Soybean Breeding Aiming at increasing Productivity and Root-Knot Nematode Resistance

In Brazil, the root-knot nematode (Pratylenchus brachyurus) has gained importance, whatever because of the damage caused to soybean crops or because of its broad dispersion and incidences in producing areas. Therefore, this invention aimed at developing a new cultivar resistant to the major soybean diseases as well as to the root-knot nematode. As a result, we developed a soybean cultivar designated UFUS 8301. Generations were advanced by the single seed descent method. Value for Cultivation and Use assays were carried out during a 3-year period (2010/13). Distinctness, uniformity, and stability experiments were carried out during a 2-year period (2011/13). We used the reproduction factor (RF) statistics to assess damage and reproductive potentials of P. Brachyurus; analysis of variance tested differences between means. We accepted the null hypothesis there was no difference between UFUS 8301 and the parameter of resistance Crotalaria spectabilis. UFUS 8301 was found distinct from any other cultivar, homogeneous to the descriptors that had identified it and stable through generations. UFUS 8301 presented 19% oil and 39% protein on the seeds, and yield (3687.5 kg ha-1) above Brazilian national average.

Brazilian soybean-breeding programs have been able to increase agricultural diversification with new well-adapted crops, bringing new lucrative opportunities for farmers. The Ministry of Agriculture is responsible for both protection and registration of new cultivars. However, to be produced and marketed in the country, it is previously required to be included in the National Register of Cultivars, regardless of whether the new variety is protected or not.
Value for Cultivation and Use (VCU) and Distinctness, Uniformity, and Stability (DUS) tests are mandatory as part of the application process to register, protect and add a new soybean cultivar on the National Register of Cultivars. VCU assays are established to assess differences in productivity, biological and chemical features and technological characteristics, resistance to pests and diseases, and other commercially important traits. These assays must be carried out for a minimum two years to compare the performance of candidate varieties with varieties already on the National Register of Cultivars. Also, The DUS assays are performed to provide evidence that the candidate cultivar subject to protection is distinct from other(s) whose descriptors are well known, also is homogeneous within generations and stable to the same traits over successive generations.
In general, soybean genetic improvement is a process of production of the variability of desired traits intending selection of superior genotypes and seeds multiplication for commercial purposes. Several selection methods have been used to identify progenies who possess the most useful combination of wanted features. Most often, these include pedigree selection, by visual screening of the bestappearing families in each generation; single-seed descent, by advancing one seed or pod from each plant the next generation to develop homozygous lines; and bulk breeding, where a population is advanced in bulk until later generations with no artificial selection, when nearly homozygous lines are selected for yield assay [3].
In the soybean-breeding program at the Federal University of Uberlândia, to develop any new cultivar, we first define our goals according to problems and weaknesses of the current germplasm. In this context, the root nematode Pratylenchus brachyurus has gained importance in Brazil, whatever because of damage caused to soybean crops or for its broad dispersion and high incidence in producing areas, such as Cerrado.
A root-knot nematode survey in the north of Mato Grosso, in the 2016/2017 harvest and 2017-second crop, has pointed to the increasing of nematode populations numbers in all municipalities; all of 3328 samples analyzed were found positive for Pratylenchus sp. presence [4]. P. brachyurus, like many other plantparasitic nematodes, are microscopic worms that can be damaging to many crops. Across the world, many billions of dollars have been lost due to the damaging abilities of nematodes in cash crops [5]. In the Brazilian soybean fields, P. brachyurus has become increasingly common. Lima et al. [6] assessed P. brachyurus populations throughout Tocantins state in fields grown off-season and found this pathogen on 82% of samples, with densities ranging from 23 to 20,400 nematodes per 200 cm 3 soil, or 10 g root samples.
Therefore, our goal with this invention was developing a soybean cultivar able to meet producers need as resistance against major soybean diseases, including to the root-knot nematode, in addition to high oil and protein contents along with high grain yield. As a result, we developed a new cultivar designated UFUS 8301, which presents comparative advantages over others in high productivity and P. brachyurus resistance.

