Month: January 2025

All sensor signals were logged at 20min intervals and averaged every hour. The results presented are the averages of four replications. Xylem sap uptake into the fruit was determined based on the daily average xylem sap flow measurements from 15 and 30 DAP.Extractions were accomplished at the end of the irrigation cycle, before 08:00h. Soil solution was extracted by adding 450ml of the nutrient solution, without Ca2+, to each plant pot, and collecting the drained leachate. After collecting the soil leachate, plants were decapitated 15 cm above the soil level and the pots containing the roots plus stem stump were used to extract the stem xylem sap. Two fruit and two middle leaves were harvested at the end of the irrigation cycle from each replicate plant with the entire peduncle and pedicel attached and kept in a sealed plastic bag for xylem sap extraction. Xylem sap extraction was accomplished by placing the plant pot, fruit, or leaf blade inside a pressure chamber , while the cut end of the stem, peduncle, or pedicel was exposed to the outside of the chamber through a seal. After sealing, the pressure inside the chamber was increased up to 0.8MPa with N2. The initial xylem sap moving out of the stem, peduncle, or pedicel cut end was blotted dry to reduce Ca2+ contamination from the cut. The following 100 μl was collected over a period of 5min and used to determine the Ca2+ concentration in the xylem sap . The plant pots were pressurized in a custom-built chamber large enough to hold a 9.5 litre pot, with a two-part lid in order to allow assembly around the stem of an intact plant .

The custom-built chamber was also used to pressurize the roots of whole plants to induce guttation on leaf blades, growing bags which were collected for Ca2+ quantification. The guttation samples represent the xylem sap extracted from leaf blades without any contamination from a cut surface. Apoplastic water-soluble Ca2+ was extracted from the blossom-end pericarp tissue of tomato fruit as previously described by De Freitas et al. .The Ca2+ concentration in the soil solution, xylem sap, and apoplastic solution was determined with an Ultra-M micro Ca2+-selective electrode . A standard Ca2+ calibration curve was used to determine the Ca2+ concentration in the samples. The Ca2+ concentration in leaf and fruit tissues was determined in freeze-dried leaf blades, as well as pericarp tissues manually cut from the peduncle and blossom regions of the fruit. Freeze-dried samples were subjected to microwave acid digestion and analysed by inductively coupled plasma atomic emission spectrometry . Calcium accumulation was quantified by subtracting the total middle leaf and fruit Ca2+ contents observed at 15 DAP from the total middle leaf and fruit Ca2+ contents observed at 30 DAP. Calcium accumulation was also estimated by multiplying the quantified xylem sap Ca2+ concentration observed in the middle leaf pedicel and fruit peduncle by its respective daily average xylem sap flow rate observed at 15 and 30 DAP.BER was completely suppressed by spraying the whole plants weekly with ABA during fruit growth and development, compared with water-sprayed fruit that reached a 36% incidence of BER at 30 DAP . Dipping the fruit in solutions containing ABA prevented BER development at 15 DAP, but ABA-dipped fruit reached a 16% incidence of BER at 30 DAP. Control fruit dipped in water had a 39% incidence of BER at 30 DAP. The electrolyte leakage of fruit pericarp tissue was lower in response to whole-plant and fruit-specific ABA treatments at 15 DAP . At 30 DAP, only the whole-plant ABA treatment had lower electrolyte leakage in fruit pericarp tissue. SWP was less negative in response to whole-plant ABA treatment at 15 and 30 DAP compared with all other treatments .

