Desarrollo y transferibilidad de los microsatélites en Prunus y su aplicación en estudios de variabilidad

The Prunus genus belongs to the Rosaceae family and includes stone fruit crops such as peach (P. persica), apricot (P. armeniaca), European plum (P. domestica), Japanese plum (P. salicina), sweet cherry (P. avium) and sour cherry (P. cerasus), as well as almond (P. dulcis), a species cultivated for its seeds. This work aims to develop simple-sequence repeat (SSR) or microsatellite markers in almond and Japanese plum, the only two diploid Prunus species lacking these markers when this thesis began, and to study their variability in a collection of cultivars of each species. In addition, we studied the transferability of the microsatellites obtained from Prunus in other cultivated rosaceous species, including six Prunus species, and three other genus: apple (Malus x domestica), pear (Pyrus comunis) and octoploid strawberry (Fragaria x ananassa). To develop new microsatellite markers, we used two methods: one from enriched DNA genomic library (for sequences CT/AG), for which we obtained 31 SSRs in almond and 27 in Japanese plum, and another using the available sequences of ESTs (expressed sequence tags) of almond (22 SSRs) and peach (25 SSRs). All these microsatellites were polymorphic in a set of eight cultivars of their respective species. We used the obtained markers in an extensive collection of almond varieties (30) to study their genetic variability using 47 microsatellites derived from this species (25 genomic and 22 derived from ESTs). A similar study was conducted in 38 varieties of Japanese plum with 27 genomic SSRs obtained in this species. These markers were highly variable in both species, with an average of 7.3 alleles per locus in almond and 7.2 in Japanese plum, allowing us to distinguish individually all the studied genotypes. Our data indicated that the SSRs of the same species are more variable than those developed in other related species. In addition, in almond we found that the microsatellites derived from ESTs, and particularly those located in coding regions, were less variable than those obtained from genomic sequence. The grouping of the studied varieties in function of their genetic distance (dendrogram) or their population structure was quite similar both in almond and Japanese plum. The almond varieties were grouped by their geographical origin and their flowering time, whereas the Japanese plum varieties, of recent origin and largely developed in the United States, were clustered according to the breeding programs of the different States they were obtained. A total of 145 Prunus SSRs [25 genomic from almond, peach and Japanese plum, 25 ESTs derived from peach, 22 ESTs derived from almond and 23 derived from apricot (10 genomic and 13 from ESTs)] were chosen to study transferability, all polymorphic and identifying a single locus in the origin species. These microsatellites were studied in eight varieties of the following nine species: almond, peach, European plum, Japanese plum, apricot, sweet cherry, apple, pear and strawberry. Eighty-three percent (83%) of these markers amplified bands of the expected size in the other Prunus species and 63.9% were polymorphic, indicating the high transferability within this genus. This transferability decreased as the genetic distance between the species origin of the SSR and the studied species increased. Thus, only 16.3% of the tested SSRs were transferable to species of other rosaceous genera (apple, pear and strawberry). No significant differences were detected between microsatellites of different origins (genomic and ESTs) regarding their transferability, nor their capacity to detect variability. From the studied SSRs, 31 amplified and were polymorphic in all tested Prunus species. Twelve, selected to cover the whole genome, were proposed as the universal set for the analysis of variability in Prunus

Two Loci, RiAF3 and RiAF4, Contribute to the Annual-Fruiting Trait in Rubus

Most Rubus species have a biennial cycle of flowering and fruiting with an intervening period of winter dormancy, in common with many perennial fruit crops. Annual-fruiting (AF) varieties of raspberry (Rubus idaeus and Rubus occidentalis L.) and blackberry (Rubus subgenus Rubus) are able to flower and fruit in one growing season, without the intervening dormant period normally required in biennial-fruiting (BF) varieties. We used a red raspberry (R. idaeus) population segregating for AF obtained from a cross between NC493 and ‘Chilliwack’ to identify genetic factors controlling AF. Genotyping by sequencing (GBS) was used to generate saturated linkage maps in both parents. Trait mapping in this population indicated that AF is controlled by two newly identified loci (RiAF3 and RiAF4) located on Rubus linkage groups (LGs) 3 and 4. The location of these loci was analyzed using single-nucleotide polymorphism (SNP) markers on independent red raspberry and blackberry populations segregating for the AF trait. This confirmed that AF in Rubus is regulated by loci on LG 3 and 4, in addition to a previously reported locus on LG 7. Comparative RNAseq analysis at the time of floral bud differentiation in an AF and a BF variety revealed candidate genes potentially regulating the trait.info:eu-repo/semantics/publishedVersio

Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars

Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: “Tuono”, “Cristomorto”, and “Nonpareil”. Descendants from “Tuono” or “Cristomorto” number 76 (sharing 34 descendants), while “Nonpareil” has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the Sf allele for self-compatibility, the mean relatedness coefficient was 0.125, with “Tuono” as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress.info:eu-repo/semantics/publishedVersio

Construction of an almond linkage map in an Australian population Nonpareil × Lauranne