II.
MATERIALS AND METHODS UFUS 8301 was derived from the crossing between IAC-8.2 x MG / BR 46 (Conquista) on the Capim Branco farm, located in Uberlândia, MG, Brazil; Latitude 18º 52' 94'' S, Longitude 48º 20' 45'' O, Altitude 835 m. Generations were advanced by the single seed descent (SSD) method. In this approach, each generation has only one single seed randomly selected from each plant, which is bulked to grow the next generation. In general, this procedure refers to planting a segregating population, harvesting a sample of one seed per plant, and using this one-seed sample to produce the next generation. Other methods as single-pod descent (SPD) and bulk methods (BM) produce redundant inbred lines that are descended from either the same F2 or F3 plant. However, single seed descent (SSD) has the advantage of minimizes the amount of genetic variability, although SSD method requires more time than SPD and BM [7].
Manual crosses produced hybrid seeds (F1). The F1s were grown on the field and allowed to self-pollinate to produce F2 seeds. One single seed from each F2 plant was collected and bulked to plant the F3 generation. F3 seeds were advanced by single seed descent (SSD) method until F6. Superior plants F6 were grown in the field, and the best lines were selected and assessed in progeny tests (F7). Progeny tests were carried out in a randomized block design, with three replications. Final yield assays were conducted in many locations for a 2-year period, and promising advanced breeding lines were thoroughly tested and compared to appropriate standards in environments representative across several places in the Brazilian states Minas Gerais (MG), Goiás (GO) Mato Grosso (MT),   We have assessed agronomic traits: number of days to flowering, plant height at flowering, number of days to maturity, plant height at maturity, first pod height, number of nodes on the main stem at maturity, number of pods with one, two and three seeds per plant, total number of pods per plant, number of seeds per pod and grain yield. We computed the number of days from the emergence to maturation when 95% of pods were dried; and the vegetative cycle by the number of days from emergence to flowering (50% of flowering plants); maturity was reported in the first day on which 95% of the pods turned brown. We measured first pod insertion hight from the soil level to the insertion of the first pod. We have assessed pod dehiscence using a scale ranging from 0 (no dehiscence) to 10 (complete dehiscence), and lodging resistance from 1 (no lodging) to 5 (all plants are prostrate) [8].
We assessed yielding per useful plots area to evaluate the productivity of cultivars and lineages; standardized to 13% of humidity and transformed into kilograms per hectare [9]. Oil and protein were assessed by NIR spectrophotometry and results were reported on an as is percentage basis.
The Scott-Knott test grouped means at 5% probability level. Experiments that had presented Coefficients of Variation higher than 20% were not computed in the yielding by region analysis.

Pratylenchus brachyurus resistance
We evaluated UFUS 8301 P. brachyurus resistance in a completely randomized design, with three replicates. The experiment was carried out from November 2013 to February 2014. A pure population of P. brachyurus inoculum was multiplied and maintained at greenhouse conditions in soybean plants. We have extracted P. brachyurus isolates from soybean roots through the method proposed by Coolen & D'Herde [10].
To assess damage and reproductive potentials of P. Brachyurus populations, we have measured the reproduction factor (RF). This statistics has been widely used in nematological studies to define resistance and susceptibility of plants to nematodes. According to Taylor & Sasser [11], since the reproductive ability on a given host is directly related to resistance nematode reproduction that can be used to measure root-knot nematode resistance. The FR is the ratio, R = final nematode population/initial nematode population, where R ≤ 1 indicates an inefficient host [12]. In our experiment, the RF was assessed 30, 60 and 90 days after inoculation. BRS MT Pintado was setting as the parameter of susceptibility, and Crotalaria spectabilis as the parameter of resistance. Santos et al. [13] have shown that the best moment for assessing the FR of soybean genotypes under greenhouse conditions range from 75 to 90 days afterward inoculation with P. brachyurus.
Analysis of variance (ANOVA) was used to test the differences between RF means. The null hypothesis stated there was no difference between the observed values and the expected value given by C. spectabilis, which is a poor-host Pratylenchus spp. The use of C. spectabilis in succession or rotation with soybean has been the most effective measure for the control of root-knot and rootlesion nematodes in infested areas [13,14]. RF means were grouped by Scott-Knott at 5 % of probability level to find out the homogeneous groups, whenever the situation had led to a significant F-test [16].