Leaf stomatal conductance progressively increased from the base, middle, to the top regions of the plant, and was lower in the whole-plant ABA treatment at 15 and 30 DAP compared with all other treatments . Whole-plant water spraying, as well as water and ABA dip treatments, had similar stomatal conductance for the basal, middle, and top leaves. Based on the stomatal conductance analysis, the plant ABA uptake was considered high with whole-plant ABA treatment, and no significant ABA movement from the fruit into the plant was observed based on changes in stomatal conductance in response to fruit-specific ABA dip treatment . Plant water loss was reduced by the whole-plant ABA treatment at 15 and 30 DAP , but was similar in all other treatments, including the ABA fruit dip. Whole-plantat 15 and 30 DAP . The water spray treatment had the highest sap flow during most of the daylight period at 15 and 30 DAP . The average flow of xylem sap moving into the fruit during a 24h irrigation cycle was substantially higher on plants sprayed with ABA, compared with all other treatments , with the same diurnal pattern as seen in the leaves . At 15 DAP, fruit on water-sprayed plants, as well as water- and ABA-dipped fruit had a reverse flow of xylemic sap from the fruit back to the plant, starting in the late afternoon until the next irrigation cycle in the morning . The ABA-dipped treatment had a slightly higher sap flow to the fruit than the two water treatments at 15 and at 30 DAP . Fruit on plants sprayed with ABA had no reverse xylemic sap flow throughout the irrigation cycle at 15 DAP, but had the same diurnal pattern, with the lowest flows occurring during the night period . The diurnal pattern of fruit xylem flow at 30 DAP was similar to that at 15 DAP, but the magnitude was substantially reduced, again with no discernible reverse flow . Spraying tomato plants with ABA resulted in higher total fruit water uptake used for growth and lower fruit water uptake through the phloem from 15 to 30 DAP .

The estimated phloem sap solute concentration uptake into the fruit from 15 to 30 DAP was higher in ABA-sprayed plants than in non-sprayed plants .The Ca2+ concentrations in the soil solution and in the main stem xylem sap were similar among all treatments at 15 and 30 DAP. The average Ca2+ concentration in the soil solution among treatments was 1.41±0.09 mM at 15 DAP and 1.08±0.14 mM at 30 DAP. The average Ca2+ concentration in the main stem xylem sap was 0.72±0.04 mM at 15 DAP and 0.63±0.05 mM at 30 DAP. There was no statistical difference among treatments in Ca2+ concentrations in the xylem sap of basal, middle, or top leaves at 15 or 30 DAP. The same results were obtained when Ca2+ was determined on an independent set of plants under the same treatments using the leaf guttation method . The average xylem sap Ca2+ concentrations were 0.71±0.06 mM and 0.86±0.02 mM in top leaves, 0.77±0.03 mM and 0.87±0.02 mM in middle leaves, and 0.64±0.03 mM and 0.81±0.02 mM in basal leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in the peduncle xylem sap was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, nursery grow bag compared with all other treatments , and water-soluble apoplastic Ca2+ was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, compared with all other treatments . Fruit dipped in ABA solution had slightly higher water-soluble apoplastic Ca2+ than fruit dipped in water and fruit from plants sprayed with water at 15 DAP. The Ca2+ concentration in the top and middle leaves was statistically lower in response to whole-plant ABA treatment compared with all other treatments at 15 and 30 DAP . The Ca2+ concentrations in ABA-sprayed plants were 8.7±0.21 and 8.1±0.09mg g DW–1 in top leaves and 17.5±0.52 and 16.1±0.63mg g DW–1 in middle leaves at 15 and 30 DAP, respectively. The Ca2+ concentrations in all other non-ABA-sprayed plants were 13.0±0.36 and 13.0±0.15mg g DW–1 in top leaves and 25.1±0.96 and 23.9±0.81mg g DW–1 in middle leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in basal leaves was similar in all treatments at 15 DAP , and statistically lower in plants sprayed with ABA than all other treatments at 30 DAP. The Ca2+ concentration in fruit tissue collected at thepeduncle and blossom ends of the fruit was higher in ABAsprayed plants at 15 and 30 DAP . Fruit dipped in ABA had a higher Ca2+ concentration at the blossom-end tissue at 15 DAP, compared with water-dipped fruit and fruit of water-sprayed plants . Ca2+ accumulation was lower in the leaf and higher in the fruit of ABA-sprayed plants than in the other treated plants and fruit from 15 to 30 DAP . Ca2+ accumulation in leaf and fruit quantified by tissue analysis was similar to the estimated Ca2+ accumulation based on the Ca2+ concentration in the xylem sap and xylem sap flow rates into leaf and fruit tissues . The average relative humidity and air temperature from 15 to 30 DAP inside the greenhouse, where