Background: Despite a high genetic similarity to peach, almonds (Prunus dulcis) have a fleshless fruit and edible kernel, produced as a crop for human consumption. While the release of peach genome v1.0 provides an excellent opportunity for almond genetic and genomic studies, well-assessed segregating populations and the respective saturated genetic linkage maps lay the foundation for such studies to be completed in almond. Results: Using an almond intraspecific cross between ‘Nonpareil’ and ‘Lauranne’ (N × L), we constructed a moderately saturated map with SSRs, SNPs, ISSRs and RAPDs. The N × L map covered 591.4 cM of the genome with 157 loci. The average marker distance of the map was 4.0 cM. The map displayed high synteny and colinearity with the Prunus T × E reference map in all eight linkage groups (G1-G8). The positions of 14 mapped gene-anchored SNPs corresponded approximately with the positions of homologous sequences in the peach genome v1.0. Analysis of Mendelian segregation ratios showed that 17.9% of markers had significantly skewed genotype ratios at the level of P < 0.05. Due to the large number of skewed markers in the linkage group 7, the potential existence of deleterious gene(s) was assessed in the group. Integrated maps produced by two different mapping methods using JoinMap® 3 were compared, and their high degree of similarity was evident despite the positional inconsistency of a few markers. Conclusions: We presented a moderately saturated Australian almond map, which is highly syntenic and collinear with the Prunus reference map and peach genome V1.0. Therefore, the well-assessed almond population reported here can be used to investigate the traits of interest under Australian growing conditions, and provides more information on the almond genome for the international community.Iraj Tavassolian, Gholmereza Rabiei, Davina Gregory, Mourad Mnejja, Michelle G Wirthensohn, Peter W Hunt, John P Gibson, Christopher M Ford, Margaret Sedgley, and Shu-Biao W

Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars

[ES]: Reducir la pérdida de variabilidad genética es un reto en los programas de mejora debido al repetido uso de un escaso número de genotipos. Para estudiar la variabilidad genética del almendro en su mejora a nivel mundial se utilizaron datos genealógicos de 222 variedades y selecciones provenientes de Argentina, Australia, Francia, Grecia, Israel, Italia, Rusia, España y EE. UU. Se calculó la consanguinidad, las relaciones por parejas y la contribución genética para todos los genotipos. Los resultados señalan dos principales líneas de mejora basadas en tres cultivares ‘Tuono’–‘Cristomorto’ y ‘Nonpareil’. Existen 75 descendientes directos (compartiendo 30) de ‘Tuono’ o ‘Cristomorto’ mientras que ‘Nonpareil’ tiene 72 descendientes directos. El coeficiente medio de consanguinidad de los genotipos analizados fue 0,036, con 13 presentando una elevada consanguinidad. Los programas de mejora de EE. UU. (0,06), Francia (0,05) y España (0,03) mostraron consanguinidad. De acuerdo con su contribución genética, las variedades modernas de Israel, Francia, EE. UU., España y Australia, se basan en seis, cinco, cuatro, cuatro y dos genotipos fundadores principales respectivamente. Entre el grupo de 65 genotipos con el alelo Sf de autocompatibilidad, el coeficiente medio de relación fue de 0,133, con ‘Tuono’ como principal fundador (23,75% de la contribución genética total).[EN]: Loss of genetic variability is a challenge increasing when breeding due to the repeated use of a reduced number of founders. Pedigree data of 222 almond cultivars and selections were used to study worldwide genetic variability in modern programs from Argentina, Australia, France, Greece, Israel, Italy, Russia, Spain and USA. Inbreeding coefficients, pairwise relatedness and genetic contribution were calculated. The results reveal two mainstream breeding lines based in three cultivars: ‘Tuono’–‘Cristomorto’ and ‘Nonpareil’. Direct descendants from ‘Tuono’ or ‘Cristomorto’ account to 75 (sharing 30 descendants), while ‘Nonpareil’ has 72 direct descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.036, with 13 genotypes presenting a high inbreeding coefficient. Breeding programs from USA (0.06), France (0.05) and Spain (0.03) showed inbreeding. According to their genetic contribution, modern cultivars from Israel, France, USA, Spain and Australia, trace back to six, five, four, four and two main founders respectively. Among the group of 65 genotypes with the Sf allele for self–compatibility, the mean relatedness coefficient was 0.133, with ‘Tuono’ as main founder (23.75% of total genetic contribution).Peer reviewe

Providing molecular breeding expertise in a wide range of crops to a private sector company

Trabajo presentado al Internal Seminar of the Centre de Recerca Agrigenómica (CRAG), celebrado el 13 de septiembre de 2019

Image_1_Inheritance of esters and other volatile compounds responsible for the fruity aroma in strawberry.JPEG

1 figure.Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB).Peer reviewe

Table_4_Inheritance of esters and other volatile compounds responsible for the fruity aroma in strawberry.XLSX

Supplementary table 2. VOCs relative content in parental and grandparental lines and 'FC50xFD54' population for six harvests.Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB).Peer reviewe

Image_4_Inheritance of esters and other volatile compounds responsible for the fruity aroma in strawberry.JPEG

1 figure.Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB).Peer reviewe

Table_5_Inheritance of esters and other volatile compounds responsible for the fruity aroma in strawberry.XLSX

Supplementary table 3. p-value from Shapiro-Wilk test using VOCs relative content and log2 transformed content in 'FC50xFD54' population.Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB).Peer reviewe