Washington Tree Fruit Research Commission

Research Reports

Inheritance of chilling-dependent pear fruit ripening (2014)

FINAL PROJECT REPORT
WTFRC Project #
YEAR 0/0
Organization Project #
Title:Inheritance of chilling-dependent pear fruit ripening
PI:Richard L. Bell
Organization:USDA, ARS 304-725-3451 Ext. 353 richard.bell@ars.usda.gov 2217 Wiltshire Road Appalachian Fruit Research Station Kearneysville, WV 25430
 PDF version of report

ORIGINAL OBJECTIVES

  1. Determine the segregation of an absolute and quantitative requirements for fruit chilling in the two crosses by evaluating fruit ripening after 7 days at 20°C following 0, 30, and  60 days at  -1°C.
  2. Determine the genetic composition of the parents and selected seedlings with no, moderate, and high chilling requirements for allelic variants of ACS genes, and determine the relationship between genotype and chilling-requirement.

 

Significant findings

·                                 Seedlings segregated for chilling-independence (n=19) and chilling-dependence (n=13) for fruit ripening.  This ratio, tested by chi-square analysis, is consistent with genetic control by a single gene or genetic locus. 

·                                 There were no genetic differences among the parents and the seedlings for the ACS1 andACS2 using an agarose gel detection system and the ACS1 and ACS2 allele-specific primers reported by El-Sharkawy et al. (2004).  These results were duplicated in the laboratory of Nahla Bassil at the USDA, ARS, National Clonal Germplasm Repository in Corvallis, Oregon.  Dr. Bassil also tried genotyping an additional 7 cultivars and breeding selections, including ‘Passa Crassane’ and ‘Old Home’, which El-Sharkawy reported were chilling-dependent and chilling-independent, and which he reported to differ in their ACS2 genotypes. She did not find any differences.  These results are also consistent with results obtained by Dr. Amit Dhingra of Washington State University.  However, using a capillary electrophoresis system, Dr. Bassil detected a small fragment (81 base pairs) of ACS1b which may be associated with chilling-dependence.

Results and discussion

In 2013, the presence or absence of a chilling requirement for pear fruit ripening was determined for 32 seedlings of the cross ‘Beurre d’Anjou’ (high chilling requirement) and US76128-009 (low chilling requirement).  Seedlings segregated for chilling-independence (n=19) and chilling-dependence (n=13) (Table 1).  This ratio is statistically consistent with a 1:1 segregation indicating single gene control.  With data from 2011, a total of 45 seedlings have been evaluated.  Among those seedlings which exhibited chilling-dependence, five required 4 weeks of chilling, three required 8 weeks of chilling, and five did not ripen after 8 weeks of chilling, and apparently require more than 8 weeks. The chilling requirement of an additional five could not be accurately determined due to insufficient fruit for all treatments.  The genetic control of quantitative difference in chilling requirement could not be determined from this type of analysis, but is probably due to different genes than that controlling the chilling dependence versus independence.     

No differences in ACS1 and ACS2 genotypes were detected using an agarose gel detection system and the ACS1 a/b and ACS2 a/b allele-specific primers reported by El-Sharkawy et al (2004) (Table 2).  We concluded that new primers designed from the ACS1 and ACS2 gene and/or promoter sequences are needed to genotype pears for these genes involved in ethylene biosynthesis and to answer the question of whether they control fruit chilling response in a qualitative or quantitative manner.   Similar results were obtained in the laboratory of Dr. Nahla Bassil, who collaborated on the study.  The DACS1a primer amplified a larger fragment than reported by El-Sharkawy et al. (2004).  The primers for DACS1b, DACCS2a, and DACS2b amplified products similar to those reported by El-Sharkawy, but no polymorphisms were observed in ‘Old Home’ and ‘Passa Crassane’, the two pear cultivars used in his study, nor in seven other pear cultivars which varied in date of maturity, and presumably in chilling requirement (Table 3).  However, polymorphic products of low molecular weight were found with the DACS1b (73 bp, 75 bp, 79 bp and 81 bp) and DACS2b (109 bp and 115 bp) primers were found (Table 4).   The 81 bp DACS1b fragment was associated with those cultivars and selections which had a chilling requirement.  The significance of this fragment must be verified with additional cultivars, selections and segregating seedling populations of known chilling requirement. 

REFERENCES

El-Sharkawy, I., B. Jones, L. Gentzbittel, J.-M. Lelièvre, J. C. Pech and A. Latché. 2004. Differential regulation of ACC synthase genes in cold-dependent and –independent ripening in pear fruit.  Plant Cell and Environment 27:1197-1210.  

Table 1. Segregation of chilling-independence versus chilling-dependence1. 