the tomato plants were grown, oscillated from 58.2% and 27.8 °C during the day up to 77.8% and 18.2 °C during the night, respectively . The VPD increased from 0.5 kPa at 05:30h to 1.6 kPa at 14:30h, decreasing thereafter . The number of Safranin-O-stained vascular bundles in the placenta and pericarp tissues at the peduncle and blossomend regions of the fruit was higher in response to whole-plant and fruit-specific ABA treatments at 15 DAP . The number of stained vascular bundles decreased in all treatments from 15 to 30 DAP, and all treatments showed a similar number of stained vascular bundles in the placenta and pericarp tissues at the peduncle and blossom-end regions of the fruit at 30 DAP . The fruit growth rate was higher in ABA-sprayed plants compared with all other treatments at 15 and 30 DAP . All treatments showed a positive fruit growth rate during a 24h period at 15 and 30 DAP . For all treatments, the fruit growth rate was higher at 15 DAP than at 30 DAP . The average fruit weight was also higher in ABA-sprayed plants at 15 and 30 DAP . Fruit Ca2+ uptake, both directly quantified and estimated based on the product of fruit xylem sap uptake and fruit peduncle xylem sap Ca2+ concentration, was 6-fold higher in ABA-sprayed plants compared with water-sprayed controls . A much smaller increase in Ca2+ uptake was found in ABA-dipped fruit, but, again, this was consistent for both directly quantified and estimated values . The sprayed and dipped ABA/water ratios for fruit growth rate were 1.41 and 1.15, respectively .Previous studies showed that weekly spraying of tomato plants with ABA prevented BER development in the fruit, while water-sprayed plants reached a 30–45% incidence of BER at 40–45 DAP . At that time, possible mechanisms through which ABA increased fruit Ca2+ concentration and reduced fruit susceptibility to BER were suggested based on estimations of fruit xylem sap uptake and Ca2+ concentration in the xylem sap .Water uptake in leaves comes exclusively from xylem vessels, while water uptake into the fruit comes from both phloem and xylem vascular tissues . Treating the whole plant with ABA reduced stomatal conductance, which resulted in lower plant water loss, lower soil water uptake and xylemic water movement into the leaves, as well as higher SWP and increased xylemic water movement into the fruit. Considering that Ca2+ concentrations in the soil solution and stem xylem sap were similar among all treatments, the observed lower Ca2+ accumulation in ABA-sprayed plants was due to lower soil solution uptake triggered by lower leaf transpiration rates . Our results also estimate a higher solute concentration in the phloem sap moving into the fruit of ABA-sprayed plants . Although ABA reduced stomatal conductance and this would be expected to decrease leaf photosynthesis , the improved plant water status associated with ABA application may have caused compensatory physiological effects in other areas, such as reduced carbon partitioning to roots and/or improved carbon transport rates, resulting in higher solute concentration in the phloem sap, compared with the other treatments. The non-ABA-sprayed plants had an average fruit phloem sap uptake of 1.04ml fruit–1 d–1 and an average phloem sap solute concentration of 144.3mg ml–1 .

Fruit ripening has been studied for decades, yet there are still many unanswered questions about the timing and coordination of the biological processes related to this developmental program. Much of this research has been done in the model for fleshy fruit ripening, tomato , and has utilized the spontaneous single ripening mutants Cnr , nor , and rin . Each of these mutations produces pleiotropic defects to ripening and occur in or near genes encoding the transcription factors SPL-CNR, NAC-NOR, and MADS-RIN, belonging to the SQUAMOSA promoter binding protein-like , NAM, ATAF1/2, CUC2 and, MCM1, AG, DEF, SRF TF families, respectively. Each TF family functions in diverse developmental processes and have distinct spatiotemporal expression patterns . These mutants were used to study ripening under the assumption that the mutations cause a complete loss of function to the corresponding protein. Recently, it has been discovered that the nor and rin mutations produce proteins that are still functional and gain the ability to negatively regulate their targets . In nor, the two base pair deletion truncates the protein but still produces a functional DNA-binding and dimerizing NAC domain . In rin, a large deletion creates a chimeric protein with the neighboring gene MACROCALYX , producing a functional protein with suppression activity . The Cnr mutation is also thought to be a gain of function mutation, although the mechanism has yet to be understood . The Cnr mutation results from hypermethylation upstream of the gene near the promoter and has been shown to inhibit the genome-wide demethylation cascade associated with normal tomato ripening .

