Pea Accessions Research Articles

The First Report on the Application of ISSR Markers in Genetic Variance Detection among Butterfly Pea (Clitoria ternatea L.) Accession in North Maluku Province, Indonesia

Butterfly pea (Clitoria ternatea L.) is a leguminous plant with several potential health benefits. The scientific name is derived from its origin on Ternate Island, North Maluku. Therefore, this study aimed to analyze the genetic variability in butterfly pea using Intergenic Simple Sequence Repeat (ISSR) markers in North Maluku. Field surveys, collection trips, and habitat studies of butterfly pea plants were conducted on Ternate, Tidore, Halmahera, and Morotai Islands. Genetic diversity was analyzed based on molecular data from the ISSR method. The molecular analysis results obtained using PCR-ISSR on 18 accessions showed a low degree of similarity. Among these, 15 accessions from Ternate, Tidore, Morotai, and Halmahera were in group A, while 3 from Ternate, Tidore, and Halmahera were in group B. All accessions exhibited a genetic similarity level of 0.709, indicating significant diversity. The arrangement among accessions on the dendrogram was similar to the phylogenetic tree, showing separation and spread at 0.608–0.924 based on the Jaccard coefficient. The results suggested that C. ternatea probably originated from Ternate, and subsequently spread to Tidore, Halmahera, and Morotai due to its use as a herbal medicine and ornamental plant. This information could be used as the basis for butterfly pea conservation and cultivation activities in Indonesia, specifically in Ternate Island, North Maluku.

Digital imaging of morphometric characteristics of pigeon pea (<i>Cajanus cajan</i>) for genotype discrimination

Digital seed imaging was utilized in this study to evaluate the morphometric characteristics of pigeon pea for genotype discrimination. One hundred seeds in each of the pigeon pea accession were subjected to digital imaging analysis using the WinSEEDLE™ software to differentiate the seed metric characters. Seeds were placed on a lighting hood and were automatically analyzed by the scanner and the image of the seed was recorded by the WinSEEDLE™. The procedure was repeated three times. The parameters observed were projected area, straight length, curve length, straight width, curve width, volume circle, and projected perimeter. Data collected were subjected to analysis of variance, principal component analysis and product moment correlation coefficients between the parameters were obtained. The result revealed that the pigeon pea accessions were highly variable in all the seed metric traits evaluated. Accessions NSWCC-34 and NSWCC-32 showed superiority values in most of the seed metric traits evaluated. Accessions NSWCC-32 had 28.35 mm2 , 6.99 mm, 7.37 mm, 5.23 mm and 5.28 mm in seed projected area, seed straight length, curve length, straight width and curve width, respectively while NSWCC-34 recorded 28.34 mm2 , 6.75 mm, 7.10 mm, 5.22 mm and 5.27 mm for the same evaluated traits. Most of the seed traits showed significant positive and strong association. The study, therefore, recommends that accessions NSWCC-34 and NSWCC-32 as well as those traits with high contribution to the major variation (seed projected area, seed straight length, curve length, straight width, curve width and volume circle) could be included in the seed improvement programme for improved seed quality in pigeon pea.

Assessment of Genetic Diversity and Protein Content of Scandinavian Peas (Pisum sativum)

We produced homogeneous lines of 227 pea accessions from the Nordic Genetic Resource Center via single seed descent. The genetic diversity among these, mostly Scandinavian accessions, was investigated using three microsatellite markers, A9, AC58 and AA5. The microsatellites were highly informative and separated 153 of 194 accessions on a Neighbor Joining topology. The high polymorphism information content (PIC) values between 0.87 and 0.91 indicated that the gene bank material contains a large number of pea accessions with different breeding histories. The peas were grown in the field for two years and seed protein content showed variation between 9.3% and 34.1% over the years and accessions, respectively. The mean thousand seed weight was 152.05 g. More than 10 accessions had a protein content above 28%, showing that the collection has potential as breeding nursery for high-protein pea cultivars.

