance in 2000 and 2002, respectively. Two major QTL on chromosomes 3.04 and 10.04 were consistently detected over two environments, explaining 7.2% to 26.9% and 15.3% to 15.8% of the phenotypic variance.

Media summary

QTL identified for SCMV resistance will facilitate germplasm improvement and breeding effort for resistance to SCMV in China.

Key words

Sugarcane mosaic virus, resistant gene, SSR marker, genetic linkage map, quantitative trait loci.

Introduction

Sugarcane Mosaic Virus (SCMV) transmitted by the aphid is the most important viral disease of maize in China, causing 10% to 15% yield losses annually. SCMV, formerly known as maize dwarf mosaic virus (MDMV) strain B, is more prevalent in China and Europe, while MDMV is widespread in southern parts of the USA (Cheng et al. 2001; Melchinger et al. 1998; Louie et al. 1991). Insecticides are not effective to control SCMV because of the non-persistent mode of virus transmission, while development and cultivation of resistant hybrids are recognized to be the best way to control SCMV. Studies on resistance to MDMV and SCMV have been conducted with US and European maize germplasm. One to five genes for MDMV resistance were identified in US maize inbreds based on segregation analyses, and one dominant major gene was located on chromosome 6S by RFLP markers (McMullen et al. 1989; Louie et al. 1991). Genetic analyses suggested one to three genes involved in resistance to SCMV in European maize germplasm (Melchinger et al. 1998), and two major genes, Scm1 and Scm2, were mapped on chromosome arms 6S and 3L, respectively (Melchinger et al. 1998; Xia et al. 1999). So far, information on the genetic basis of resistance to SCMV in the background of Chinese maize germplasm is still lacking. Preliminary investigation suggested that one to polygenes conferred resistance to SCMV (Cao et al. 1987; Wu et al, 2002). In the present study, we describe the quantitative trait loci (QTL) conferring SCMV resistance using 184 F3 families from the cross Huangzao4Ye107. The objectives of this study were to 1) estimate the number and genomic position of major and minor genes involved in SCMV resistance, 2) determine their gene effects, and (3) to identify molecular markers linked to these QTL.

Materials and Methods

Plant materials and field trial

Two Chinese maize inbred lines, Huangzao4(P1), which is resistant to SCMV, and Ye107(P2), which is highly susceptible to SCMV, were crossed. A random of 184 F2 individual plants was selected and genotyped by SSR markers to construct the genetic linkage map, and the corresponding F3 families were phenotyped for SCMV resistance in a randomized complete block design with two replicates in field conditions under artificial inoculation at Tangshan in 2000, and at Beijing in 2001. The plot consisted of one row 4m long and 0.65m wide, and was over-planted and thinned to 20 plants.

SCMV inoculations and scoring

Virus inocula were prepared from typical infected seedlings of susceptible line of Mo17. Leaves with mosaic symptoms were homogenized in 0.01 M phosphate buffer (pH 7.0) in 1:10 dilution. Plants at 4- to 5- leaf stage were rubbed with inoculum containing carborundum two times with a one week interval. Plants were recorded for virus symptoms at weekly intervals, beginning at 7-10 days after the initial inoculation, and ending at 20 days after pollination. The rating system was taken on a plant severity scale of 0 (leaf symptomless) to 3 (severe stunt with few ears formed) (Lin, 1989). Disease Index (%) =∑(No. of infected plantsrated scale)100 / (total plantsmaximum scale). The disease index mean of F3 families across two replicates was used for QTL analysis.

SSR analyses

Genomic DNA was extracted from two top leaves of each 20-day-old F2 plant using a modified CTAB procedure. The PCR reactions were performed using a PTC-200 Thermal Cycler (MJ Research, Watertown, MA). The DNA extraction, PCR amplification program, electrophoresis and silver staining method were adopted from the protocols of Hoisington et al (1998).

Statistical and QTL analyses

The segregation at each marker locus was checked by appropriate Chi-square test for the expected Mendelian segregation ratio. Estimates of the proportion of parental genome for each individual were obtained according to Paterson et al. (1988). Linkage analysis of SSR markers was conducted by multipoint analysis using MAPMAKER version 3.0. Mapping of QTL and estimation of effects were performed by composite interval mapping (Zeng 1994). A LOD threshold of 2.8 was chosen for declaring a putative QTL significant. A QTL position was determined at the local maximum of the LOD plot curve in the region under consideration. The proportion of phenotypic variance explained by a single QTL was calculated as the square of the partial correlation coefficient. Each QTL was represented by a 20-cM interval with the local LOD maximum as center.

Results

Linkage of SSRs

The 184 F2 individuals from the cross of Huangzao4Ye107 were genotyped by 102 SSR markers that were polymorphic between the two parental lines. Allele frequencies did not deviate significantly from the segregation ratio (1:1) at any marker locus. The proportion of Huangzao4 and Ye107 genome among the 184 F3 families was 50.9% and 49.1%, respectively, which followed the expected segregation distribution (1:1). The observed genotype frequencies deviated significantly (P<0.01) from the expected ratio (1:2:1) at 12 of the 102 marker loci. The genetic linkage map was constructed containing 89 marker loci and covering 1543 cM on ten chromosomes with an average interval of 17.3 cM (Fig.1). The order of marker loci in the linkage map was in good accordance with that in the SSR bin map (Maize DB, 2002).

