Euphytica 44 : 1 63-166, 1989 .©1989 Kluwer Academic Publishers. Printed in the Netherlands .

Linkage relationship between two genes conferring resistance to peanutstripe virus and soybean mosaic

S.H . Choi', S.K. Green and D.R . LeeAsian Vegetable Research and Development Center, Tainan, Taiwan, R . 0 . C.; 'Present address:Department of Plant Pathology, Institute of Agricultural Sciences, R . D.A ., Suweon, Korea ;

Received 14 June 1988 ; accepted in revised form 2 October 1988

Key words: Glucine max, soybean, peanut stripe virus, resistance, genetics, linkage

Summary

The possible linkage relationship between hypocotyl color, leaflet shape, resistance to soybean mosaic virus(SMV) and to a soybean isolate of peanut stripe virus (PStV-isolate PN) was examined in two soybean linesAGS 129 and Ankur .

Hypocotyl color, leaf shape and reactions to SMV-G1 and PStV were found to be inherited monogenically,with purple hypocotyl color, ovate leaf shape and resistance to both of the viruses being dominant . Thereactions to SMV and PStV were conditioned by genes with 9 ± 2 .4 percent recombination as couplingphase. They were inherited independently from hypocotyl color and leaf shape .

Introduction

A soybean (Glycine max (L.) Merr.) line, AGS129, was previously found to possess resistance toboth SMV strain G1 and to a new potyvirus(AVRDC, 1985, Green et al ., 1986) . In previousevaluations of AVRDC soybean accessions andbreeding lines (AVRDC, 1985) for resistance tothese 2 viruses it was found that those lines whichwere immune to SMV-G1 were also immune to thesoybean isolate of PStV (PStV, isolate S-PN) .

The objective of the present study was to deter-mine whether the gene(s) conferring resistance tothese 2 viruses are linked and whether linkage rela-tionships exist with morphological characteristics,such as hypocotyl color and leaf shape, which mightprovide convenient selection criteria in breedingprograms .

Materials and methods

The soybean cv . Ankur orginating from India, andAVRDC breeding line AGS 129, were used asparents . Ankur is susceptible to both SMV-G1 andthe soybean isolate of PStV (S-PN) and has greenhypocotyl color and ovate leaf shape . AGS 129 isresistant to both viruses and has purple hypocotylcolor and lanceolate leaf shape .

Both viruses which have previously been isolatedfrom field-grown soybean in Taiwan, were propa-gated on G. max cvs. Rampage or Ankur . Inoculawere prepared from freshly harvested infectedleaves, homogenized at a ratio of 1 : 3 (w/v) with0.1 M potassium phosphate buffer, pH 7 .0. The ho-mogenate was strained through four layers ofcheesecloth, and inoculated to unifoliolate leavespreviously dusted with carborundum (600 mesh) .

Symptom appearance was observed visually for

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50 days . At 16 days after inoculation, the two sideleaflets of the first trifoliolate leaf of the parents,Fl, backcross and F2 generations were tested forpresence of virus by double antibody sandwich en-zyme linked immunosorbent assay (DAS-ELISA)according to a standard procedure (Clark &Adams, 1977) . In case of mixed virus inoculations asecond DAS-ELISA test was performed at 30 daysafter the inoculation, using the two side leaflets ofthe 2nd trifoliolate leaf. ELISA values were mea-sured photometrically with a Titertek MultiskanMC reader .

The reactions were classified into two categories :resistant (symptomless and negative ELISA reac-tions or necrosis regardless of ELISA reaction) andsusceptible (mosaic symptoms and positive ELISAreaction) . Hypocotyl color was recorded at 15 daysafter sowing. Leaf shape was determined on thefully developed 2nd trifoliolate leaflets by the leaflength and width ratio . Leaves with a ratio above2.01 were classified as lanceolate .

The experiment was conducted in the green-house in three separate sets . Set 1 was inoculatedwith the soybean isolate of PStV and set 2 wasinoculated with SMV-G1 . Set 3 was inoculatedwith a mixture of both viruses .

The data from set 3 were combined with those ofset 1 to determine the reaction to PStV and withthat of set 2 to determine the reaction to SMV.Pooled data from set 1 and set 2 were used toanalyze the inheritance of hypocotyl color and leafshape. The linkage between genes governing resist-ance to PStV and hypocotyl color or leaf shape wastested in set 1 . The linkage between genes govern-ing resistance to SMV and hypocotyl color of leafshape was tested in set 2. The combined segre-gation for reaction to PStV and SMV was analyzedin set 3 .