Morphological characterization and in vitro digestibility of seven Lathyrus sativus (grass pea) accessions originating from Eurasia, Africa, and Canada

Lathyrus sativus is considered as an important economical source of high-quality proteins and adaptability to global climate change. To detect accessions useful for future breeding strategies, the genetic variability among and within seven Lathyrus sativus accessions was assessed by morphological characters and the in vitro digestibility study. An important genetic diversity was found using various morphological traits. The significant heterogeneity revealed by variance analysis (ANOVA) and correlation test could be elucidated by the different geographical origins of the studied accessions and the natural selection of grass pea. The Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) exhibited the accumulation of several genotypes in the same cluster which confirm that these genotypes are not dispersed according to their origin. Whereas, the individuals of grass pea from Ethiopia were collected in distinct group indicating that these samples are close each other’s and are different from the other accessions. In the same aim, the in vitro digestion model showed that most of the proteins were digested after 60-min incubation except the β-lathyrin protein. The different digestion kinetics found could be used as a method to distinguish different crop accessions. In fact, the densitometry analysis of β-lathyrin protein band showed that grass pea from Ethiopia, Germany, Bangladesh, and Morocco were more digested than grass pea from Canada, Afghanistan, and Greece in gastric phase, and Grass pea from Afghanistan and Germany were more digested than the rest of the samples in duodenal phase.

AMMI and GGE Biplot Analysis of Yield and Related Traits among Selected Mini-core Pigeonpea (Cajanus cajan L. Millsp.) Accessions

Pigeon pea [Canjanus cajan (L.) Millsp.] is an orphan crop that has remained greatly unimproved in quality and quantity in Africa even though it has great potential of supplying food to the growing population. Its yield is affected by various biotic and abiotic factors and a lack of varieties with broad adaptability to a range of environments. It is therefore important to select accessions that are stable in different environments for yield and yield components. A randomized complete block design layout was used to carry out a multi-locational trial using 107 pigeon pea accessions. Data was taken for the total number of pods per plant at maturity, pod length, seed weight, seeds per pod, and seed yield per plant. Additive main effects and multiplicative interaction (AMMI) with genotype and genotype by environment interaction (GGE) biplot were used to interpret the data. The mean squares for the PCA explained by the first two principal components account for 100% of the G x E interaction. The results revealed that the differences in the yield and yield components across the three locations were mostly due to the genotypes of the pigeon pea and to some extent the genotype by environment interaction. Six accessions were observed to be high-yielding and stable across all three environments.

Vegetable peas (Pisum sativum L.) diversity: An analysis of available elite germplasm resources with relevance to crop improvement

Aim of study: To determine the amount of diversity in pea breeding materials with the objective to classify a set of potential parents carrying novel/economic variations that could be used in future breed pea varieties. Area of study: ICAR–Indian Institute of Vegetable Research, Varanasi. Material and methods: A total of 45 pea accessions were analysed for phenotypic and molecular diversity using 17 agro-morphological traits and 52 SSR markers. Main results: All traits under investigation showed considerable genetic variation. The genotypes exhibited 6.7, 2.7 and 12-fold variation for traits viz., pods/plant, 10-pod weight and yield/plant, respectively. Among 52 SSR markers, 22 were found to be polymorphic. A total of 90 allelic variants were detected, with an average of 2.7 alleles/locus. PIC and D-values for markers AA135 (0.79 and 0.81) and PSMPSAD51 (0.7 and 0.74) were the highest, while AB40 (0.19 and 0.2) had the lowest. Two principal components PC1 and PC2 explained 46.96 and 23.96% of total variation, respectively. The clustering based on agro-morphological traits differentiated 45 individuals into three mega clusters, while SSR markers-based clustering classified these accessions into four groups. Research highlights: Based on their uniqueness, we identified a set of genotypes (VRPD-2, VRPD-3, PC-531, ‘Kashi Nandini’, ‘Kashi Udai’, ‘Kashi Mukti’, ‘Arkel’, VRPE-101, ‘Azad Pea-3’, EC865944, VRPM-901 and VRP-500) harbouring genes for various economic traits. The findings presented here will be extremely useful to breeders who are working on improvement of peas through selective introgression breeding.

Lathyrus sativus Resistance Against the Existing and Emerging Pathogens Erysiphe pisi and E. trifolii: A Case of Commonalities or Total Discrepancy?