Previously, these TFs were regarded as master regulators of ripening; however, nursery pots given the new information about the nature of the mutations in Cnr, nor, and rin, it is less clear the precise roles the TFs are playing in ripening . The nor and rin mutants have been utilized in breeding for developing tomato hybrids with extended shelf life or extended field harvest depending on their purpose for the fresh market and processing tomato industries . Hybrids between elite varieties and the ripening mutants have a delayed ripening progression, but with the tradeoff of decreased fruit quality attributes, such as color, taste, and aroma . Although there are some publications dedicated to evaluating the physiological characteristics of mutant or hybrid fruit , up to this point, much of what we know about the ripening mu-tations is based on controlled greenhouse experiments with limited fruit and few ripening stages examined. A complete dataset of phenotypic data produced from large-scale field trials evaluating fruit ripening and senescence is lacking to provide information relevant to breeding, particularly in the new context of the molecular mechanisms behind the nor and rin mutations. The Cnr mutant provides a unique opportunity to study the role of epigenetics in fruit ripening but is not used in breeding because the mutant phenotype is dominant. It has been suggested that Cnr fruit undergo normal growth and development ; however, fruit appear different from wild type even before ripening, with a smaller size, alterations in cell wall enzyme expression, and earlier chlorophyll degradation . To better utilize Cnr as a tool for studying fruit development and ripening, a broader understanding of the physiological and transcriptomic alterations in this mutant is necessary. These spontaneous single mutants need to be reevaluated as tools to understand the wide-ranging biological processes regulated by each TF.

Previous literature has generally assumed that the mutations block ripening, resulting in similar processes affected . This study demonstrates that each mutant has a unique ripening phenotype, resulting from a combination of inhibited and delayed developmental processes. We integrated phenotypic data with gene expression data and hormone measurements in the Cnr, nor, and rin mutants across ripening and senescence to characterize the extent and timing of the ripening defects. Tomatoes grown under field conditions were assessed for fruit traits over multiple seasons. We then performed a transcriptomic analysis to gain more definition of the timing in which mutant fruit deviated from WT in their development and to determine specific molecular functions altered in each mutant. Due to their pivotal role in regulating ripening, we focused on defects in hormone networks, including biosynthesis and accumulation. We analyzed the influence of each mutation on the expression of the other TF throughout ripening and senescence. Finally, to better understand the combined genetic effects of the mutants on fruit ripening, we generated homozygous double mutants of Cnr, nor, and rin and used phenotyping and transcriptional data to evaluate the relationships between the mutants.Tomato plants of c.v. ‘Alisa Craig’ and the isogenic ripening mutants Cnr, nor, and rin were grown in randomized plots under standard field conditions in Davis, CA, United States, during the 2016, 2017, 2018, and 2020 seasons. Fruit tagged at 10 days post-anthesis , which corresponds to 7 mm in fruit diameter, were harvested at stages equivalent to the WT fruit. Fruit were sampled at the mature green , turning , red ripe , and overripe stages, corresponding to 37, 45, 50, and 57 dpa, respectively. The term “RR” is used throughout the manuscript to refer to the 50 dpa stage of all genotypes, even when the mutant fruit do not turn red. Fruit stages for each of the mutants were further validated by external color analysis . Double mutant fruit were generated through reciprocal crosses: Cnr × nor, nor × Cnr, Cnr × rin, rin × Cnr, nor × rin, and rin × nor. Fruit were selfed after the initial cross to generate an F2 segregating generation. The double mutants were initially selected in the F2 generation through genotyping and phenotyping. At least two additional generations after F2 were obtained through selfing to ensure the stability of the double mutations and to perform the experiments in this study.