Distinctness, uniformity and stability (DUS)
DUS We evaluated the descriptors hypocotyl color, type of growth, pubescence color, flower color, pod color, the shape of the seed, integument color, and peroxidase reaction.

III.
RESULTS AND DISCUSSION We found UFUS 8301 distinct from any other cultivar, homogeneous to the descriptors that had identified it, as well stable through successive generations. The number of atypical plants found in the DUS experiments was equal to 3. Conquista was found the most similar cultivar to UFUS 8301; therefore, it was used for differentiation purposes; traits that differentiate them both are described in Table 2. UFUS 8301 presented determined growth and lodging resistance. Excessive vegetative growth might lead to lodging. Soybean lodging can reduce yield potential and increase harvest losses as lodged plants are more difficult to cut and gather into the combine [17]. Buzzello et al. [18] found a negative correlation between lodging and grain yield, and a positive correlation between plant height and lodging. UFUS 8301 has also shown to be pod dehiscence resistant. Pod dehiscence (shattering) is a significant source of yield loss of mechanically harvested soybeans. Harvesting shattering-susceptible soybean varieties in dry weather conditions can lead to seed losses of 50 to 100% [19].
Pereira Júnior et al. [20] described standard values of insertion of the first pod greater than 14 cm. However, according to Almeida et al. [21], the first pod insertion height should be higher than 10 cm because it is desirable to mechanical harvesting since that would avoid losses of uncollected pods due to its low insertion height. UFUS 8301 first pod height ranged from 10 to 11 cm (Table 3). Val et al. [22] assessed agronomic traits of 30 soybean genotypes in Jaboticabal, SP, 2012/2013 harvest, and observed first pod heights ranging from 5.40 to 20.73 cm; in particular, for Conquista, the most similar cultivar to UFUS 8301, that was equal to 13.07 cm.
As soybean is a photoperiod-sensitive and selfpollinated species, days to flowering and maturity, duration of flowering-to-maturity and plant height are crucial for soybean adaptability and yield [25]. The photoperiod influences soybean cultivars causing changes in behavior depending on the latitude, i.e., in Brazil, Arantes et al. [24] observed Conquista reaches maturity of about 130 days in Uberaba, MG (latitude 19 ° S); but in Sorriso, MT (latitude 15 ° S), it reached maturity at 110 days. UFUS 8301 has shown the life cycle of 118 to 129 days. Soybean flowering period is relatively extensive (ranging from 30 to 40 days) and overlaps with the formation of pods and seeds, which makes it resist short periods of drought during flowering [26]. Sudhanshu et al. [27] found that both days to 50% flowering and plant height have a direct effect on reducing seed yield. UFUS 8301 has shown 42 to 46 days to 50% flowering (Table 3).
Soybean yielding depends on numerous traits contributing to production, which might have their action linked. Therefore, the selection practiced on one characteristic may simultaneously bring change in the other related feature. Path coefficient analysis has shown that seed yield/plant is a positive and significant association with biological yield, pods per plant and 100seed weight, indicating that an intense selection for these characters will improve seed yield in soybean. Among these traits, 100 seed-weight exhibited the highest positive direct effect on seed yield [26]. The average weight of 100 seeds may vary depending on the sowing season and locality [24]; UFUS 8301 average 100-seed weight ranged from 15g to 14g (Table 3).