The Chi-square (x2) test was used to determinethe inheritance of each character in the backcrossand the F2 population . The linkage among charac-ters was calculated from the F2 data by the productratio method (Immer and Henderson, 1943) .

Results and discussion

All plants of the susceptible parent Ankur showedmosaic symptoms after inoculation with either vi-rus alone or a mixture of both viruses and in eachcase, the inoculated virus(es) could be detected byELISA. However, none of the plants of the resist-ant parent AGS 129 showed any distinguishablesymptoms when inoculated with either virus aloneor a mixture of both viruses and in each case, theinoculated virus(es) could not be detected by EL-ISA. All Fl plants and some F2 plants reacted withnecrosis when inoculated with PStV alone or amixture of PStV and SMV (Table 1) . Only PStVcould be detected by ELISA in necrotic F2 plants,while both viruses were detected in some F2 plantswith mosaic, resulting from inoculation with a mix-ture of the two viruses . The F2 and backcross plantsshowing necrosis after inoculation with PStV aloneor a mixture of PStV and SMV were classified asresistant, since necrosis was found to be expressedby the presence of a dominant allele in the hetero-zygous condition (Choi et al ., 1989) . The gene con-ferring resistance to PStV-SPN is suggested to be asingle incompletely dominant allele in the cross ofAnkur X AGS 129 (Choi et al., 1989). However,only mosaic was produced and necrosis was absentwhen inoculated with SMV-G1 alone .

The segregation for individual traits in the F2population from the cross of Ankur with AGS 129is shown in Table 1 . The segregation of 232 resist-ant to 85 suspectible plants with respect to thesoybean isolate of PStV gave a good fit to theexpected ratio of 3 resistant to 1 susceptible, in-dicating a monogenic control, with resistance ofAGS 129 being dominant .

In the case of SMV-G1, the ratio of 166 resistantto 53 susceptible plants also gave a good fit to theexpected 3 resistant to 1 susceptible ratio (Table 1) .None of the plants in the F2 population reacted

Set no . Virus inoculation Generation testedfor virus

1 PStV (isolate S-PN) P, Fl, F22 SMV (strain G-1) P, F23 Mixture of PStV (S-PN) P, BC1F1, F2

and SMV (G-1)

with necrosis when inoculated with SMV-Gl, in-dicating a single completely dominant allele inAGS 129 for resistance to SMV-Gl .

The combined segregation ratio in the F2 pop-ulation for hypocotyl color and reaction to PStVfitted the expected 9 : 3 : 3 : 1 ratio (Table 2) . Simi-lar results were obtained for reactions to SMV andhypocotyl color, indicating independent relation-ships between the genes governing hypocotyl colorand those conferring resistance to SMV and PStV .

When the combined segregation for leaf shapeand reaction to PStV of the F2 population wastested for goodness of fit, a ratio of 9 : 3 : 3 : 1 (Ta-ble 2) was obtained. Similar results were obtainedfor leaf shape and reaction to SMV (Table 2) .

Table 1 . Segregation of characters in Ankur X AGS 12 F2 population after inoculation with SMV-G1 and the soybean isolate of PStV

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P and G pertain to purple and green hypocotyl color, respectively .Pr and Sp refer to resistant and susceptible reaction to PStV .Rs and Ss refer to resistant and susceptible reaction to SMV-G1 .0 and L refer to ovate and lanceolate leaflet shape, respectively .

46

The data from set 3, inoculated with mixture of PStV and SMV, were combined with those of set 1, inoculated with PStV alone throughELISA test .n The data from set 3 were combined with those of set 2, inoculated with SMV alone through ELISA test .The data from set 1 and set 2 were combined .

Table 2 . F2 phenotypic classes and their frequencies on four different pairs of characters in the cross Ankur X AGS 129

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These findings show that the gene for leaf shape isinherited independently from the gene(s) condi-tioning the reaction(s) to PStV and SMV .