Three seasons of data were collected for the Cnr/nor fruit while only one season of data was collected for the rin/nor and Cnr/rin crosses.The mutant lines were genotyped for their respective mutations. For nor, the Phire Plant Direct PCR Kit was used to extract DNA and amplify the region of the gene containing the 2 bp mutation using the primers listed inSupplementary Table 1 . The PCRs were run on a SimpliAmp Thermal Cycler with the following conditions denaturation: 99°C for 5 min; 35 cycles of 98°C for 5 s, 56°C for 25 s, and 72°C for 25 s; with a final extension of 72°C for 1 min. The PCR products were purified using Wizard SV Gel and PCR Clean-Up System and then sequenced with Sanger technology to confirm the absence of the two nucleotides. For rin, the Phire Plant Direct PCR Kit was used to extract DNA and perform end-point PCRs using primers specific for the mutant and WT alleles . The following PCR conditions were used for the WT allele primers: denaturation 99°C for 5 min; 35 cycles of 98°C for 5 s, 55°C for 25 s, and 72°C for 25 s; with a final extension of 72°C for 1 min. The PCR conditions for the mutant allele primers were: denaturation 98°C for 5 min; 40 cycles of 98°C for 5 s, 58°C for 25 s, and 72°C for 25 s; with a final extension of 72°C for 1 min. The PCR products were visualized as bands using a 1 The Cnr epimutation was genotyped by bisulfite sequencing. Extracted DNA was treated with the Zymo Gold bisulfite kit . Bisulfite treated-DNA was PCR amplified for the CNR promoter region containing the methylation changes using the primers listed in Supplementary Table 1 . The following PCR conditions were used: 94°C for 2 min; 40 cycles of 94°C for 30 s, 54°C for 30 s, and 60°C for 45 s, plastic planters and a final extension of 60°C for 10 min. The PCR products were then Sanger sequenced and compared to the same region amplified in untreated controls with primers . The following conditions were used to amplify the untreated DNA: 95°C for 2 min; 35 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 1 min, and a final extension of 72°C for 10 min. To ensure mutants were homozygous for the locus, we confirmed the double mutants by allowing the plants to self for at least two additional generations and checking that the progeny were not segregating for any fruitphenotypes.We determined DEGs from the MG and RR stages to identify specific molecular functions altered in Cnr, nor, and rin fruit. First, we compared the ripening mutants to the WT at each stage and obtained a total of 16,085 mutation-related DEGs across all comparisons . Like the PCA suggested , Cnr MG fruit presented the largest amount of mutation-related DEGs , while nor and rin MG had considerably fewer DEGs when compared to the WT counterpart . At the RR stage, large differences between each mutant and WT were observed, with Cnr RR fruit displaying once again the largest differences in the amount of mutation-related DEGs . The large number of mutation related DEGs shown by Cnr fruit further supports our hypothesis that the Cnr mutation more broadly affects fruit development and that nor and rin appear to be more ripeningspecific mutations. We examined molecular functions based on KEGG annotations that were significantly enriched among the mutation-related DEGs for each Cnr, nor, and rin fruit at MG and RR . Large differences in enriched functions were detected in the Cnr MG fruit, which mainly corresponded to alterations in carbohydrate and amino acid metabolism, chlorophyll, and carotenoid biosynthesis, and interestingly many processes related to DNA replication and repair. The lack of green color in Cnr MG fruit could be explained by lower expression of photosynthesis and carbon fixation genes.