Powdery mildew on Lathyrus sativus (grass pea) is commonly caused by Erysiphe pisi, the causal agent of pea powdery mildew. E. trifolii could also pose an additional threat to grass pea, as it does to pea (Pisum sativum). In order to understand the potential threat and the availability of resistance sources, the response to both pathogens was analyzed on a worldwide germplasm collection of 189 grass pea accessions. Infection type and disease severity (DS) of grass pea accessions, independently inoculated with E. pisi and E. trifolii, were evaluated under controlled conditions. A wide range of responses were detected, with the previously uncharacterized partial resistance to E. trifolii in grass pea detected less frequently and uncorrelated with partial resistance against E. pisi. To test for the lack of correlation at the genetic level, an exploratory association mapping study was undertaken by statistically combining grass pea collection DS scores against both pathogens, with 5,651 previously screened genotype-by-sequencing-based single nucleotide polymorphisms (SNP). Mostly different genetic regions in grass pea were identified as being associated with the response to E. trifolii and E. pisi, anticipating an independent genetic basis that requires further validation in larger germplasm collections, with higher SNP densities. This study proposes common and unique partial resistance components against two different powdery mildews, implying the need for complementary approaches to introduce resistance to both pathogens into new grass pea varieties. The identified sources of resistance and predicted genomic targets will assist in breeding for resistance to multiple powdery mildews.

Genetic Diversity Assessment of Spanish and Some Endangered Tunisian Pea (Pisum sativum L.) Accessions Based on Microsatellite Markers (SSRs).

In the current investigation, 28 accessions of Spanish and Tunisian peas were characterized by eight SSR polymorphic markers to assess their genetic diversity. Many methods have been applied to evaluate these relationships including diversity indices, analysis of molecular variance, cluster analysis, and population structure. The means of diversity indices, the polymorphism information content (PIC), the allelic richness, and the Shannon information index were 0.51, 3.87, and 0.9, respectively. These results revealed a large polymorphism (84.15 %) which produced a higher degree of genetic distance amongst the accessions. The unweighted pair group approach with arithmetic mean divided the collection of these accessions into three major genetic clusters. Therefore, this article has clearly demonstrated the usefulness of the SSR markers that can significantly contribute to the management and conservation of pea germplasm in these countries, as well as to future reproduction.

Ovule Number and Flower Size in Pea (Pisum sativum L.): Variation, Heritability, and Correlation with Some Components of Productivity

The seed yield in pea (Pisum sativum L.) depends on numerous environmental and genotypic factors. The ongoing climate changes draw one’s attention to genetics and variation of underexplored reproductive traits. This study focuses on ovule number (ON) and flower size expressed as a length of flag petal (FL), in terms of their heritability, variability, and correlation with 1000 seed mass (TSM), seed number per pod, and seed/ovule ratio. A set of pea accessions was planted over several years in field conditions. Some of these accessions were also grown in a glasshouse. The chosen values were scored on living plants or while harvesting seeds. Nonparametric statistical methods were applied. Heritability of ON and FL was studied in five hybrid F1/F2 combinations. We found a relatively low (ca. 0.5) broad sense heritability of both ON and FL. Among other traits, TSM and ON reproduced best over the years. FL exhibited no reproducible correlations with other traits and cannot be used as a predictor of productivity. Water deficit and heat stress reduce seed yield in pea due to both seed abortion and development of fewer ovules. The differential and heritable ability to retain ON may become a basis for breeding pea cultivars adapted to changing climate.

In Vitro Screening for Salinity Tolerance in Garden Pea (Pisum sativum L.)

Soil salinity is one of the abiotic stress factors gaining importance in recent years due to the changing climate and rising temperatures. This possesses a serious risk to food security worldwide and a range of adaptations and mitigation strategies are required. Crop improvement through breeding is a possible solution to overcome salinity stress. In this respect, a study was designed to screen a collection from garden pea accessions to salinity tolerance in in vitro conditions. We analyzed the effects of four salinity levels (0, 50, 100, and 200 mM NaCl) on seed germination, seedling emergence, shoot and root lengths, and plant fresh weight in 22 garden pea genotypes. Data showed that more of the pea genotypes are able to tolerate 50 mM NaCl concentration. The increasing of salt levels to 100 and 200 mM NaCl caused a significant decrease in germination and reduced the length and weight of shoots and roots. Among the genotypes, varieties Uspeh 72, Paldin, and Flora 6 were highly sensitive to salinity stress, while varieties Prometey, Musala, and Zornitsa were distinguished as more tolerant. The results depicted that salinity treatments had a more negative effect on plant growth than on seed germination, which is probably due to the in vitro conditions in which the experiments were conducted. The studied accessions of garden pea were sensitive to salt stress; nevertheless, some tolerant accessions were identified.