                                                _________Chilling-Dependent                                 

Chilling-Independent              4 weeks           8 weeks           8 weeks+        

           

            19                                     5                       2                     6    

____________________________________________________________

Total    19                                                            13

____________________________________________________________           

1 Chi-square analysis detected no deviation from the hypothesized 1:1 ratio (Pr> ChiSq = 0.37).  

Table 2. Amplified fragment sizes (in number of DNA base pairs) for ‘Old Home’

and ‘Passe Crassane’_(Experiment 1)___________________________________
                                     ____________________ACS gene and allele________       Cultivar                                 ACS1a             ACS1b            ACS2a                ACS2b             

Old Home                               ~900                405                  266                  437

Passe Crassane                        ~900                405                  266                  437

_________________________________________________________________

  

Table 3. Amplified fragment sizes (in number of DNA base pairs) for eight pear cultivars

 (Experiment 2).

________________________________________________________________________

                                                                        DACS1                       DACS2          
CPYR Number and Cultivar     Maturity           Expected  Observed     Expected  Observed                 

1057    Angelica di Saonara    Early                785/405    405             437          437/266

1165    Buerre Bosc                Late                 785           405             266         437/266      

148      Abate Fetel                 Intermediate    785/405    405             437/266   437/266

77        Doyenne du Comice   Late                 785           405             266          437/266

448      Pautalia                       Early                785/405    405             437          437/266

431      Old Home                   Intermediate    785/405    405             437/266   437

441      Passe Crassane            Late                 785           405             266         437

__________________________________________________________________________


 

Table 4.  Amplified fragments sizes based on capillary electrophoresis using M13-tagged primers._

_________________________DACS1b              DACS2a               DACS2b_______

CPYR Number and Cultivar   

                                     Expected  Observed   Expected  Observed    Expected Observed    

441      Passe Crassane      421          73, 81          266           317             489          109

 

63        Anjou                   421          79, 81          266           317             489         109

 

431      Old Home            421          79, 81          266           317             489         109, 115

 

US78307-045     421          79, 81          266           317             489         109

 

US84907-166     421          75, 79          266           317             489         109

 

US76128-009     421          75, 79, 81    266           317             489         109

 

Sunrise                421          79                266           317             489         109

 

NJ B9 R1 T117   421          75, 79          266           317             489         109

___________________________________________________________________________

 

EXECUTIVE SUMMARY

Winter pears, such as ‘Beurré d’Anjou’, ‘Buerré Bosc’ and Doyenné du Comice’, require a period of cold storage and/ or ethylene exposure to initiate normal ripening.  The inability to ripen, while perhaps contributing to long storage life, poses challenges to the pear industry in their ability to deliver a ready-to –eat product to market.  The development of new cultivars with which are more amendable to post-harvest manipulation requires knowledge of the inheritance of the post-harvest chilling requirement.  In addition, knowledge of the underlying genomic basis for the trait and the genetic composition of important cultivars and breeding parents will improve the ability to breeding new cultivars with desirable post-harvest ripening.  Preliminary data for a seedling population derived from hybridizing ‘d’ Anjou’ (chilling –dependent with long requirement) and US76128-009, a chilling-dependent selection with a short requirement) suggested that chilling-dependence is a dominant trait, and that the amount of chilling is variable, likely determined by an unknown number of  genes.  The purpose of this study was to determine the inheritance of the absolute requirement for chilling as well as to characterize the nature of the quantitative variation in chilling requirement.     

Summary of findings 

Approximately half of the 45 seedlings exhibited chilling independence for fruit ripening, while the others exhibited chilling dependence, with the amount of chilling required varied from 4 to 8 weeks or more.  This finding is consistent with genetic control by a single gene.   It had been hypothesized that the genes ACS1 and ACS2 controlled chilling-dependence.  Using gene primers published by El-Sharkawy, we found no relationship of these genes with chilling dependence/independence.  We were unable to duplicate El-Sharkawy’s results with the cultivar he used in his study, ‘Old Home’ and ‘Passe Crassane’.  We hypothesized that different primers designed from the gene sequences would  be necessary  to further investigate the relationship.  Subsequent communication from other researchers confirmed that these genes do not control chilling dependence/independence.  We did find a possible relationship of a small DNA fragment (81 base pairs) with chilling dependence. 

Future directions  

Evaluation of additional seedlings would enhance the robustness of the analysis of the genetic control of chilling dependence, especially the quantitative variability in the amount of chilling needed to initiate normal ripening. We plan to do that in the growing season.  In addition, new seedlings of the cross between ‘Anjou’ and ‘Harrow Delight’, which has not chilling requirement, will be generated by hybridization.  We plan to determine the relationship between genotype, global gene expression patterns, and chilling-requirement using advanced genomic approaches, Pnome and RNA-Seq.  We will validate the association of the 81 bp fragment with chilling independence in additional pear cultivars and seedlings.  The overall goal will be to develop new primers or markers designed based on the gene sequences derived from the European pear genome sequence.  

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