The nor MG and rin MG fruit showed few alterations compared to WT and were mainly noted in amino acid metabolism and plant hormone signal transduction. In contrast, at the RR stage, the three ripening mutants showed significant alterations across multiple molecular pathways that range from primary and secondary metabolism to transcription, translation, and signaling processes. We proceeded to mine the mutation-related DEGs for key genes known to affect the fruit traits evaluated in the ripening mutants: color, firmness, TSS, and acidity. We selected five carotenoid biosynthesis genes involved in fruit pigmentation, six genes encodingcell wall degrading enzymes that promote fruit softening, four genes related to sugar accumulation and transport that impact the fruit’s TSS, and one gene that regulates the levels of citric acid then affecting the fruit’s acidity . At the MG stage, we observed that Cnr fruit showed significantly lower expression than WT for several of these key genes, consistent with our phenotypic data , including firmness related enzymes and carotenoid biosynthesis genes. MG fruit from the three ripening mutants showed significantly lower gene expression in an important invertase in fruit , which may contribute to the lower levels of TSS observed in all the mutants. At the RR stage, most of the fruit trait-associated genes surveyed in the ripening mutants had a significantly lower expression than WT, in support of the phenotypic data and reinforced by the numerous functional enrichments among the mutation-related DEGs . The critical carotenoid biosynthesis gene that encodes PHYTOENE SYNTHASE 1 was significantly lower expressed than WT in the mutant fruit across all stages, accounting for the lack of red pigmentation at the RR stage. Also, downstream genes in the pathway encoding Lycopene β-cyclases were highly expressed in the mutants at the RR stage, suggesting that not only was less lycopene being produced but more was being metabolized. CWDEs were negatively affected across all genotypes, with Cnr having the most mutation-related DEGs in this category. We were interested in examining if the Cnr, nor, and rin mutant fruit displayed altered ripening progression or if they were completely inhibited or delayed in ripening events. We performed another set of differential expression analyses comparing RR against MG fruit for WT and each of the mutants to reveal ripening-related DEGs. As anticipated, WT had the largest number of ripening-related DEGs , while nor showed almost no change between the two ripening stages with only 89 DEGs detected . Cnr and rin had fewer ripening-related DEGs compared to WT but still exhibited significant changes during the transition between stages with 5,788 and 2,799 DEGs, respectively. Although Cnr showed the most differences from WT in mutation-related DEGs , it had the largest number of ripening-related DEGs in common with WT fruit .

When combined with the state’s $10.4B value of milk production, alfalfa accounts for a significant portion of California’s agricultural GDP. In addition to its advantages as a forage, alfalfa also provides a host of beneficial ecosystem services: as a legume, alfalfa requires no nitrogen fertiliser, instead it fixes atmospheric nitrogen through a symbiotic relationship with rhizobia, a nitrogen fixing bacterium; the perennial nature of alfalfa improves soil health allowing fields to recover from frequent tilling and prevents topsoil loss; long roots can access water and nutrients deep in the soil profile and increase soil organic matter throughout; and alfalfa is an important insectary, hosting a diversity of beneficial insects . Yield is the most important trait for profitable alfalfa production, yet somewhat inexplicably, yield improvement in alfalfa has stalled over the last ~30 years . In addition to the lack of yield improvement and despite the significant economic and environmental benefits of alfalfa, the area cultivated has been in steady decline both nationally and in California since a peak in 1960 . Already disadvantaged by the distribution of federal subsidies, of which commodity row crops receive billions each year, alfalfa faces stiff competition for inclusion in crop rotations. Increasing yield is therefore imperative to curb the rate of decline in alfalfa area, not only to continue to support California’s significant livestock industry, but also for the benefits alfalfa provides to agricultural ecosystems. Historically, breeders have relied on traditional breeding methods to increase biomass yield in alfalfa, with little success in recent years . However, black flower buckets with the advent of new breeding technologies, such as genomic selection and high throughput phenotyping, this lack of yield gain may be approached in a new light.