Variability and assessment of interrelationships among yield and yield-related characters of pea accessions under the influence of high temperature

ABSTRACT Understanding the response and variability of pea accessions traits to high temperatures is an excellent strategy for breeding pea for heat tolerance. This research aimed to determine character variability and interrelationships among yield and yield-related traits as responses to pea grown under daily high temperature. Ninety-four pea accessions were grown under greenhouse conditions in Malaysia and Indonesia in the 2020–2021 season. During flowering up to the physiological maturity stage, the average daily maximum temperature was 31.6°C–32.4°C in Malaysia and 26.5°C–27.6°C in Indonesia. Heat stress reduced grain yield in Malaysia by 40% when the daily maximum temperature was above 32°C compared to yield in Indonesia. The number of seeds per pod, number of seeds per plant, filled pods per plant and number of ovules per pod were positively correlated with grain yield in Malaysia and Indonesia, respectively. A negative correlation was observed for percent aborted flower and aborted seed with grain yield. The genotype by trait biplot identified accessions B32, A19, G76, A11 and D44 as potentially promising under high-temperature conditions with high yield supported by plant height.

Comprehensive compositional assessment of bioactive compounds in diverse pea accessions

Pea (Pisum sativum L.) is an important legume crop providing a good source of protein, vitamins, minerals and bioactive compounds with health benefits for humans. In this study, an improved method for simultaneous analysis of multiple phytoestrogens among 100 pea accessions was developed. Ipriflavone, (a synthetic isoflavone), was used as an internal standard for the semiquantitative analysis of 17 phytoestrogens including isoflavone aglycones and conjugates, allowing direct analysis of isoflavones in their naturally occurring forms. This comprehensive dataset demonstrated that the isoflavones varied greatly and some accessions tended to have high levels of multiple phytoestrogens among the 100 accessions analyzed. Isoliquiritigenin followed by glycitein were the predominant compounds detected in the accessions and showed the highest correlation with the total phytoestrogens content. Secoisolariciresinol content was consistently higher in yellow cotyledon peas than in green cotyledon peas, whereas the contents of coumestrol, genestein and secoisolariciresinol were significantly correlated with seed coat color. The total phenolics and saponins showed a wide range of variability among the accessions with higher concentrations of total phenolics observed in seeds with pigmented seed coat or yellow cotyledon seeds, suggesting the synthesis of saponins and phenolics are significantly affected by metabolic pathway genes controlling cotyledon color or seed coat color. This study profiled the variability of bioactive compounds of pea seed quality traits in diverse pea accessions and provides an immense resource for continued research, breeding and selection of genotypes for a wide range of applications.

Aktivnost inhibitora tripsina u semenu sastrice (Lathyrus sativus L.)

Grass pea (Lathyrus sativus) is a valuable grain legume known for its high protein content and rich essential amino acid profile. Its exceptional characteristics such as drought tolerance, high adaptability to extreme conditions, disease resistance, and low cultivation inputs, make it particularly suitable for cultivation by resource-poor farmers. However, the potential use of grass pea is limited due to the presence of anti-nutritional factors, including protease inhibitors, especially trypsin inhibitor. This study aimed to develop a rapid and reliable method for measuring TI activity in seeds of grass pea and to investigate the influence of genotype and environment on trypsin inhibitor (TI) activity in seeds of grass pea. A set of 25 grass pea accessions from seven European countries was cultivated in Slovenia at the Agricultural Institute of Slovenia, and a set of 12 grass pea accessions from the Serbian gene bank was grown in Serbia at the Institute of Field and Vegetable Crops. The TI activity varied considerably among the grass pea accessions studied, with values ranging from 26.7 to 90.3 TUI/mg. To further evaluate the effects of environmental conditions on TI activity, eight grass pea accessions originating from Serbia were grown in both Slovenia and Serbia. The TI activity of the accessions grown in Slovenia ranged from 26.7 to 81.0 TUI/mg, while the activity of the accessions grown in Serbia ranged from 40.3 to 57.0 TUI/mg. The correlation of TI activity between grass pea accessions grown in Slovenia and those grown in Serbia was 0.39, with genotype diversity being the largest contributor (55.9%). This study provides a valuable insight into the variability of TI activity in grass pea and shows the possible influence of environmental conditions on this trait. However, since the data are only from a one-year field trial, further studies are needed to fully evaluate the influence of different environmental factors on TI activity.