UFUS 8301 advantage to the producers was confirmed by comparing its productivity over other materials (Table  4). Besides, crop average national productivity estimated by the CONAB [28], from 2011 to 2013, recorded 2220.95 kg / ha 1 ; the average productivity of UFUS 8301 in the same period was 3687.5 kg ha -1 representing a productivity increase of 1.66 times. Modern soybean is one of the world's most important crops mainly because of its high protein (40%) and oil (20%) content [29]. However, soybean oil and protein  The diseases are among main factors limiting the increase of soybean yield. In recent years, many soybean diseases and plagues have already been reported for the crop; however, its incidence and severity depend on factors such as climate, cultivars, pathogen inoculum potential, soil structure and fertility, plant vigor, among others [30]. We found UFUS 8301 resistant to the virus VMCS (soybean mosaic virus), and to bacterium X. axonopodis pv. glycines (bacterial pustule) and P. syringae pv. glycinea (bacterial blight). Bacterial pustule and bacterial blight can occur in all soybean-producing regions. However, most of the cultivars in use are genetic resistance to these bacteria; therefore, nowadays, these are considered minor diseases problem [30]. Also, UFUS 8301 was resistant to C. sojina (frogeye leaf spot) and P. gregata. In Brazil, some C. sojina breeds have already been detected; fortunately, most soybean cultivars in use are genetic resistance to these breeds. However P. gregata, the fungus that causes the disease known as brown stem rot can be severe in soils with reduced fertility and when temperature and moisture are favorable for soybean seed development [30].
UFUS 8301 was also resistant to D. phaseolorum f. sp. meridionalis (stem canker) and F. solani (fusarium root rot). Stem canker is a fungus adapted to regions with higher temperature; all Brazilian soybean cultivars currently in use are resistant to this pathogen. The fusarium-root-rot damages vary and depend on the intensity of the inoculum and environmental conditions; however, the decline in yield in some soybean producing regions might be evident [30]. We also found UFUS 8301 moderate resistant to powdery mildew. The fungus M. diffusa, which is common in plants grown in greenhouses, causes this disease; however, the vast amount of spores formed on the leaf surface lately is easily disseminated by the wind, and this disease is gaining economic importance, leading to the need for chemical control in many soyproducing regions [30].
Regarding the root-knot nematodes M. incognita and M. javanica, UFUS 7401 have not shown resistance; however, it has proved to be unfavorable or poorly adapted to the P. brachyurus reproduction. There was no significate difference, by the Scott Knott test at 5% probability, between UFUS 8301 and C. spectabilis ( Table  6).  25.80 a Averages followed by the same letter constitute a homogeneous group by the Scott Knott test at 5% probability; b CV = Coefficient of Variation.

Parameter of Resistance Cultivars RF
There has been considerable progress in our knowledge of microorganisms and push-pull plants (e.g., C. spectabilis) that contribute to the biocontrol of nematodes [31]. According to Monteiro [32], despite Crotalaria species are generally unfavorable or poorly adapted to the reproduction of Pratylenchus spp., some breeds can multiply or remain in these plants as these reactions vary as species and their populations in both plants, and parasites. Therefore, this might explain our Crotalaria RF results superior to one (Table 6).

IV. CONCLUSION
We developed a new soybean cultivar with high productivity and resistant to P. brachyurus; that aspects represent comparative advantages over others cultivars current in use in Brazil. UFUS 8301 has been included in the National Registry of Cultivars (RNC) under P. brachyurus resistant status, register number 33899. Seed production has begun in by the Federal University of Uberlândia (UFU), Uberlândia -MG 2014, in compliance with the regulations of MAPA. Recommended cropping from October 20 to December 10 in growing seasons in Minas Gerais, Mato Grosso, Bahia, Piauí, São Paulo and Goiás states. Ideal population density is 240 to 270 thousand plants per hectare. Suggested production systems are no-till and conventional tillage.