This PhD project investigates the incorporation of modern breeding tools and methodologies into an existing breeding program to address the lack of yield improvement in alfalfa.Alfalfa is believed to have two centers of origin, Asia Minor/Caucasia and Central Asia . Following domestication, alfalfa quickly spread throughout the ancient world due to its adaptability to diverse climates and soils. The expansion of civilizations and the Silk Road trade routes played a pivotal role in the dissemination of alfalfa, enabling its introduction to regions such as Europe, North Africa, and eventually the Americas. Alfalfa exists naturally as the Medicago sativa complex that includes a range of diploid and autotetraploid subspecific taxa . Cultivated alfalfa primarily refers to the subspecies sativa and is an autotetraploid , perennial, outcrossing legume with polysomic inheritance . It has a basic chromosome number of eight and a genome size of 800-1000 Mb. Commercial alfalfa stands typically last three to eight years depending on variety, soil, climate, and cultural practices. Pure stands are sown at high density with typical sowing rates ranging from 16-22 kg ha-1 . Plant density starts high with up to 800 plants m-2 three months after planting and then steadily declines. In established alfalfa stands, plant density has limited effect on dry matter yield per hectare until plant numbers fall below 40 plants m-2 , wherein yields decrease and growers should consider retiring the stand. Alfalfa can grow to heights above one metre and has a deep root system that reaches beyond six metres in depth when grown in deep, well drained, moist soils . Alfalfa plants generally have a single deep taproot, with variation in the number and size of lateral roots. Following establishment, alfalfa forms a crown at the top of the root system. After defoliation, alfalfa regrowth occurs from buds located on the crown and from axillary buds at nodes from the remaining above ground stubble . This regrowth cycle allows for multiple cuttings during the growing season for up to eight years before forage yields decline below economic thresholds.

Fall dormancy is an important characteristic of alfalfa that defines a population’s fitness for specific agricultural environments. Fall dormancy is a plant’s response to decreasing photoperiod and temperature and is associated with a slowing and eventual cessation of growth through the dormant period . This trait is closely related to the ability for populations to avoid winter kill and is under complex quantitative genetic control . Alfalfa varieties are classified into different fall dormancy groups, typically ranging from 1 to 11, with lower numbers referring to dormant varieties and higher numbers representing reduced fall dormancy. Non-dormant varieties tend to be higher yielding, but may be lower quality and less persistent, therefore appropriate variety selection is of key concern to the grower.Californian agricultural regions have a broad range of climates and soils and can be divided into five main growing regions: the Central Valley, Intermountain, Low Desert, High Desert, and Coastal regions . Statewide average dry matter yields for alfalfa are 15-17 MG ha-1 from an average of 6-7 harvests per year although this varies with up to 12 cuts on stands grown in the Low Desert and as few as 3 on fields in the Intermountain region . The Central Valley is the most significant area accounting for 70 percent of the state’s alfalfa production . It has a Mediterranean climate characterized by hot, dry summers and cool winters . Rainfall totals range from 20-46 cm annually, falling predominantly in the cooler months from November through to March. The Central Valley has fertile, deep, alluvial soils, although some areas suffer from high salinity. Varieties grown in the Central Valley are predominantly semi-dormant to nondormant however, in the northern tip of the valley dormant varieties are grown for better persistence in heavy soils and greater forage quality. The Low Desert region in Southern California contains 17 percent of alfalfa production.

An area with extremely low rainfall and high temperatures . Soils are generally heavy and like in the Central Valley, high salinity is a significant issue. The Low Desert is also an important alfalfa seed production area. The final area of significance for alfalfa production is the Intermountain region in Northern California accounting for approximately 10 percent of production . This area is more temperate, located at high elevation with warm summers and cool winters . Rainfall averages about 51 cm per year falling in the cooler months. Freezing winters necessitate the use of dormant cultivars to prevent winter kill. Almost all the alfalfa grown in California is irrigated. Check-flood surface irrigation is the most common in the Central Valley and Low Desert regions, while sprinklers are the preferred method in the Intermountain area. Although a significant crop in California, the area of land cultivated in alfalfa has been in steady decline since a peak in 1960. Over the last 12 years hectarage has decreased by more than 40 percent from 390,000 ha in 2011 to 235,000 ha in 2022 . The predominant end-use of alfalfa grown in California is hay for dairy cattle. California surpassed Wisconsin as the number-one dairy state in 1993 and now produces more than 21 percent of milk in the United States . At least 75 percent of alfalfa grown in California is used to supply the dairy industry.The breeding goals for alfalfa are characteristic of most plant improvement programs. They include increasing yield, enhancing nutritive value, and improving biotic