Identification of Fungi Associated with Different Pigeon Pea Seed Accessions from Iran

A species of Leguminosae family is Cajanus cajan (L.) Millsp. (Pigeon pea) is an herbaceous, annual, self-pollinated legume pulse crop. The pigeon pea is mainly cultivated in many countries of South Asia due to its nutritious quality and a good source of minerals and protein. Seeds are a common carrier of plant pathogens, which act as the primary source for the distribution of diseases. Contaminated seeds can often create defecated germination and poor seedling vigour. They lose viability much faster than non-contaminated seeds. A large number of mycoflora were reported to be associated with the pigeon pea seeds. To assess the seed health and quality of some pigeon pea accessions this experiment was conducted at the genetic department and national plant gene bank of Iran, seed, and plant improvement institute in 2021. In the experiment, a total of 52 accessions from the pigeon pea collection were used for laboratory tests. During this study different species of Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp., and Rhizopus spp. were found along with germinating seed and seedlings of contaminated pigeon pea. The most identified species on the contaminated pigeon pea seeds were Rhizopus spp., with 50%, and the least were Alternaria spp. and Aspergillus spp., with 7.2%. The other species Penicillium spp., and Fusarium spp., were measured on 21.4% and 14.2% of the contaminated seeds respectively. On contaminated pigeon pea seeds, 20 to 100% reduction in seed germination has been measured. Paying attention to seed health is the fundamental strategy for the integrated management of the disease for durable crop production and seed conservation.

Phenotypic traits differentiating the genetic resources of pea (Pisum sativum L.) by the type of use.

The paper presents an analysis of the data obtained for pea accessions from the VIR collection studied at the Adler Experiment Station in the setting of the Krasnodar Territory in 2017-2019. It was for the f irst time that these accessions were studied for a set of phenotypic traits. The object of the study was a sample of 494 pea accessions originated from 43 countries and 18 regions and territories of the Russian Federation. The work was carried out in compliance with the methodological guidelines developed at VIR. Statistica 13.3 software was employed for statistical data processing. An assessment of four qualitative, 10 quantitative and four phenological traits in the accessions made it possible to differentiate them by the type of use, that is, as dry, forage and garden peas. The varieties differing in the type of use signif icantly differed by the values of such traits as stem length, number of pods per plant, number of nodes to the f irst f lower, number of f lowers in the inf lorescence, the maximum number of seeds per pod, pod length, and a narrower pod of forage pea compared to that of dry and garden peas. The average values of these traits were recorded for the peas with different types of use. The maximum difference was noted between garden and forage pea varieties. Dry pea varieties occupied an intermediate position. The complex of phenotypic traits identif ied determines the differences between three types of pea use, which is important when selecting the initial material for breeding appropriate varieties.

Genetic diversity of pea (Pisum sativum L.) genotypes differing in leaf type using SNP markers

A collection of 46 pea (Pisum sativum L.) accessions, mostly from Europe, were analysed for genetic diversity using the GenoPea 13.2 K SNP Array chip. Of these accessions were 24 nomal-leaved and 22 semi-leafless. Principal components analysis (PCA) separated the peas into two groups characterized by the two different leaf types, although some genotypes were exceptions and appeared in the opposite group. Cluster analysis confirmed the two groups. A dendrogram showed larger genetic distances between genotypes in the normal-leafed group compared to semi-leafless genotypes. Both PCA and cluster analysis show that the two leave types are genetically divergent. So normal-leaved peas are an interesting genetic resource, even if the breeding goal is to develop semi-leafless varieties.

Biochemical Characterization of Rare and Threatened Local Populations of Peas (Pisum sativum L.) Cultivated in the Arid Region of Southern Tunisia.