Phenotype analysis of resistance to SCMV

Both parents Huangzao4 and Ye107 differed significantly for resistance to SCMV in 2000 and 2001(Fig.2). Huangzao4 was resistant, while Ye107 was highly susceptible to SCMV, with disease index of 16.8% and 90.5%, respectively. The mean disease index across all 184 F3 families was 42.4% and 64.6% in 2000 and 2001, respectively. The coefficients of skewness and kurtosis suggested that the disease index of F3 families in both years distributed normally and could be adopted for QTL analysis.

QTL analyses of resistance to SCMV

With the method of composite interval mapping, three QTL conferring resistance to SCMV were identified on chromosomes 3.04, 6.01, and 10.04 at Tangshan in 2000, adjacent to markers phi053, phi423796 and phi062, respectively (Table1, Fig.1). The Huangzao4 contributed all three QTL alleles conferring resistance to SCMV. The largest QTL was located in the region of chromosome 3.04, which could explain 19.5% of phenotypic variance and showed an additive gene effect. The other two QTL identified on chromosomes 6.01 and 10.04 could account for 7.1% and 7.4% of phenotypic variance, respectively, and both displayed partial dominant effects.

Figure 1 The SSR linkage map based on the F2 population derived from the cross Huangzao4Ye107
The putative QTL for SCMV resistance identified in 2000 and 2001, respectively

Figure 2 Distribution of disease index of 184 F3 families in 2000 and 2001

At Beijing in 2001, five QTL were found with significant effects for resistance to SCMV on chromosomes 1.01, 3.04, 5.05, 6.01 and 10.04 (Table 1, Fig.1). Three QTL on chromosomes 3.04, 6.01 and 10.04 were detected consistently in both years, while the other two QTL were found on chromosomes 1.01 and 5.05 in 2001, adjacent to markers umc1160 and umc1822, respectively. The largest QTL was also located on chromosome 3.04, followed by that on chromosomes 10.04 and 1.01, which could explain 26.9%, 15.8% and 15.2% of phenotypic variance, respectively. Both QTL on chromosomes 3.04 and 10.04 showed significant additive effects, while the other three QTL on chromosomes 1.01, 5.05 and 6.01 displayed dominant, over-dominant and partial dominant gene effects, respectively.

Table 1 QTL analyses for resistance to SCMV in 184 F3 families from the cross of Huangzao4Ye107

Year

QTL

Chromosome

SSR loci

Map distance+

LOD

Genetic effect++

Gene action+++

Phenotypic variance

a

d

[d/ a]

 

RQTL-2

3.04

phi053

─ 2.0

9.82

-11.39

1.37

-0.12

A

19.48

2000

RQTL-4

6.01

phi423796

+ 1

3.87

-5.06

-2.66

0.53

PD

7.06

 

RQTL-5

10.04

phi062

─ 5.3

3.61

-4.81

-3.05

0.63

PD

7.37

 

RQTL-1

1.01

umc1160

─ 12.9

3.31

-7.89

9.02

-1.14

D

15.18

 

RQTL-2

3.04

phi053

─ 2

14.64

-17.47

3.52

-0.20

A

26.86

2001

RQTL-3

5.05

umc1822

─ 0.9

4.04

4.97

-9.80

-1.97

OD

5.90

 

RQTL-4

6.01

phi423796

+ 1

3.73

-6.08

-2.11

0.35

PD

5.72

 

RQTL-5

10.04

bnlg292c

+ 12

6.89

-12.97

-1.32

0.10

A

15.76

+ The distance is between the nearest SSR locus and the maximum LOD locus of the QTL. The positive sign (+) shows
QTL identified below the nearest SSR locus, while the negative sign (─) shows QTL above the nearest SSR locus.
++
a: additive effect, d: dominant effect, [d/a]: dominant degree.
+++
A: additive (d/ a =0-0.2), PD: partial dominant (d/ a =0.21-0.80), D: dominant (d/ a =0.81-1.20), OD: over-dominant (d/ a >1.20)

Conclusion

The characterization of QTL affecting resistance to SCMV was investigated using a linkage map with 89 SSR marker loci and the phenotypic data of F3 families derived from the cross Huangzao4Ye107. Three QTLs conferring resistance to SCMV were consistently identified in the region of chromosomes 3.04, 6.01 and 10.04 in 2000 and 2001. While two more QTL on chromosomes 1.01 and 5.05 were found only in 2001. The Huangzao4 contributed all QTL alleles conferring SCMV resistance except the one identified on chromosome 5.05 in 2001. Both major QTL on chromosomes 3.04 and 10.04 showed significant additive gene effects, while the other three QTL on chromosomes 1.01, 5.05 and 6.01 displayed dominant, over-dominant and partial dominant gene effects, respectively. By using the molecular markers linked to these QTL, a marker aided selection approach could be established and investigated in germplasm improvement of maize for SCMV resistance.

Acknowledgement

The research was supported by International Joint Research Project (2003-Q03) and National High- technology Program (2001AA211111).

References

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