and abiotic stress tolerance . The majority of desired traits are complex and quantitatively inherited; however, some pest and disease resistance mechanisms are likely under simple genetic control.Alfalfa breeding programs are based on recurrent phenotypic selection, with or without progeny testing. They are designed to increase the frequency of desirable alleles for quantitatively inherited traits, while maintaining genetic variability for continued genetic improvement . Although self-fertilization is common, alfalfa suffers from severe inbreeding depression which is prohibitive to the production of hybrids, french flower buckets thus commercial cultivars are marketed as synthetic populations generated by crossing different numbers of selected genotypes . As a consequence of the structure of the alfalfa genome, cross pollination and severe inbreeding depression, cultivars exhibit high levels of genetic variation . Significant gains have been made in most traits of interest in alfalfa. Forage quality has improved, demonstrated by the release of new high-quality cultivars . Current cultivars exhibit resistance to a suite of pests and diseases including bacterial wilt , Verticillium wilt , Fusarium wilt , anthracnose , Phytophthora root rot , Aphanomyces root rot , root-knot nematodes , stem nematode , spotted alfalfa aphid , pea aphid , and others . However, there has been little to no improvement in yield, for which a variety of explanations have been proposed. Perennial forage breeders are at a disadvantage compared to those working with annual grain crops when it comes to yield improvement due in part to its perennial nature requiring multiple years of evaluation before selection can be made, the negative genetic correlation between forage quality and forage yield, and the inability to make gains in the harvest index that is possible in grain crops, as all above ground biomass is harvested . A lack of forage breeders and limited resources constrain the size and scope of breeding trials, reducing their efficacy in improving traits with low heritability, such as yield.

The emphasis by alfalfa breeders on pest and disease resistance and greater persistence may lead to a realization of yield potential but it is not increasing yield per se. Perhaps the most important reason for the lack of yield improvement in alfalfa is that breeders have not been explicitly selecting for increased yield potential under commercial production conditions. Yield is often selected indirectly based on evaluation of vigor on spaced plants or on short family rows rather than on measurements of yield on plots grown as a densely sown sward . Although little can be done to address issues such as the inability to increase the harvest index of alfalfa, we do have the ability to modify our trial methodology and to utilize modern technology such as genomic selection and high throughput phenotyping to improve yield potential in alfalfa. Measuring yield on plots instead of individual plants provides a better proxy of commercial production systems, and the incorporation of remote sensing, high throughput phenotyping and genomic selection allow for better allocation of resources within a breeding program and can help to speed up the rate of genetic gain through shorter selection cycles.Genomic selection is a form of marker-assisted selection where the breeding value for a given trait of a genotype can be estimated from many markers distributed across the entire genome . MAS is based upon the establishment of a tight linkage between a molecular marker and the chromosomal location of the gene governing the trait to be selected in a particular environment . This is useful when working with traits controlled by a few large-effect loci, however this is not the case for many important traits in plant breeding, including biomass yield. GS expands on this idea and guides selection based on the cumulative impact of many small-effect loci that are in linkage disequilibrium with genetic markers . To estimate breeding values for a genotype, first a model must be developed from a training population that exhibits variation for the trait of interest. This training population is both genotyped and phenotyped to develop a model which optimizes the chromosomal pattern of alleles. This model then allows breeders to estimate the genetic potential of unobserved genotypes based solely on marker information. Because it relies only on a plants genotype, which can be determined when the plant is still very young, genomic selection allows significantly shorter selection cycles than are required under recurrent phenotypic selection, thus has the potential to significantly increase the rate of genetic gain in an alfalfa breeding program. Early methods of GS in plant breeding used gene chip arrays and were mostly adopted by well-resourced breeding programs in major crop species . As high-throughput sequencing costs have decreased, sequencing based approaches have become more viable and GS is now being explored in a wide range of crops. Genotyping-by-sequencing is currently the most popular reduced-representation approach for genomic selection . It is a reduced-representation approach that uses restriction enzymes to cut the genome at fixed points to show good overall coverage. Samples are sequenced using high throughput next-generation sequencing platforms. A variety of software programs can then be used for genotype calling and identification of polymorphisms for downstream analysis . Because genome coverage is incomplete, causal loci are unlikely to be identified, but the polymorphisms are likely to be in linkage disequilibrium with loci that are causal.