Pea (Pisum sativum L.) is an important legume for human and animal consumption, cultivated in many temperate regions of the world. In the present investigation, the seeds of twelve pea accessions collected from the arid regions of southern Tunisia, characterized by different colors of flowers and seed coats, were evaluated for their proteins, phenolic compounds and antioxidant activities. The protein content determined by the Bradford method ranged from 46.91 to 151.08 mg/g DW. The content of total phenol, total flavonoid and condensed tannin, ranging from 36.85 to 102.52 mg GAE/100 g DW, 6.95 to 48.11 mg QE/100 g DW, and 12.37 to 70.43 mg CE/100 g DW, respectively. These parameters were characterized respectively by the following three methods, the Folin-Ciocalteu reagent, the aluminum chloride and the vanillin assay. The LC-ESI/MS analysis identified the presence of 8 phenolic acids and 9 flavonoids which quinic acid, protocatechuic acid, hyperoside, and quercetin-3-O-rhamnosidewere detected as the most abundant compounds. Moreover, the antioxidant activity of pea seeds was evaluated by the ABTS+and DPPH tests. Indeed, DPPH and ABTS anti-free radical activity values ranged from 1.92 to 14.11 μmoL TEAC/g DW and 2.3 to 14.95 μmoL TEAC/g DW, respectively. The variability across the assessed traits revealed significant differences as computed by ANOVA tests. Else, the results showed significant correlations between seed coat color, seed shape, flower color and phenolic compounds. Therefore, genotypes characterized by a purple flower, brown seed coat, and wrinkled form had the highest levels of phenolic compounds and antioxidant activity. Correlation analysis revealed that phenolic compounds presented a strong positive correlation with antioxidant activities, while being negatively correlated with protein content. UPGMA cluster and Principal component analysis (PCA) revealed two main groups. This work highlights the nutritional value of P. sativum seeds as a good source of natural antioxidant compounds that may be useful in the food and pharmaceutical industries.

Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics

Complete and accurate reference genomes and annotations provide fundamental resources for functional genomics and crop breeding. Here we report a de novo assembly and annotation of a pea cultivar ZW6 with contig N50 of 8.98 Mb, which features a 243-fold increase in contig length and evident improvements in the continuity and quality of sequence in complex repeat regions compared with the existing one. Genome diversity of 118 cultivated and wild pea demonstrated that Pisum abyssinicum is a separate species different from P. fulvum and P. sativum within Pisum. Quantitative trait locus analyses uncovered two known Mendel’s genes related to stem length (Le/le) and seed shape (R/r) as well as some candidate genes for pod form studied by Mendel. A pan-genome of 116 pea accessions was constructed, and pan-genes preferred in P. abyssinicum and P. fulvum showed distinct functional enrichment, indicating the potential value of them as pea breeding resources in the future.

Large-Scale Heat-Tolerance Screening and Genetic Diversity of Pea (Pisum sativum L.) Germplasms.

Pea (Pisum sativum L.) is an important legume crop. However, the yield of pea is adversely affected by heat stress in China. In this study, heat-tolerant germplasms were screened and evaluated in the field under multi-conditions. The results showed that heat stress could significantly affect pea yield. On the basis of grain weight per plant, 257 heat-tolerant and 175 heat-sensitive accessions were obtained from the first year’s screening, and 26 extremely heat-tolerant and 19 extremely heat-sensitive accessions were finally obtained in this study. Based on SNaPshot technology, two sets of SNP markers, including 46 neutral and 20 heat-tolerance-related markers, were used to evaluate the genetic diversity and population genetic structure of the 432 pea accessions obtained from the first year’s screening. Genetic diversity analysis showed that the average polymorphic information content was lower using heat-tolerance-related markers than neutral markers because of the selective pressure under heat stress. In addition, population genetic structure analysis showed that neutral markers divided the 432 pea accessions into two subpopulations associated with sowing date type and geographical origin, while the heat-tolerance-related markers divided these germplasms into two subpopulations associated with heat tolerance and sowing date type. Overall, we present a comprehensive resource of heat-tolerant and heat-sensitive pea accessions through heat-tolerance screenings in multi-conditions, which could help genetic improvements of pea in the future.

Identification of resistance sources against Fusarium udum (Race-2) in wild accessions of pigeon pea for strengthening the pre-breeding program

Identification of resistance sources against Fusarium udum (Race-2) in wild accessions of pigeon pea for strengthening the pre-breeding program