When tested for reaction to the soybean strain ofPStV and SMV-G1 the combined segregation inthe F2 population and the backcross progeny withthe susceptible parent Ankur failed to segregate inthe expected 9 : 3 : 3 : 1 and 1 : 1 : 1 : 1 ratio, respec-tively (Table 3) ; linkage of these two characters istherefore suggested . Presence of both dominantalleles on the same chromosomes in the resistantparent AGS 129, and a deficiency of plants in therecombinant classes and an excess in the parentalclasses in the F2 and backcross population (Table3) indicates the presence of a coupling phase link-

Characters Phenotypes' and their frequencies x2 (9 :3 : 3 : 1 ratio)

Value P

Hypocotyl color & reaction to PStV P-Rp-91 P-SpSp 33 GGRp-20 GGSpSp 14 5 .285 0.25-0 .10Hypocotyl color & reaction to SMV-G1 P-Rs-30 P-SsSs 12 GGRs-11 GGSsSs 7 3.281 0.50-0 .25Leaf shape & reaction to PStV O-Rp-87 O-SpSp 37 LLRp-24 LLSpSp 10 2.945 0.50-0 .25Leaf shape & reaction to SMV-G1 O-Rs-33 O-SsSs 15 LLRs-8 LLSsSs 4 2.222 0.75-0 .50

Characters Classes and their frequencies Ratio tested Chi-square

resistant susceptible value P

Reaction to PStV-PN' 232 85 3 :1 0.556 0.50-0 .24

Reaction to SMV-Gl°(symptomless and necrosis)166

(mosaic)53 3 :1 0 .075 0.90-0 .75

Hypocotyl color'(symptomless)purple

(mosaic)green 3 :1 0 .153 0.75-0 .50

166 52Leaf shape` ovate lanceolate 3 :1 1 .768 0.25-0.10

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Table 3. Frequencies of phenotypic classes in F2 and BC of the cross Ankur X AGS 129 with respect to reaction to SMV-G1 and thesoybean isolate of PStV

aThe combined segregation was analyzed in set 3, inoculated with mixture of PStV and SMV, through ELISA test . Rp, Rs, Sp and Ssrefer to resistance to the soybean isolate of PStV, resistance to SMV-Gl, susceptibility to the soybean isolate PStV and susceptibility toSMV-Gl, respectivelly .

Table 4. Linkage test between genes conferring resistance to the soybean strain of PStV and SMV-Gl in the F2 population from the crossAnkur X AGS 129

Parents

Number of F2 plants'

a

b

Ankur X AGS 129

116

5

' a: Resistant to both PStV and SMV (symptomless as resistant parent of AGS 129 and necrosis resulted from heterozygosity to PStV) ; b :Resistant to PStV, susceptible to SMV (recombinant) ; c : Susceptible to PStV, resistant to SMV (recombinant) ; d : Susceptible to bothPStV and SMV (parental type as Ankur) .

age between the genes conferring resistance toPStV S-PN and SMV G-1 .The calculated recombination percentage be-

tween genes governing the reaction to PStV andSMV in the observed F2 population from the dou-ble heterozygous F1 parent was 9 ± 2 .4 as couplingphase (Table 4) by the product ratio (ratio of cross-over to parental types) method (Immer & Hen-derson, 1943). It was however not possible in thepresent study to determine the linkage group towhich the two genes, conditioning reaction toPStV-PN and SMV G-1 belong .

c

9

References

Asian Vegetable Research and Development Center, 1987 .1985 Progress Report, Shanhua, Tainan, R.O.C. 470 p .

Clark, M.F. & A .N. Adams, 1977 . Characteristics of the micro-plate method of emzyme-linked immunosorbent assay for thedetection of plant viruses. J . Gen . Virol . 34: 475-483 .

Choi, S.H ., S.K. Green, D .R. Lee & J .Y. Yoon, 1989 . In-completely dominant single resistance gene for peanut stripevirus in soybean line AGS 129 . Euphytica 40 : 193-197 .

Green, S.K ., D.R. Lee, D .E. Lesemann & H .J . Vetten, 1986 .The occurrence of peanut stripe virus on peanut and soybeanin Taiwan . Plant Protection Bulletin, Taiwan (R.O.C.) 28 :445-446.

Immer F.R. & M.T. Henderson, 1943 . Linkage studies in bar-ley. Genetics 28: 419-440 .

d

total

Recombination % Phase

29

159

9 ± 2.4

Coupling

Generation Frequencies in phenotypes Ratio tested Chi-square

Rp-Rs- Rp-SsSs SpSpRs- SpSpSsSs value P

F2 116 5 9 29 9 :3 :3 :1 79.635 < 0 .01BC (Ankur X Fl) 17 0 4 11 1 :1 :1 :1 21 .250 < 0 .01BC (AGS 129 x Fl) 28 0 0 0 1 :0 :0 :0